MEDICINSKI GLASNIK Official publication of the Medical Association of Zenica-Doboj Canton, Bosnia and Herzegovina Volume 6 Number 2, August 2009.

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1 MEDICINSKI GLASNIK Official publication of the Medical Association of Zenica-Doboj Canton, Bosnia and Herzegovina Volume 6 Number 2, August ISSN

2 Published and copyright by: Medical Assotiation of Zenica-Doboj Canton; Address: Zenica, 72000, Bulevar kralja Tvrtka I 4, Bosnia and Herzegovina; tel./fax: ; ljkozedo@bih.net.ba, web site: http// For ordering information please contact: Tatjana Žilo, ljkozedo@bih.net.ba; Access to this journal is available free online trough: The Journal is indexed by EMBASE (Exerpta Medica), Scopus, Science Citation Index Expanded (SciSearch ), and Journal Citation Reports/Science Edition, EBSCO; ISSN Printed by: EG - ING & Graphic and web design studio B Panel Zenica, Armije BiH 2, info@bpanel.ba, tel ,

3 Medicinski Glasnik Official Publication of the Medical Association of Zenica-Doboj Canton Bosnia and Herzegovina Editorial Board Editor-in-chief Selma Uzunović-Kamberović Zenica, Bosnia and Herzegovina Technical editor Harun Drljević Zenica, Bosnia and Herzegovina Editors Adem Balić, Tuzla, Bosnia and Herzegovina Dubravka Bartolek, Zagreb, Croatia Branka Bedenić, Zagreb, Croatia Asja Čelebić, Zagreb, Croatia Josip Čulig, Zagreb, Croatia Filip Čulo, Mostar, Bosnia and Herzegovina Jordan Dimanovski, Zagreb, Croatia Branko Dmitrović, Osijek, Croatia Davorin Đanić, Slavonski Brod, Croatia Lejla Ibrahimagić-Šeper, Zenica, Bosnia and Herzegovina Tatjana Ille, Belgrade, Serbia Vjekoslav Jerolimov, Zagreb, Croatia Mirko Šamija, Zagreb, Croatia Ines Drenjančević-Perić, Osijek, Croatia Sven Kurbel, Osijek, Croatia Snježana Pejičić, Banja Luka, Bosnia and Herzegovina Belma Pojskić, Zenica, Bosnia and Herzegovina Asja Prohić, Sarajevo, Bosnia and Herzegovina Velimir Profozić, Zagreb, Croatia Zlatko Puvačić, Sarajevo, Bosnia and Herzegovina Radivoje Radić, Osijek, Croatia Amira Redžić, Sarajevo, Bosnia and Herzegovina Suad Sivić, Zenica, Bosnia and Herzegovina Sonja Smole-Možina, Ljubljana, Slovenia Vladimir Šimunović, Mostar, Bosnia and Herzegovina Adrijana Vince, Zagreb, Croatia Jasmina Vraneš, Zagreb, Croatia Živojin Žagar, Zagreb, Croatia Secretary: Tatjana Žilo; Proofreaders: Aras Borić (Bosnian, Croatian, Serbian), Glorija Alić (English), Cover: Jasmin Kukavica

4 MEDICINSKI GLASNIK Official Publication of the Medical Association of Zenica-Doboj Canton, Bosnia and Herzegovina Volume 6, Number 2, August 2009 Free full-text online at: and (DOAJ, Directory of Open Access Journals) Review 147 Značenje nastanka mikrobnog biofilma u patogenezi i liječenju kroničnih infekcija Significance of microbial biofilm occurence in the pathogenesis and treatment of chronic infections Jasmina Vraneš, Vladimira Leskovar Original article 166 Effect of inoculum size of Enterobacteriaceae producing SHV and CTX-M extended-spectrum β-lactamases on the susceptibility to β-lactam combinations with inhibitors and carbapenems Branka Bedenić, Jasmina Vraneš, Nataša Beader, Ines Jajić-Benčić, Vanda Plečko, Selma Uzunović-Kamberović, Smilja Kalenić 173 Comparison of the frequency and the occurrence of antimicrobial resistance among C. jejuni and C. coli isolated from human infections, retail poultry meat and poultry in Zenica-Doboj Canton, Bosnia and Herzegovina Selma Uzunović-Kamberović, Tina Zorman, Ingrid Berce, Lieve Herman, Sonja Smole Možina 181 Atelektaza pluća i infekcije donjih dišnih puteva u djece na odjelu za intenzivno liječenje Lung atelectasis and lower respiratory tract infections in children in the intensive care unit Nada Mladina, Devleta Hadžić, Amela Selimović 188 Evaluacija dijagnostičke vrijednosti Interleukina-6 i C-reaktivnog proteina iz krvi pupčanika u prepoznavanju rane infekcije terminske novorođenčadi male porođajne mase Diagnostic value of Interleukin 6 and C-reactive protein from umbilical cord blood in recognition of early infection in term newborns with low birth weight Almira Ćosićkić, Fahrija Skokić, Selmira Brkić 197 Alterations in body weight and biochemistry in patient treated with different psychotropic drugs in a clinic in Istanbul Aliye Ozenoglu, Serdal Ugurlu, Huriye Balci, Gunay Can, Funda Elmacıoglu, Yeltekin Demirel, Engin Eker 203 Klinička revizija lipidnog statusa kod tipa 2 dijabetesa na nivou timova obiteljske medicine u općini Zenica, Bosna i Hercegovina Audit of lipids control level for Diabetes Mellitus type 2 patients done by Family Medicine Teams in Zenica, Bosnia and Hercegovina Larisa Gavran, Selmira Brkić 211 Učestalost pušenja i nikotinska ovisnost kod medicinskih radnika Incidence of smoking and nicotine depedence among medical workers Željko Martinović, Cvita Martinović, Mladen Čuturić 218 Smoking is the most frequent risk factor for cardiovascular diseases in Croatian Western region: findings of the Croatian health survey Đulija Malatestinić, Nena Rončević, 1 Henrietta Benčević-Striehl, Suzana Janković, Vladimir Mićović

5 227 Influence of different glass fiber reinforcements on denture base polymer strength (Fiber reinforcements of dental polymer) Denis Vojvodić, Dragutin Komar, Zdravko Schauperl, Asja Čelebić, Ketij Mehulić, Domagoj Žabarović 235 Influence of cast surface finishing process on metal-ceramic bond strength Ketij Mehulić, Martina Lauš-Šošić, Zdravko Schauperl, Denis Vojvodić, Sanja Štefančić 243 Use of digital photography in the reconstruction of the occlusal plane orientation Nikola Petričević, Marko Guberina, Robert Ćelić, Ketij Mehulić, Marko Krajnović, Robert Antonić, Josipa Borčić, Asja Čelebić 249 A three-dimensional evaluation of microleakage of the class V cavities restored with flowables Paris Simeon, Silvana Jukić-Krmek, Goranka Prpić-Mehičić, Ivica Smojver, Ivica Anić, Ivica Pelivan 256 Oral health of the Croatian army recruits in 2001 Tomislav Badel, Jadranka Keros, Vjekoslav Jerolimov, Nikša Dulčić, Snježana Restek Despotušić 261 Security perception of a portable PC user (The difference between medical doctors and engineers): a pilot study Krešimir Šolić, Vesna Ilakovac Case report 265 Akutna bruceloza udružena sa Coombs-pozitivnom autoimunosnom hemolitičkom anemijom i diseminiranom intravaskularnom koagulacijom Acute brucellosis associated with Coombs-positive autoimmune hemolytic anemia and disseminated intravascular coagulation (DIC) Nerma Mušić Erratum Pneumolabirint Pneumolabyrinth Đenad Hodžić 271 Sphenochoanal polyposis Ivana Pajić-Penavić, Davorin Đanić, Ljubica Fuštar-Preradović 274 Primary extranodal Natural Killer/T-cell lymphoma of the ethmoid sinus masquerading as orbital cellulites Davorin Đanić, Ana Đanić Hadžibegović, Ivana Mahovne 277 Knee disarticulation Ognjen Živković, Antun Muljačić, Renata Poljak-Guberina 280 Izvanmaternična trudnoća izliječena metrotreksatom Extrauterine pregnancy treated with Metrotrexat Ljiljana Bilobrk Josipović, Branka Lovrinović, Anton Galić Medicinski Glasnik is indexed by EMBASE (Exerpta Medica), Scopus, Science Citation Index Expanded (SciSearch ), Journal Citation Reports/Science Edition and EBSCO

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7 REVIEW Značenje nastanka mikrobnog biofilma u patogenezi i liječenju kroničnih infekcija Jasmina Vraneš 1, 2, Vladimira Leskovar 2 1 Katedra za medicinsku mikrobiologiju, Medicinski fakultet Sveučilišta u Zagrebu, 2 Služba za mikrobiologiju, Zavod za javno zdravstvo Dr. Andrija Štampar ; Zagreb, Hrvatska Corresponding author: Jasmina Vraneš, Zavod za javno zdravstvo Dr. Andrija Štampar, Mirogojska cesta 16, Zagreb, Hrvatska Phone: ; Fax: ; jasmina.vranes@stampar.hr SAŽETAK Sposobnost adherencije bakterija na biotičke i abiotičke površine, funkcioniranje kao zajednica, te međusobna komunikacija bakterijskih stanica, od posebne su važnosti u nastanku kroničnih infektivnih bolesti. Sesilna zajednica mikroorganizama, danas poznata kao biofilm, povezuje se s brojnim bakterijskim infekcijama. Ključne karakteristike biofilm-infekcija jesu perzistencija infekcije, te rezistencija na antimikrobne lijekove i obranu imunološkog sustava domaćina. Napretkom tehnologije i primjenom novih mikroskopskih i molekularnih metoda u proučavanju ultrastrukture i funkcionalnim odnosima unutar biofilma, nastoji se pronaći novi terapijski pristup u kontroli biofilm-infekcija, koje su jedan od najvećih izazova 21. stoljeća. Ključne riječi: biofilm, kronične infekcije, patogeneza, liječenje Originalna prijava: 06. april 2009.; Korigirana verzija: 08. april 2009.; Prihvaćeno: 03. maj Med Glas 2009; 6(2):

8 Medicinski Glasnik, Volumen 6, Number 2, August 2009 UVOD U prirodi mikroorganizmi mogu egzistirati kao planktonski organizmi - individualne stanice koje slobodno plivaju u tekućem mediju; ili u obliku sesilne zajednice - biofilma. Biofilm je izrazito rasprostranjen način života u okolišu, gotovo na svakoj granici vode i zraka, te zemlje i vode (1, 2). U protekla dva desetljeća definicija biofilma se neprestano mijenjala jer svako novo istraživanje nadograđuje postojeće znanje o stvaranju, strukturi, sazrijevanju, te rezistenciji biofilma. Objedinjenjem spoznaja o već poznatim karakteristikama, te novootkrivenim fiziološkim osobinama, biofilm je danas definiran kao sesilna zajednica mikroorganizama čije su stanice ireverzibilno povezane sa supstratom i međusobno, te uklopljene u izvanstanični matriks polisaharidnih polimera koji su same stvorile, a ispoljavaju izmijenjen fenotip uslijed promijenjene brzine razmnožavanja i transkripcije gena koje ne uočavamo u planktonskih organizama (3). Formiranje biofilma započinje kondicioniranjem neke površine polimerima iz vodenog okoliša što omogućuje adherenciju mikroorganizama (2). Površine na kojima se može naći biofilm jesu, primjerice, metal, plastika, kamen, čestice zemlje, medicinski implantati, te tkiva (2). Nakon početnog, reverzibilnog vezivanja planktonskih stanica za površinu supstrata, slijedi stvaranje stabilne veze posredovane adhezinima na staničnoj stijenki bakterija, a potom proliferacija, te nakupljanje bakterijskih stanica u višeslojne stanične nakupine i stvaranje izvanstaničnog polisaharidnog matriksa. Održavanje takve višestanične zajednice bilo bi teško bez postojanja međustanične komunikacije bakterijskih stanica, posredovane malim signalnim molekulama sa sposobnošću difuzije u izvanstanični okoliš (4). Nakupljanje signalnih molekula u okolišu omogućuje svakoj pojedinoj bakterijskoj stanici procjenu stanične gustoće, odnosno ukupnog broja bakterija, a ta se pojava naziva detekcija kvoruma (engl. quorum sensing). Signalne molekule u gram-negativnih bakterija jesu N-acil-homoserinski laktoni (AHL), a u gram-pozitivnih su mali ili modificirani peptidi (Slika 1) (4). Koncentracija signalnih molekula postignuta pri točno određenoj, kritičnoj gustoći stanica, postaje dovoljna za aktivaciju gena uključenih u sintezu faktora virulencije ili sekundarnih metabolita (4, 5). Maturacija biofilma odvija se u pet razvojnih stadija (6). Prvi stadij je reverzibilno povezivanje, u kojem se planktonske stance tek kratkotrajno povezuju sa supstratom, a nakon toga slijedi drugi stadij, ireverzibilno povezivanje, u kojem se planktonske stanice čvrsto vežu na površinu supstrata i gube svojstvo pokretljivosti (6). Treći stadij jeste maturacija I za koji je karakteristično stvaranje izvanstaničnog matriksa, te povećavanje i višeslojnost mikrokolonijâ (6). Mikrokolonije, najčešće gljivolikog ili stupićastog izgleda, svoju maksimalnu veličinu dosežu u stadiju maturacije II (6). Proces maturacije biofilma završava petim stadijem, disperzijom, u kojem se između mikrokolonija formiraju vodeni kanalići, a same mikrokolonije mijenjaju svoj oblik u školjkasti, uslijed izdvajanja bakterijskih stanica smještenih u središnjem dijelu mikrokolonija u potrazi za novim i boljim izvorom hranjivih tvari (6). Biofilm može činiti samo jedna bakterijska vrsta, no češće se sastoji od više vrsta bakterija, ili bakterija i gljiva (1, 3). Jednom pričvršćeni za površinu, mikroorganizmi biofilma mogu, svojim aktivnostima, doprinositi ili štetiti zbivanjima u okolišu, ovisno o uvjetima koji u njemu vladaju. Poznato je da ove sesilne zajednice sudjeluju u razgradnji brojnih onečišćivača okoliša, stvaranju i razgradnji organske tvari, te u kruženju dušika, sumpora i drugih elemenata u prirodi (7-10). Prije saznanja o postojanju, strukturi i karakteristikama biofilma, biotehnolozi su već koristili prednosti biofilma u sustavima za pročišćavanje voda, pri uklanjanju opasnih tvari koje su kontaminirale zemlju i podzemne vode, te pri ekstrakciji plemenitih metala iz rudače (11-13). No, pozitivna djelovanja biofilma daleko su Slika 1. Stvaranje biofilma (Alma Šimunec Jović, 2007.) 148

9 Vraneš et al Biofilm i kronične infekcije rjeđa od štetnih koja uzrokuju ogromne ekonomske gubitke u agrokulturi i industriji, te mnogobrojne probleme u medicini. Biofilm se povezuje sa stafilokoknim mastitisom u goveda (14), biokorozijom (deterioracijom metalnih materijala u prisutnosti biofilma) vodenih sustava u industriji (15), te naftnih cjevovoda (16), kao i problemima u industriji prehrambenih i papirnatih artikala (17-18). U medicini biofilm se povezuje s brojnim kroničnim infekcijama (1), te različitim infekcijama biomaterijala poput kateterâ, protezâ, implantatâ, te drugih medicinskih naprava od metala ili plastičnih polimera koji se nalaze u ljudskom organizmu (1, 19). Prema procjenama Centra za kontrolu bolesti i prevenciju iz Atlante, biofilm se povezuje s gotovo dvije trećine bakterijskih infekcija (3). Brojnost, kroničan tijek, te rezistencija na antimikrobnu terapiju, tek su neke od značajki biofilm-infekcija, koje su jedan od najvećih izazova medicine 21. stoljeća. REZISTENCIJA BIOFILMA Perzistencija infekcija uzrokovanih mikrobnim biofilmom kontinuirano motivira istraživače u potrazi za jedinstvenim mehanizmom rezistencije biofilma, kako na antimikrobne lijekove, tako i na dezinfekcijska sredstva (3). Poznato je da su bakterije, unutar biofilma, puta rezistentnije na djelovanje antimikrobnih lijekova nego planktonske stanice, što govori u prilog postojanja mehanizma rezistencije kojeg potencijalno posjeduju svi patogeni, no koji se ispoljava samo pri tvorbi biofilma (20, 21). Proučavanjem biofilma takav mehanizam još nije utvrđen, niti su otkriveni mutanti u planktonskim kulturama koji bi govorili tomu u prilog (21), pa se trenutno rezistencija biofilma objašnjava tek hipotezama. Hipoteze starijeg datuma, kao moguće mehanizme rezistencije, navode oslabljenu difuziju antimikrobnih lijekova u biofilm (20, 22) ili promjene u mikrookolišu biofilma koje utječu na brzinu razmnožavanja mikroorganizama, odnosno djelotvornost antimikrobnih lijekova u dubini biofilma (20, 22). Oslabljena difuzija antimikrobnih lijekova objašnjava se dvojako; s jedne strane, interakcijom molekula s egzopolisaharidnim matriksom biofilma, koji djeluje poput molekularnog sita na velike molekule ili kao ionski izmjenjivač na hidrofilne, pozitivno nabijene molekule antimikrobnih lijekova (1, 22, 23); te, s druge strane, enzimskom razgradnjom, kojom se biofilm štiti od molekula, kao, primjerice, vodikovog peroksida ili β-laktamskih antimikrobnih lijekova (20, 23). Kao što je vidljivo, primjenjivost ove hipoteze ovisna je o prirodi samog lijeka, no nisu svi antimikrobni lijekovi visokoreaktivne molekule poput, primjerice, aminoglikozida, pa ih većina, vjerojatno, ipak penetrira u biofilm (23). Biofilm je prostorno heterogeničan (24). S porastom debljine slojeva stanica u biofilmu, mijenjaju se i uvjeti mikrookoliša, poput dostupnosti hranjivih tvari, prisutnosti kisika ili kiselih otpadnih metaboličkih tvari (20, 22, 25). Sve to utječe na brzinu razmnožavanja bakterijskih stanica, koje, u takvim uvjetima, prelaze u stacionarnu fazu u kojoj su manje osjetljive na baktericidno djelovanje antimikrobnih lijekova, osobito onih čije je ciljno mjesto djelovanja sinteza makromolekula (26). Novija hipoteza jeste hipoteza perzistera. Većina stanica biofilma, kao i planktonske stanice koje se oslobađaju s površine biofilma, osjetljive su na antimikrobnu terapiju (23, 27). Naprotiv, samo mala frakcija stanica (0,1-10% svih stanica biofilma) neosjetljiva je na produženu izloženost ili više koncentracije antimikrobnih lijekova, te perzistira usprkos terapiji (26). Premda su perzisteri otkriveni još sredinom 20. stoljeća u planktonskim kulturama (21), o njihovoj se prirodi još uvijek malo zna. Za sada je poznato da perzisteri nisu mutanti (21), te da ne predstavljaju poseban stadij staničnog ciklusa bakterija (23). Pretpostavlja se da je riječ o fenotipskoj varijanti koja je, iz divljeg tipa, spontano ušla u stanje promjenjivih fenotipskih obilježja (26). Mehanizam nastanka perzistera još je manje poznat. Do sada je utvrđeno da ove stanice ne nastaju kao odgovor na antimikrobnu terapiju (21, 27), te da bakterijske signalne molekule, kojima se detektira stanična gustoća, nemaju nikakva utjecaja na stvaranje perzistera (21, 27). U novije vrijeme pretpostavlja se da u kontroli stvaranja perzistera sudjeluju dva procesa. Jedan 149

10 Medicinski Glasnik, Volumen 6, Number 2, August 2009 je promjena razine specifičnih perzister-proteina (27), ovisno o fazi razvitka biofilma (21), a drugi je kontroliranje razine ekspresije tih proteina, koja ovisi o gustoći stanica (27), te o faktorima mikrookoliša, poput niske koncentracije supstrata (26). Misli se da perzister-proteini induciraju prelazak bakterija u stanje mirovanja, tzv. dormant fazu (27), što rezultira tolerancijom na antimikrobne lijekove uslijed utišane ekspresije gena u biosintetskim putevima ovih stanica, a time i onemogućavanjem djelovanja lijekova na njihova ciljna mjesta (21, 27). Ako znamo da perzisteri postoje i u planktonskim kulturama (23, 27), onda se nameće pitanje kako perzisteri biofilma doprinose njegovoj visokoj rezistenciji. Odgovor leži u sinergističkom djelovanju imunološkog sustava i antimikrobnih lijekova. Dok imunološki sustav uništava i uklanja zaostale planktonske perzistere (21, 23, 27), spram perzistera biofilma je nemoćan jer su ovi zaštićeni egzopolisaharidnim matriksom (23, 27) i nakon što se smanji koncentracija lijeka (21, 27) ili prekine terapija uslijed nestanka simptoma bolesti (23), perzisteri obnavljaju biofilm, oslobađaju se nove planktonske stanice i simptomi bolesti se vraćaju (21, 23). ULOGA BIOFILMA U NASTANKU KRONIČNIH INFEKCIJA U LJUDI Akutne bakterijske infekcije, koje izazivaju planktonski oblici patogenih bakterija, stoljećima su odnosile ljudske živote. Razvoj antimikrobnih lijekova i cjepiva omogućio je stjecanje kontrole nad ovim akutnim bolestima, te se može reći da one sada, uglavnom, pripadaju prošlosti. Sredinom prošlog stoljeća započela je postantibiotska era, odnosno era biofilm-infekcija. Ove infekcije perzistiraju mjesecima i godinama, izmjenjuju se periodi odsutnosti kliničkih simptoma infekcije s akutnim egzacerbacijama, manje su agresivne od akutnih infekcija, te pogađaju umjereno kompromitirane osobe (28). Uzročnici biofilm-infekcija jesu bakterijske vrste koje se nalaze u okolišu ili koje nalazimo kao komenzale ljudskog organizma (3, 29), a koje već stoljećima, u svom prirodnom okolišu, postoje u obliku biofilm-zajednice. Biofilm se, u ljudskom organizmu, razvija na vitalnom ili nekrotičnom tkivu, te na inertnim površinama različitih biomaterijala (29). Bez obzira radi li se o kroničnoj infekciji ili o infekciji biomaterijala, ove biofilm-infekcije imaju iste kliničke karakteristike, a to su spor nastanak na jednom ili više mjesta u organizmu, te kasna pojava simptoma bolesti (29). Nekoliko karakteristika biofilm čini jedinstvenim u procesu nastanka infekcije. Prva od njih jeste mogućnost disperzije stanica ili agregata biofilma, što može rezultirati nastankom embolusa, ili pak infekcijom mokraćnog ili krvožilnog sustava (2, 30). Disperzija pojedinih stanica nastaje uslijed odvajanja stanica-kćeri iz aktivno-razmnožavajućih stanica, te kao posljedica promjene dostupnosti hranjivih tvari ili detekcije kvoruma (2), a odvajanje manjih dijelova biofilma nastaje pod utjecajem brzine protoka tekućine u neposrednoj blizini površine biofilma (2, 30). Dok pojedine stanice, ubrzo nakon odvajanja, poprimaju fenotip planktonskih stanica, čini se da čestice biofilma zadržavaju određene karakteristike biofilma, poput antimikrobne rezistencije (2, 30). Antimikrobnom terapijom eradiciraju se planktonske stanice oslobođene iz biofilma, što rezultira povlačenjem simptoma akutne egzacerbacije bolesti, no ne uspjeva se uništiti sesilna zajednica (2, 29, 30). Rekurirajući simptomi biofilm-infekcije u konačnici će nestati tek nakon uklanjanja biofilma kirurškim zahvatom ili odstranjenjem inficiranog biomaterijala (29, 30). Slijedeća jedinstvena karakteristika biofilm- infekcija jeste rezistencija biofilma na stanični i humoralni odgovor imunološkog sustava domaćina. U početku se smatralo da rezistenciji pridonosi interferencija egzopolisaharidnog matriksa biofilma s kemotaksijom polimorfonuklearnih leukocita (PMN) (31), te s prodorom baktericidnih i opsonizirajućih protutijela (28, 31). Danas je poznato da aktivirani PMN penetriraju u biofilm, ulaze u vodene kanaliće, te penetriraju u mikrokolonije, no tamo ne uspijevaju fagocitirati stanice biofilma koje se nalaze u njihovoj neposrednoj blizini (32). Mehanizam ove neuspješne fagocitoze tek predstoji objasniti (32). Opsonizirajuća protutijela, dio humoralne obrane, u slučaju biofilm- 150

11 Vraneš et al Biofilm i kronične infekcije infekcije, dijele sudbinu polimorfonuklearnih leukocita. Iako prodiru u dubinu biofilma (33), u matriks ulaze u interakciju s antigenima koji su tamo u suvišku, te uslijed toga ne dospijevaju do antigena na površini stanica biofilma, što onemogućuje njihovo opsonizirajuće djelovanje i posljedično uništavanje stanica biofilma (33). Perzistencija kroničnih infekcija, nemogućnost detekcije mikrobnog uzročnika standardnim mikrobiološkim tehnikama kultivacije u većini slučajeva, te neuspjeh antimikrobne terapije i imunološkog sustava domaćina u eradikaciji uzročnika, naveli su neke znanstvenike na pomisao da kronične infekcije uzrokuje biofilm. Ovu tezu valjalo je potkrijepiti istraživanjem sesilnih zajednica in situ, što, uslijed nedostatka raspoloživih metoda, nije bilo ostvarivo sve do unazad dvadesetak godina. Razvoj i primjena konfokalnog skenirajućeg laserskog mikroskopa (engl. confocal scanning laser microscope, CSLM) omogućila je istraživanje ultrastrukture biofilma (1-3), a nove molekularne tehnologije, poput primjene fluorescentnih rrnk-usmjerenih proba, te fluorescentne in situ hibridizacije (FISH), doprinijele su identifikaciji i kvantifikaciji uzročnikâ biofilma, te razumijevanju njihovih međusobnih funkcionalnih odnosa (1). Mogućnost vizualizacije i proučavanja biofilma na biomaterijalima, te u humanim uzorcima, rezultirala je intenzivnim istraživanjima brojnih infekcija i njihove povezanosti sa stvaranjem biofilma (13). Danas se biofilm, uz infekcije biomaterijala, povezuje s brojnim kroničnim infekcijama. Najčešće kronične infekcije, te njihove uzročnike, prikazuje Tablica 1. PERIODONTITIS U periodontalne bolesti ubraja se cijeli niz poremećaja potpornog tkiva zubi, od kojih pobolijeva gotovo 90% svjetske populacije. Najblaži oblik periodontalne bolesti je gingivitis, reverzibilna upala gingive (desni), a najteži, kronični periodontitis, kronična destrukcija periodontalnog tkiva (gingive, periodontalnog ligamenta i alveolarne kosti) koji može rezultirati gubitkom zubi (3). Primarno mjesto periodontalne infekcije jeste prostor između korijena zuba Tablica 1. Najčešći uzročnici kroničnih infekcija povezanih s biofilmom (The most common etiology of chronic infections involving biofilm) Kronična infekcija Chronic infection Endokarditis nativnih valvula Native valve endocarditis Periodontitis Periodontitis Cistična fibroza Cystic fibrosis Kronični bakterijski prostatitis Chronic bacterial prostatitis Kronični cistitis Chronic cystitis Inficirani bubrežni kamenci Infected kidney stones Kronični otitis media Chronic otitis media Mišićno-koštane infekcije Musculoskeletal infections Nekrotizirajući fasciitis Necrotizing fasciitis Infekcije bilijarnog sustava Biliary tract infection Osteomijelitis Osteomyelitis Meloidoza Meloidosis Kronične rane Chronic wounds Uobičajen bakterijski patogen biofilma Common biofilm bacterial pathogen Streptokoki viridans grupe Viridans group streptococci Gram-negativne anaerobne bakterije Gram-negative anaerobic bacteria Pseudomonas aeruginosa Pseudomonas aeruginosa Gram-negativne enterobakterije Gram-negative enterobacteria Uropatogena Escherichia coli Uropathogenic Escherichia coli Ureaza-producirajuće enterobakterije Ureasa-producing enterobacteria Haemophylus influenzae, Streptococcus pneumoniae Haemophylus influenzae, Streptococcus pneumoniae Gram-pozitivni aerobni koki Gram-positive aerobic cocci Streptococcus pyogenes Enterobacteriaceae Različite bakterijske i gljivične vrste Various bacterial and fungal species Pseudomonas pseudomallei Pseudomonas pseudomallei Različite aerobne i anaerobne bakterijske vrste Various aerobic and anaerobic bacterial species 151

12 Medicinski Glasnik, Volumen 6, Number 2, August 2009 i gingive, koji se naziva subgingivalna pukotina, a koji se, s progresijom bolesti, može produbiti u periodontalni džep. Inače, smatra se da periodontitis nastaje kao posljedica poremećaja ekvilibrija u dentalnom plaku (34). Dentalni plak je mikrobni biofilm kojeg sačinjavaju mikroorganizmi normalne flore usne šupljine (34). Iako je dentalni plak najproširenija sesilna zajednica u ljudi, klinički se znakovi periodontitisa javljaju samo u onih kod kojih kronično prisustvo dentalnog plaka izaziva pojavu imunološkog odgovora i upale (35). To je uvjetovano osjetljivošću domaćina ili promjenom uvjeta u mikrookolišu usne šupljine (36). Osjetljivost domaćina određuju genetski i okolišni faktori, te stečene navike poput pušenja (35). Iako je upalni odgovor domaćina protektivan, destrukcija gingivalnog i periodontalnog tkiva može nastati ukoliko je preslabo ili prejako izražen (35). Destrukciji tkiva doprinosi i ekspresija faktora virulencije mikroorganizama u trenutku kada se, uslijed povećanog protoka tekućine u gingivalnim pukotinama i dotoka hranjivih tvari, te povišenja ph, mijenja mikrookoliš u oralnoj šupljini (34, 36). Nastanak dentalnog plaka odvija se u tri koraka. Neposredno, nakon čišćenja površine zubi, izloženi dijelovi cakline bivaju obloženi proteinskim kondicionirajućim filmom, koji služi kao supstrat ranim bakterijskim kolonizatorima (1, 2, 37). Predominantni kolonizatori ranog biofilma su streptokoki (38), poput bakterija Streptococcus gordonii, S. sanguis, S. oralis, S. parasanguis i brojnih drugih (1, 38). Čimbenici koji favoriziraju streptokoke, kao inicijalne kolonizatore, jesu ekspresija adhezinâ koji prepoznaju receptore na kondicionirajućem filmu; sposobnost da, kao jedine izvore hranjivih tvari, metaboliziraju komponentne sline; te izrazita sposobnost intra- i inter-generičke koagregacije (38, 39). Procesom koagregacije, međustaničnog prepoznavanja i adherencije genetički različitih bakterija, te metaboličkim interakcijama drugih bakterija sa streptokokima, rani biofilm ubrzo postaje multigenerička mikrozajednica koju čine aerobne i aerotoleratne bakterijske vrste rodova Gemella, Actinomyces, Veillonella, Haemophilus, te Neisseria (38, 40). Skenirajućim elektronskim mikroskopom uspješno je prikazana dinamika stvaranja oralnog biofilma (39). Pojedinačne i manje nakupine stanica uočavaju se već nakon četiri sata, mikrokolonije nakon osam sati, a nakon 12 sati na caklini se može uočiti monosloj bakterija. Ključnim posrednikom procesa koagregacije između ranih i kasnih kolonizatora u dentalnom plaku smatra se bakterija Fusobacterium nucleatum (41). Također se smatra kako ova gram-negativna bakterija promovira stvaranje anaerobnog mikrookoliša koji striktnoanaerobnim, kasnim kolonizatorima, omogućuje preživljavanje u aerobnoj atmosferi (41). Kasni kolonizatori su gram-negativne anaerobne bakterije, poput Porphyromonas gingivalis, Tanerella forsythensis i Treponema denticola (42). Apikalnom progresijom supragingivalnog plaka nastaje subgingivalni plak, a promjenom integriteta spojnog epitela dolazi do postepene kolonizacije površine zuba i stvaranja periodontalnog džepa. U studijama bakterijske etiologije subgingivalnog biofilma, a time i periodontitisa, najčešće su bile korištene klasične metode kultivacije i molekularne identifikacijske metode, koje su, kao periodontalne patogene, identificirale proteolitičke vrste bakterija, koje su već navedene kao kasni kolonizatori dentalnog plaka (42). Kloniranjem i sekvencioniranjem gena bakterijske 16S rrna u uzorcima subgingivalnog plaka osoba s periodontitisom, otkrilo se kako bakterije P. gingivalis, T. forsythensis i T. denticola čine tek manji dio zajednice, a kako dominiraju bakterije Peptostreptococcus sp. i Filifactor sp. Prevencija nastanka periodontitisa bazira se na kontroli nastanka oralnog biofilma, no za sada su se mehaničko odstranjivanje i kemijska kontrola pokazali neuspješnim (34). Nove strategije prevencije nastanka oralnog biofilma, poput interferencije transdukcije signala u biofilmu, modifikacije površine zubi, zamjena potencijalno patogenih s genetski modificiranim, manje virulentnim mikroorganizmima, te imunizacija, tek su početak nastojanja kontrole oralnog biofilma (34). Koja će od ovih strategija biti uspješna u prevenciji i kontroli oralnog biofilma pokazat će budućnost. 152

13 Vraneš et al Biofilm i kronične infekcije KRONIČNI OTITIS MEDIA Nakon obične prehlade, otitis media (OM) ili upala srednjeg uha, najčešći je razlog posjeta liječniku u dječjoj dobi. Do svoje treće godine života, gotovo 75% dječje populacije doživjet će najmanje jednu akutnu epizodu OM-a. Kronični OM dijeli se u dva podtipa - rekurentni (ROM) i eksudativni otitis media (eng. otitis media with effusion, OME) (43, 44). Dijagnoza rekurentnog OM-a postavlja se nakon tri ili više epizoda OM-a, tijekom šest mjeseci, između kojih nema kliničkih znakova bolesti (43, 44), a eksudativnog, ukoliko sekrecija perzistira duže od tri mjeseca (43). Kronični OM najčešći je uzrok provodnog oštećenja sluha u dječjoj dobi, što može imati za posljedicu otežan razvoj govora, a potom i socijalizacije djeteta (44). Primjena antimikrobnih lijekova u liječenju kroničnog OM-a, vrlo često rezultira terapijskim neuspjehom. Ukoliko eksudat u srednjem uhu perzistira dulje od šest tjedana, kirurškim se zahvatom u bubnjište postavlja cjevčica za kontinuiranu drenažu. Dugo vremena kronični OM smatrao se isključivo upalnim procesom usmjerenim spram zaostalih bakterijskih metabolita, a eksudat sterilnim (45). Tek od godine, otkrićem bakterija u eksudatu (43), patogeneza kroničnog OM-a objašnjava se bakterijskom infekcijom (46). Klasičnim metodama kultivacije u aspiratu eksudata srednjeg uha, tek u 20-40% slučajeva, može se dokazati bakterijski uzročnik kroničnog OM-a (43). Prema učestalosti, to su bakterije Haemophylus influenzae (8-20%), Streptococcus pneumoniae (4-10%) i Moraxella catarralis (2-8%), te manje zastupljeni patogeni, poput bakterija Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pyogenes i Pseudomonas aeruginosa (43, 46). Primjena visokoosjetljivih tehnika molekularne biologije, kao što je reakcija lančane polimeraze (engl. polymerase chain reaction, PCR), u detekciji bakterijske DNK tri najčešća patogena u aspiratu eksudata, rezultirala je pozitivnim nalazom u 80% slučajeva kroničnog OM-a (43, 44). Isprva velika razlika u detekciji uzročnika kroničnog OM-a klasičnim i PCR metodama, objašnjavala se činjenicom da početnice (engl. primer) korištene u PCR tehnologiji umnožavaju male bakterijske DNK fragmente, koji ne moraju nužno značiti prisutnost viabilnih mikroorganizama, već predstavljaju samo intaktne fragmente DNK uništenih bakterija ili ostatke patogena u uzorku (43, 47, 48). Ubrzo su uslijedila istraživanja koja su to opovrgla. Post i suradnici dokazali su, na animalnom modelu OM, da se pročišćena bakterijska DNK, te DNK toplinom inaktiviranih bakterija ne može detektirati dulje od tri dana u eksudatu bubnjišta (49). U istom je istraživanju uočeno da ubrzo, nakon primjene antimikrobnih lijekova, više nije moguće dokazati prisutnost uzročnika klasičnim metodama kultivacije, no PCR tehnologijom DNK patogena u uzorku može se detektirati i do tri tjedna nakon terapije (49). Slijedeći korak u istraživanjima bio je dokazivanje prisutnosti uzročnika u uzorcima. To je ostvareno detekcijom bakterijske glasničke ribonukleinske kiseline (mrnk) bakterije H. influenzae u uzorcima eksudata bubnjišta bolesnika s dijagnozom OME (50). Imajući na umu da je mrnk uzročnika infekcije molekula, čiji se životni vijek mjeri u sekundama i minutama, te da se sintetizira isključivo u intaknom mikroorganizmu, zaključeno je kako se u uzorku nalazi metabolički aktivan patogen (50). Nemogućnost uzgoja uzročnika klasičnim metodama kultivacije, te rezistencija pri primjeni antimikrobnih lijekova, rezultirala je postavljanjem hipoteze o bakterijskom biofilmu kao mogućem etiološkom faktoru u kroničnom OM-u. Koncept mukoznog biofilma zaživio je tek vizualizacijom sesilne zajednice na sluznici srednjeg uha, prvo na animalnim modelima (45, 51), a potom i na bioptatima sluznice djece s kroničnim OM-om (44). Istraživački tim američkog Centra za genomske znanosti (Center for Genomic Sciences, Pittsburgh, Pennsylvania), i godine, primjenom skenirajućeg elektronskog mikroskopa (SEM), detektirao je prisutnost bakterijskog biofilma na sluznici srednjeg uha, u prvom animalnom modelu, upotrijebivši u pokusu činčile (45, 51). Skenirajući uzorke elektronskim mikroskopom, pojava prvih mikrokolonija zamijećena je već 24 sata nakon indukcije eksperimentalnog OM-a, injiciranjem bakterije H. 153

14 Medicinski Glasnik, Volumen 6, Number 2, August 2009 influenzae u bubnjište činčila (45). Prisutnost biofilma detektirala se i 21. dan nakon inokulacije, a viabilnost bakterija, unutar biofilma, potvrdila primjenom CLSM-a i diferencijalnih vitalnih boja (45). Ubrzo je detektirano stvaranje biofilma, na životinjskom modelu, i pri injiciranju bakterije P. aeruginosa u bubnjište makaki majmuna (52). Nakon studija na animalnim modelima, godine, primjenom CLSM-a, dokazan je mukozni biofilm u 46 od 50 bioptičkih uzoraka sluznice srednjeg uha, djece s OME-om i rekurentnim OM-om (44). To je bio ključni dokaz da kronična infekcija srednjeg uha nije rezultat sterilnog upalnog procesa, već indolentne bakterijske infekcije, te da se neuspjeh antimikrobne terapije i obrane imunološkog sustava domaćina, može objasniti postojanjem biofilma u patogenezi ove bolesti. KRONIČNA PLUĆNA INFEKCIJA U BOLESNIKA S CISTIČNOM FIBROZOM Cistična fibroza (CF) je kronična, genetski uvjetovana, bolest donjeg respiratornog sustava. Godine otkriven je gen za cističnu fibrozu, a njegov produkt označen je kraticom CFTR (regulatorni protein transmembranskog prijenosa u cističnoj fibrozi). Ovaj regulatorni protein djeluje kao kloridni kanal u epitelnim stanicama respiratornog sustava. U bolesnika oboljelih od cistične fibroze, izrazito je oštećen transport kloridnih iona, što za posljedicu ima dehidraciju i zgušnjavanje sluzi koja normalno prekriva respiratorni epitel, te oštećenje mukocilijarnog sustava koji pomaže u odstranjivanju inhaliranih čestica. Oštećenje ovog primarnog neupalnog obrambenog mehanizma, dovodi do začepljenja malih dišnih puteva hiperviskoznom sluzi, sekundarne akutne ili kronične bakterijske infekcije, te aktivacije upalnog obrambenog mehanizma pluća (53, 54). U slučaju perzistencije infekcije, aktivacija PMN i oslobađanje njihove DNA, doprinosi viskoznosti sekreta u dišnim putevima, a stvaranje IgG protutijela rezultira nastankom imunokompleksa, koji oštećuju plućno tkivo (53, 54). U najranijoj životnoj dobi, u ovih bolesnika, kao prvi kolonizatori dišnih puteva, dominiraju sojevi bakterija S. aureus i H. influenzae koji se, uz oštećeni mukocilijarni klirens, te povećanu ekspresiju receptora za bakterije na površini epitelnih stanica, smatraju predisponirajućim faktorima za kolonizaciju sojevima P. aeruginosa (3, 55). Prevalencija kolonizacije ovim oportunističkim patogenom, kod bolesnika odrasle dobi, gotovo je 80% (55). Dišni sustav, sinusi, te probavni trakt (pretpostavlja se uslijed gutanja sputuma), inicijalno se koloniziraju nemukoidnim sojevima P. aeruginosa, koji se tipično nalaze u okolišu (55). Interferencijom faktora virulencije (posebno toksina elastaze i alkalne proteaze) bakterije P. aeruginosa s nespecifičnom i specifičnom imunološkom obranom domaćina, početna intermitentna prelazi u perzistentnu kolonizaciju (55). Tijekom perzistentne P. aeruginosa kolonizacije, na bakterije djeluju dehidrirani i visokoosmolarni mikrookoliš u dišnom sustavu, te slobodni radikali kisika, nastali uslijed PMNodgovora na kolonizaciju, što rezultira promjenom nemukoidnog fenotipa sojeva u mukoidni, uslijed prekomjerne produkcije alginata (53-56). Alginat, nerazgranati, linearni egzopolisaharid, koji se sastoji od monomera manuronske i guluronske kiseline, glavna je komponenta matriksa mikrokolonija koje P. aeruginosa stvara u dišnim putevima, te jedini antigen ovog patogena, koji se izravno povezuje s lošom kliničkom prognozom kod ovih bolesnika (53, 55). Tranzicija sojeva u mukoidni fenotip povezuje se s indukcijom transkripcije algc, ključnog gena za biosintezu alginata, te inaktivacijskom mutacijom muca gena, koji kodira antisigma faktor algt gena (1, 56). Posljedica mutacije muca gena jeste porast razine sigma faktora, produkta algt gena, koji, za sada, nepoznatim mehanizmom, utječe na regulaciju sinteze flagela, što rezultira izostankom pokretljivosti bakterije (1, 56). Pojava mukoidnog fenotipa korelira s povećanim stvaranjem protutijela na gotovo sve antigene (uključujući i alginat) i toksine P. aeruginosa, te lošom kliničkom prognozom, što se povezuje s kroničnom upalnom reakcijom u kojoj dominiraju PMN, aktivacija komplementa i lokalna produkcija proupalnih citokina, te stvaranje imunokompleksa u kojima je glavni antigen lipopolisaharid (LPS) bakterijske stijenke (53, 55). U 154

15 Vraneš et al Biofilm i kronične infekcije prilog hipotezi da kronični endobronhiolitis, u bolesnika s CF-om, uzrokuje bakterija P. aeruginosa, koja stvara biofilm, govore elektronskomikroskopski prikazi mikrokolonija u sputumu bolesnika i uzorcima plućnog tkiva, uzetim prigodom autopsije (53, 56), te detekcija homoserinlaktonskih signalnih molekula u sputumu bolesnika (58). U proteklih desetak godina, bakterija P. aeruginosa postala je ogledni mikroorganizam za istraživanje biofilma i uočeno je kako formiranje biofilma varira s obzirom na raspoložive nutritivne izvore, te uvjete okoliša. Iako je model heterogenog biofilma trenutno prihvaćen kao model nastanka P. aeruginosa biofilma, sve je više istraživanja koja upućuju na potrebu nadopune ovog modela (58). Prema navedenom modelu, koji je uočen pri korištenju glukoze kao izvora ugljika, u adherenciji bakterija i stvaranju pojedinačnog sloja bakterija sudjeluju flagele, dok tip IV fimbrije omogućavaju kretanje bakterije po površini, te agregaciju stanica i stvaranje mikrokolonija, a na stvaranje gljivolikih višestaničnih zajednica, u procesu maturacije, utječe detekcija kvoruma (58). Korištenjem citrata, kao izvora ugljika, uočeno je da P. aeruginosa stvara ravan biofilm, koji se bitno razlikuje od prethodno opisanog heterogenog modela (58). U ovom alternativnom modelu, flagele nisu sudjelovale u adherenciji bakterija za supstrat, mikrokolonije su nastale klonalnim rastom jedne stanice, a kretanje posredovano tip IV fimbrijama rezultiralo je širenjem bakterija duž supstrata, uz izostanak većih mikrokolonijskih struktura (58). Nedavno je iz soja 57RP P. aeruginosa biofilma, izolirana i visokoadherentna, mala fenotipska varijanta, sa prekomjerno izraženim fimbrijama, koja ima povećane sposobnosti stvaranja biofilma (59). Premda se u kronično inficiranih CF bolesnika susreće jedan ili tek nekoliko genotipova P. aeruginosa, značajne fenotipske varijabilnosti izolata rezultat su učestalih promjena okoliša u plućima bolesnika, te odgovora ove bakterije na te promjene fenotipskom adaptacijom (59, 60). Otkrivanje mehanizama koji omogućuju fenotipsku adaptaciju, te daljnje istraživanje stvaranja biofilma u kronično inficiranih bolesnika, povezuje se s uspješnijom kontrolom i eradikacijom infekcije u oboljelih. U kronično inficiranih bolesnika s CF-om, trajna eradikacija P. aeruginosa gotovo je nedostižan cilj, pa su potrebne druge kratkotrajne i prijelazne terapijske mjere s ciljem smanjenja broja bakterija u sputumu, poboljšanja funkcije pluća, te odgađanja nepovratnih oštećenja plućnog tkiva (55). Dugotrajna perzistencija P. aeruginosa, te rezistencija na primjenu antimikrobnih lijekova u bolesnika s CF-om, pripisuje se mutacijama bakterija i izmjeni genskog materijala koji doprinose rezistenciji na lijekove, smanjenoj djelotvornosti lijekova u anaerobnim uvjetima, te biofilmspecifičnim mehanizmima rezistencije (61). Nemogućnost trajne eradikacije i rezistencija patogena naglašavaju potrebu za prevencijom ili odgodom uspostavljanja kronične infekcije već u fazi intermitentne kolonizacije ranom i agresivnom antimikrobnom terapijom (55). Terapijske mjere, poduzete nakon inicijalnog izoliranja P. aeruginosa, poput inhalacija kolistina ili tobramicina, te oralne primjene ciprofloksacina, uz kohortnu izolaciju bolesnika, pokazale su se uspješnim u prevenciji i epidemiologiji kronične P. aeruginosa infekcije (62). Nove spoznaje rezultirale su promjenom terapijskog pristupa u kroničnoj infekciji, pa je prethodna terapija samo akutnih egzacerbacija kronične infekcije zamijenjena kroničnom supresivnom kemoterapijom kojom se nastoji smanjiti broj i aktivnost bakterije P. aeruginosa u plućima bolesnika s ciljem poboljšanja plućne funkcije (53). Iako mehanizam djelovanja antimikrobnih lijekova u kroničnoj infekciji povezanoj s biofilmom nije razjašnjen, in vitro se primjenom kombinacije β-laktama i aminoglikozida, najčešće korištenih antipseudomonasnih lijekova, uočilo smanjenje broja bakterija u sesilnoj zajednici na svega 20%, a pri primjeni pojedinih antibiotika u subinhibicijskim koncentracijama (submik), supresija stvaranja proteaze, fosfolipaze C, te alginata u P. aeruginosa (53, 63). Antimikrobna terapija rezultira smanjenjem prisutnosti bakterijskih antigena, a time i imunološki uzrokovanog oštećenja plućnog tkiva (53). Rezultati novijih istraživanja pokazali su djelotvornost makrolida u biofilm infekcijama uzrokovanih bakterijom P. aeruginosa, 155

16 Medicinski Glasnik, Volumen 6, Number 2, August 2009 što se povezuje s njihovom protuupalnom aktivnošću, te submik učinkom, poput inhibicije adherencije, pokretljivosti bakterije, te detekcije kvoruma (64-66). Istraživanjima kronične P. aeruginosa infekcije u bolesnika s cističnom fibrozom, nastoji se otkriti učinkovita terapija koja bi prevenirala nastanak ili omogućila uništenje biofilma u ovih bolesnika. KRONIČNI CISTITIS Infekcije mokraćnog sustava (IMS), nakon respiratornih, druge su po učestalosti bakterijske infekcije. Većina se ovih infekcija javlja kod mladih i zdravih žena. Gotovo 80% žena svjetske populacije, tijekom svoga života, ima najmanje jednu IMS, a 27-44% ovih bolesnica, unutar slijedećih šest mjeseci, ima i rekurentnu epizodu bolesti, unatoč antimikrobnoj terapiji (67, 68). U gotovo 80% slučajeva IMS-a kao uzročnik se izolira uropatogena Escherichia coli (UPEC) (68, 69). Bakterijski biofilm ima važnu ulogu u patogenezi, perzistenciji i terapiji infekcija mokraćnog sustava. Naime, stvaranje biofilma u mokraćnom sustavu povezuje se s kroničnim cistitisom, te struvitnom urolitijazom. U posljednje tri godine intenzivno se proučava povezanost progresije IMS-a u perzistentnu UP- EC-induciranu infekciju, u nekoliko in vitro i animalnih in vivo modela (68, 70-72). Kao ključni faktor uspostavljanja biofilma uropatogene E. coli na abiotičkim površinama, u stacionarnim uvjetima i uvjetima hidrodinamičkog protoka, te luminalnoj površini mišjeg mokraćnog mjehura, smatra se FimH adhezin, koji se nalazi na vrhu tipa 1, manoza-senzitivnih fimbrija (71, 72). Ovaj adhezin veže manozilirane ostatke na integralnim membranskim proteinima, uroplakinima (UP), koji su izraženi na luminalnoj strani superficijalnih epitelnih stanica mokraćnog mjehura (70). Udruživanjem četiri poznata UP molekula (UP Ia, Ib, II i III) nastaju heksamerni prsteni, a organizacijom prstena nastaju kristalni plakovi, koji prekrivaju gotovo cijelu luminalnu površinu mokraćnog mjehura sa zadaćom očuvanja integriteta epitelne membrane, te stvaranja kemijski nepropusnog sloja za difuziju u epitel (70). U mišjem modelu cistitisa, primjenom skenirajućeg i transmisijskog elektronskog mikroskopa, zamijećeno je da, jedan do tri sata nakon adheriranja bakterije na površinu epitelnih stanica, dolazi do brzog prodora UPEC-a u epitelne stanice. Prodor bakterije u stanicu posredovan je FimH adhezinom koji otpočinje signalnu kaskadu u domaćinovim epitelnim stanicama, što rezultira lokaliziranim preslagivanjem aktina, te uvlačenjem adherirane bakterije u stanicu zatvaranjem membrane oko mikroorganizma (68, 70, 72). Nakon invazije u superficijalne epitelne stanice mokraćnog mjehura, UPEC se ubrzano dijeli u citoplazmi, te u narednih osam sati, stvara slabo povezane, male nakupine bakterija, koje zadržavaju tipičnu morfologiju štapića (68, 70, 71). Uspostavljanjem ovih ranih intracelularnih bakterijskih zajednica (IBZ), bakterija stvara protektivni okoliš u kojem je zaštićena od djelovanja antimikrobnih lijekova, te domaćinova imunološkog odgovora (68, 70). Šest do osam sati nakon infekcije, dolazi do dramatične fenotipske promjene IBZ-a, te rana IBZ sazrijeva u zajednicu nalik na biofilm (70, 71). Bakterije u biofilmu poprimaju kokoidni oblik i ispunjavaju cijelu superficijalnu epitelnu stanicu; brzina rasta bakterija usporava na više od jednog sata, male nakupine bakterija povezuju se u gustu organiziranu zajednicu globularnog izgleda (70, 71). U ovom stadiju, bakterije na svojoj površini imaju izražena brojna amiloidna vlakna, koja omogućavaju stvaranje individualnog odjeljka za svaku bakteriju, te površinske molekule, poput tip 1 fimbrija i antigena 43, koje doprinose interakcijama, tijekom stvaranja mikrokolonija (68, 70). Ključni proces maturacije intracelularnog biofilma, koji tvori E. coli, jeste sekrecija kolanske kiseline, egzopolisaharida koji stvara polisaharidni matriks oko bakterijskih stanica (68, 70). Vizualizacijom ovog stadija infekcije, na površini inficiranog mišjeg mokraćnog mjehura, uočene su brojne protruzije, nastale uslijed utjecaja mase bakterijskog biofilma na staničnu membranu superficijalnih epitelnih stanica (68, 71). Dvanaest sati nakon infekcije, kokoidne bakterije, na površini IBZ-a, ponovo poprimaju morfologiju štapića, postaju 156

17 Vraneš et al Biofilm i kronične infekcije izrazito pokretne, te se odvajaju iz bakterijskog biofilma (71). U trenutku kada stignu do ruba stanice domaćina, ove bakterije izbijaju kroz staničnu membranu, te prodiru u lumen mokraćnog mjehura procesom koji se naziva izlijevanje (71). Narušavanjem integriteta domaćinove stanične membrane, ubrzo se, nakon ovih pojedinačnih stanica, u lumenu mokraćnog mjehura pojavljuju bakterije iz mnogobrojnih IBZ-a, što rezultira oslobađanjem miliona UPEC, pojave bakteriurije, te širenja i kolonizacije novih superficijalnih epitelnih stanica (70, 71). U lumenu mokraćnog mjehura, bakterije, oslobođene iz biofilma, diferenciraju se u 70 µm duge filamentozne tvorbe (71). Iako je mehanizam i svrha nastanka filamenata do danas nepoznata, pretpostavlja se da, na taj način, UPEC preživljava napad imunološkog sustava domaćina dovoljno dugo da septacijom filamenata nastane nova populacija bakterija, koja započinje slijedeći ciklus invazije i stvaranja IBZ-a u do tada neinficiranim superficijalnim epitelnim stanicama (68, 71). U nekoliko slijedećih postinokulacijskih dana, bakterije prolaze kroz višestruke cikluse stvaranja IBZ-a, no, sa svakim novim ciklusom, vrijeme koje je potrebno da IBZ prođe kroz sva četiri fenotipa intracelularnog biofilma, sve je sporije i sporije (68). U konačnici se, unutar superficijalnih epitelnih stanica, uočavaju samo male nakupine štapićastih bakterija, koje ulaze u stanje mirovanja, tzv. dormant fazu (68). U fazi mirovanja, UPEC može perzistirati u mokraćnom mjehuru mjesecima nakon inicijalne infekcije (68). Zahvaljujući IBZ koja je nalik na biofilm, UPEC uspješno odolijeva nespecifičnom imunološkom odgovoru domaćina (70, 71). Naime, lipopolisaharidi stanične stijenke UPEC-a aktiviraju Tollnalik receptore tipa 4 na membrani superficijalnih epitelnih stanica, što rezultira oslobađanjem upalnih citokina i masivnom infiltracijom mokraćnog mjehura polimorfonuklearnim leukocitima (PMN) (68). Iako u mokraćnom mjehuru, u fazi nastanka rane IBZ, polimorfonukleari migriraju prema inficiranim stanicama i pokušavaju prodrijeti u intracelularni biofilm, a kasnije pokušavaju uništiti filamentozne oblike bakterija, njihovo djelovanje na UPEC je bez uspjeha (68, 71). Osim aktivacije PMN, infekcija inducira i masivnu eksfolijaciju superficijalnih epitelnih stanica, no odljuštenje stanica u lumen mokraćnog mjehura rezultira samo smanjenjem broja bakterija, a ne i uništenjem IBZ (70). Ako odolijevanju imunološkom odgovoru domaćina pridodamo i rezistenciju biofilma na antimikrobnu terapiju, jasno je da sposobnost stvaranja IBZ-a omogućuje uropatogenoj E. coli iznimno dugu perzistenciju u mokraćnom mjehuru, a time i mogućnost nastanka kroničnih rekurentnih infekcija (68). Nakon mišjeg modela infekcije, kojim je pokazano značenje biofilma u patogenezi IMS-a, slijedećim studijama predstoji dokazati postojanje IBZ-a u bolesnika s rekurentnim cistitisom. Uz kronični cistitis, bakterijski se biofilm povezuje i s patogenezom stuvitne uro/nefrolitijaze. Struvitni kamenci nastaju kao posljedica bakterijske IMS, te premda čine svega 10-20% svih kamenaca u mokraćnom sustavu, predstavljaju pravi izazov u liječenju, uslijed brzog rasta i rekurencije koja se javlja u gotovo 50% slučajeva ove bolesti (73). Ključni faktori virulencije bakterija, uključeni u stvaranje kamenaca, jesu produkcija metaloenzima ureaze i bakterijski egzopolisaharidi (73, 74). Enzim ureaza hidrolizira ureu, koja je u urinu prisutna u velikim količinama, pri čemu nastaje amonijak i ugljični dioksid. Tako stvoreni amonijak podiže ph urina, uslijed čega topivi ioni magnezija i kalcija precipitiraju, te nastaju kamenci koji sadrže magnezij amonij fosfat (struvit) i kalcij fosfat (apatit) (74). Bakterijski egzopolisaharidi doprinose stvaranju kamenaca ubrzavanjem supersaturacije i kristalizacije iona kalcija i magnezija elektrostatskim interakcijama, te vezivanjem ovih iona za anionske grupe na njihovoj površini (73). Uzročnik koji, uz ostale, posjeduje oba navedena faktora virulencije i koji se najčešće povezuje s nastankom stuvitnih kamenaca jeste bakterija Proteus mirabilis. Pretpostavljalo se da, u procesu nukleacije kristala i nastanka kamenaca, ključnu ulogu ima sposobnost bakterija da stvaraju biofilm, pri čemu bi izvanstanični polisaharidni matriks biofilma promovirao vezivanje kristala (75). To je potaklo istraživače 157

18 Medicinski Glasnik, Volumen 6, Number 2, August 2009 da u studijama povezanosti bakterija sa struvitnim kamencima, primjene morfološke tehnike moderne mikrobiologije, poput skenirajućeg i transmisijskog elektronskog mikroskopa. Ovim je tehnikama tako dokazana prisutnost bakterija u obliku mikrokolonija, unutar glikokaliksa intersticija, jezgre i površine struvitnih kamenaca, kirurški odstranjenih iz bolesnika (76), te infekcijom induciranih u animalnim modelima (štakor, miš) (74, 77). Odolijevanje djelovanju antimikrobnih lijekova i obrani imunološkog sustava domaćina objašnjava se pozicioniranjem bakterija duboko u matriksu kamenca, odnosno stvaranjem sesilne zajednice, ukoliko se nalaze na njegovoj površini (74). Sve to doprinosi perzistenciji infekcije, daljnjem rastu struvitnih kamenaca, čestim rekurentnim infekcijama, te teškim oštećenjima, osobito bubrega. Danas se u terapiji struvitnih kamenaca upotrebljava mrvljenje kamenaca udarnim valovima ili perkutana nefrolitotomija, odnosno ureteroskopija, no u budućnosti se očekuje primjena lijekova koji penetriraju u biofilm kamenaca i uništavaju ga, te primjena inhibitora ureaze (78). KRONIČNI PROSTATITIS Prostatitis, upala prostate, najčešći je urološki problem kod muškaraca mlađih od 50 godina, te treći po učestalosti klinički entitet kod muškaraca starije životne dobi. Primjenom indeksa simptoma kroničnog prostatitisa (engl. Chronic Prostatitis Symptom Index, CPSI), razvijenog od strane američkog Nacionalnog instituta za zdravlje (NIH), kod muškaraca, u dobi od 20. do 74. godine, nađena je 10% prevalencija simptoma prostatitisa (79). Sve do godine, kada je u Bethesdi, pod pokroviteljstvom NIH-a, donešena nova klasifikacija (80), prostatitis se klasificirao u četiri klinička entiteta: akutni i kronični bakterijski prostatitis, nebakterijski prostatitis i prostatodinija. U novoj su klasifikaciji prve dvije kategorije ostale iste, no preostala dva klinička entiteta objedinjena su u kategoriju III kronični abakterijski prostatitis/kronična bol u zdjelici (81). Ova kategorija, s obzirom na prisutnost ili odsutnost upalnih stanica u uzorcima specifičnim za prostatu (eksprimat prostate i prvi mlaz urina nakon masaže prostate) (80), dodatno se dijeli na upalnu (IIIA) i neupalnu (IIIB) potkategoriju. Novost spram stare klasifikacije jeste i kategorija IV, u koju ulaze bolesnici bez simptoma i dijagnoze prostatitisa, a kod kojih je biopsijom prostate ili u specifičnim uzorcima detektirana prisutnost upale i/ili mikroorganizama (80). Kronični bakterijski prostatitis karakteriziraju rekurentne infekcije mokraćnog sustava, s nespecifičnim simptomima između simptomatskih infekcija, te perzistencija bakterija u prostati usprkos višestruko provedenim antimikrobnim terapijama (82). Najčešći uzročnici prostatitisa jesu dobro poznati gram-negativni uropatogeni poput bakterija E. coli (u gotovo 80% slučajeva), Klebsiella sp., Serratia sp., Enterobacter sp. i P. aeruginosa. Gram-pozitivne bakterije, izuzev bakterije Enterococcus faecalis, koja se smatra uzročnikom kroničnog bakterijskog prostatitisa, imaju vrlo upitnu ulogu u mikrobnoj etiologiji prostatitisa (80). Kada uzročnici uđu u kanaliće i acinuse prostate, ascenzijom iz mokraćne cijevi i/ili povratom urina u prostatične kanaliće (75), ondje se ubrzano razmnožavaju i izazivaju akutni upalni odgovor domaćina. Vitalne i umiruće bakterije, i stanice akutne upale, deskvamirane epitelne stanice i stanični detritus, ispunjavaju kanaliće, no sve dok je infekcija u akutnoj fazi, odgovarajućom antimikrobnom terapijom, moguće je eradicirati planktonske uzročnike, te suzbiti upalni proces (75). Pri subakutnoj upali ili perzistenciji bakterija unutar opstruiranih prostatičnih kanalića, bakterije mogu adherirati na epitel stijenki kanalića i ubrzo stvoriti sporadične mikrokolonije biofilma, što može dovesti do perzistentne stimulacije imunološkog sustava i posljedično kronične upale (75). Premda je klasična kvantitativna kultivacija uzoraka urina i eksprimata prostate ključna za dijagnozu kroničnog prostatitisa, kada se radi o mikrobiološkoj dijagnostici kliničkih entiteta u kategoriji III i IV, to je onda pravi klinički izazov (75). Neuspjeh detekcije bakterija kultivacijom u ovih bolesnika objašnjava se prisutnošću/ odsutnošću inhibitornih tvari u sekretima prostate, poput visoke koncentracije cinka i prostatičnog antibakterijskog faktora, te planktonskih bak- 158

19 Vraneš et al Biofilm i kronične infekcije terija u uzorcima (80, 83). Naime, planktonske se stanice teško odvajaju od sesilne zajednice i bivaju eradicirane primjenom empirijske antimikrobne terapije (80). Da antimikrobna terapija ne utječe na biofilm, te da bakterijske stanice perzistiraju u prostati, ubrzo je potvrđeno detekcijom mikroorganizama u bioptičkim uzorcima prostate u bolesnika, te elektronsko-mikroskopskim prikazom egzopolisaharidom prekrivenih mikrokolonija čvrsto adheriranih bakterija za stijenke kanalića i acinusa (84). Novi koncept mikrobiološke etiologije kroničnog prostatisa rezultirao je primjenom molekularnih tehnika u studijama prostatitisa. Primjenom PCR metode, detektirana je prisutnost 16S rrna bakterija u 77% bioptičkih uzoraka prostate (83), a potom u 65% uzoraka eksprimata prostate u oboljelih od kroničnog prostatitisa (85). Ubrzo se postavilo pitanje jesu li detektirani mikroorganizmi doista i uzročnici kroničnog prostatitisa. Prvi dokaz dobiven je usporedbom PCR rezultata dobivenih iz uzoraka prostatičnog tkiva zdravih donora prostate i bolesnika koji su bili podvrgnuti prostatektomiji (86). Dok su u grupi zdravih donora, u svim uzorcima PCR-a, rezultati bili negativni, te znakovi upale odsutni, u grupi oboljelih od karcinoma prostate i benigne hipertrofije prostate (BHP), u 70% uzoraka, dokazana je prisutnost upale i dobiven pozitivan PCR rezultat. Imajući na umu da gotovo 90% bolesnika s BHP-om ima prostatitis (86), te da prisutnost upalnih stanica u uzorku bioptata prostate dobro korelira s detekcijom bakterijskih genskih sekvenci (83), može se zaključiti da su detektirani mikroorganizmi ujedno i uzročnici prostatitisa. Bakterijski biofilm utječe i na rezultate primjene antimikrobne terapije u sindromu kroničnog prostatitisa. Iako se u početku smatralo da kronični upalni proces u prostati mijenja farmakokinetska svojstva antimikrobnih lijekova (87) i utječe na terapijski uspjeh, studijama na animalnom modelu to nije potvrđeno. Danas se nemogućnost eradikacije bakterija u kroničnom prostatitisu objašnjava sesilnim načinom života bakterija u prostati, te intrizičnom rezistencijom biofilma (75). Primjena molekularnih tehnika u detekciji i elektronskog mikroskopa u vizualizaciji, omogućili su povezivanje biofilma s etiologijom kroničnog prostatitisa, a rezultati daljnjih istraživanja trebali bi unaprijediti terapijski pristup ovom sindromu. KRONIČNE INFEKCIJE RANA Vrlo općenito, rane, prema njihovoj etiologiji, dijele se na akutne i kronične. Dok akutne rane uzrokuje eksterno oštećenje intaktne kože, kronične rane nastaju endogenim mehanizmima koji su povezani s predisponirajućim stanjima, poput metaboličke bolesti ili oštećenja arterijskog, odnosno venskog protoka, uslijed čega dolazi do oštećenja integriteta dermalnog i epidermalnog tkiva (88). Osim s kolonizacijom, prisutnost bakterija u akutnim i kroničnim ranama, povezuje se s razvojem upale, a time i procesom cijeljenja, invazivnim infekcijama i sepsom, zatajenjem organa, pa i smrtnim ishodom. Premda se u brojnim studijama mikroorganizmi, poput bakterija S. aureus, P. aeruginosa, te β-hemolitički streptokoki, najčešće povezuju s odgođenim cijeljenjem i infekcijom rana, u većini rana nalazi se polimikrobna, aerobna i anaerobna flora (88, 89). Zbog perzistencije infekcije, te rezistencije na sistemske i topičke antimikrobne lijekove, posljednjih godina pretpostavlja se kako bakterije koje koloniziraju kronične rane tvore sesilnu zajednicu, odnosno biofilm (90, 91). Sklonost bakterijskoj kontaminaciji, dostupnost supstrata, te izrazito velika površina koja podržava adherenciju bakterija, čini kronične rane gotovo idealnim okolišom za stvaranje biofilma (90). No, direktni dokazi koji bi podržali značenje biofilma u patogenezi kroničnih rana za sada su rijetki. Prvi pokušaji vizualizacije formiranja biofilma u kroničnim ranama temeljili su se na primjeni modificirane Kongo-red tehnike bojanja za prikaz polisaharida. Primjenom navedene tehnike u in vitro modelu P. aeruginosa biofilma, uočeno je kako je bakterijama, iz uzoraka rana ljudi s opeklinama, dovoljno tek sedam do deset sati za stvaranje zrelog biofilma (92). Nakon in vitro modela, ista grupa autora dokazala je postojanje biofilma i u ranama svinjskog in vivo modela (90). Uslijedio je i prikaz S. aureus biofilma u akutnim ranama pomoću skenirajuće elektronske mikroskopije (93), a naposljetku 159

20 Medicinski Glasnik, Volumen 6, Number 2, August 2009 istraživači američkog Centra za inžinjering biofilma Sveučilišta u Montani dokazali su i prisutnost biofilma u 60% uzoraka kroničnih rana (94). Upravo se stvaranje biofilma u kroničnim ranama smatra najboljim objašnjenjem zatajenja procesa cijeljenja takvih rana. Na cijeljenje rane i tijek infekcije utječe bakterijska sposobnost stvaranja stabilne, sesilne zajednice, te sposobnost domaćinova imunološkog sustava da kontrolira infekciju (91). Zajednice mikroorganizama mogu na proces cijeljenja utjecati direktno, produkcijom destruktivnih enzima i toksina, ili indirektno, promoviranjem stanja kronične upale u kojem oslobađanje slobodnih radikala i brojnih litičkih enzima negativno utječe na proliferaciju stanica, a time i na proces cijeljenja rane (91). Nadalje, djelovanje biofilma na proces cijeljenja objašnjava se njegovom smanjenom osjetljivošću na imunološki obrambeni odgovor domaćina. Poznato je da matriks biofilma inhibira kemotaksiju i degranulaciju PMN-a i makrofaga, te da PMN ne uspijevaju fagocitirati bakterije unutar biofilma. To PMN potiče na oslobađanje velike količine proupalnih enzima i citokina, te metaloproteaza matriksa (95, 96). Posljedice povišene razine endogenih metaloproteaza matriksa, te nestanka njihovih prirodnih inaktivatora u kroničnim ranama, jesu mnogobrojne. Proteolitičkim djelovanjem ovih enzima smanjuje se razina faktora rasta i citokina koji reguliraju pokretanje i rast stanica poput keratinocita, fibroblasta i stanica endotela, a dolazi i do nekontrolirane destrukcije ekstracelularnog matriksa koji, osim što koži daje snagu i otpornost, čini osnovu za prerastanje epidermisa i rast krvnih žila i živaca (96). Kako svaki od navedenih faktora sudjeluje u procesu cijeljenja u točno određenom trenutku, izostanak normalna slijeda događaja rezultira zaustavljanjem kronične rane u fazi upale i stvaranja granulacijskog tkiva. Dokazana prisutnost biofilma u kroničnim ranama i njegova povezanost s procesom cijeljenja motivirala je istraživače na razmatranje novih strategija kontrole biofilma. Jedna od ideja temelji se na prevenciji razvoja bakterijskog biofilma posredstvom komponente nespecifične imunosti laktoferina (97, 98). Laktoferin je glikoprotein sa sposobnošću vezivanja željeza, koji, pri koncentracijama nižim od onih potrebnih za uništenje ili prevenciju rasta bakterija, stimulira specijalni oblik kretanja bakterije na površini supstrata, te na taj način onemogućuje adherenciju i stvaranje mikrokolonija i nakupljanje (97, 98). Stoga se pretpostavlja da bi preparati laktoferina mogli razoriti već postojeći biofilm, što bi utjecalo na zaštitu kronične rane od infekcije. Druga mogućnost kontrole stvaranja biofilma jeste interferencija s detekcijom kvoruma. Poznato je da gram-negativne bakterije, kao signalne molekule, koriste AHL, a gram-pozitivne cikličke peptide (4). Proučavanjem strukture ovih signalnih molekula, te stvaranja, širenja i primanja signala, istraživači su, u potrazi za mogućnostima inhibicije detekcije kvoruma, pronašli brojne molekule s agonističkim, antagonističkim ili enzimskim djelovanjem na AHL, kao što su supstance nekih biljaka i gljiva koje interferiraju s AHLposredovanom komunikacijom, te halogenirane tvari furanona koje utječu na ekspresiju gena koji kontroliraju detekciju kvoruma i povećavaju osjetljivost biofilma na antimikrobne lijekove (4). Smatra se kako farmakološka inhibicija detekcije kvoruma pruža novu nadu u kontroli bakterijskog biofilma brojnih infekcija, pa tako i one u kroničnim ranama. Zaključak Svakako, nužni su novi terapijski pristupi koji bi omogućili uspješno liječenje biofilminfekcija. Potencijalna terapija, uz primjenu inhibitora detekcije kvoruma i činitelja virulencije bakterija, uključuje i enzimsku razgradnju ili oštećenje izvanstaničnog matriksa biofilma, što bi za posljedicu imalo disperziju biofilma, a time i smanjenje njegove rezistencije na djelovanje imunološkog sustava domaćina i antimikrobnih lijekova (99). Johansen i suradnici dokazali su baktericidnu aktivnost oksidoreduktaza, te učinak primjene enzima koji hidroliziraju polisaharide na uništenje bakterijskog biofilma koji tvore bakterije S. aureus, S. epidermidis, P. aeruginosa i Pseudomonas fluorescens sa abiotičkih supstrata (100). Nedavno je francuska grupa istraživača uočila uspjeh u primjeni disperzina B, 160

21 Vraneš et al Biofilm i kronične infekcije enzima koji hidrolizira poli-n-acetilglukozamin, u kombinaciji s proteazama u eradikaciji biofilma različitih stafilokoknih sojeva (101). Liječenje kroničnih infekcija mora počivati na spoznajama o karakteristikama, organizaciji i funkcioniranju biofilma, kao cjelovite multicelularne strukture u određenoj kroničnoj infekciji, a ne biti usmjereno na pojedinačne mikroorganizme kao do sada. LITERATURA Davey ME, O Toole GA. Microbial biofilms: from ecology to molecular genetics. Microbiol Mol Biol Rev 2000; 64: Donlan RM. Biofilms: microbial life on surfaces. Emerg Infect Dis 2002; 8: Donlan RM, Costerton JW. Biofilms: survival mechanisms of clinically relevant microorganisms. Clin Microbial Rev 2002; 15: Hentzer M, Givskov M. Pharmacological inhibition of quorum sensing for the treatment of chronic bacterial infections. J Clin Invest 2003; 112: Greenberg EP. Bacterial communication and group behavior. J Clin Invest 2003; 112: Marić S, Vraneš J. Characteristics and significance of microbial biofilm formation. Period Biolog 2007; 109: Gieseke A, Purkhold U, Wagner M, Amann R, Schramm A. Community structure and activity dynamics of nitrifying bacteria in a phospateremoving biofilm. Appl Environ Microbiol 2001; 67: Macedo AJ, Kuhlicke U, Neu TR, Timmis KN. Abraham WR. Three stages of a biofilm community developing at the liquid-liquid interface between polychlorinated biphenyls and water. Appl Environ Microbiol 2005; 71: Chenier MR, Beaumier D, Roy R, Driscoll BT, Lawrence JR, Greer CW. Influence of nutrients, hexadecane, and temporal variations on nitrification and exopolysaccharide composition of river biofilms. Can J Microbiol 2006; 52: Chenier MR, Beaumier D, Fortin N, Roy R, Driscoll BT, Lawrence JR, Greer CW. Influence of nutrient inputs, hexadecane, and temporal variations on denitrification and community composition of river biofilms. Appl Environ Microbiol 2006; 72: Satoh H, Yamakawa T, Kindaichi T, Ito T, Okabe S. Community structures and activities of nitrifying and denitrifying bacteria in industrial wastewater-treating biofilms. Biotechnol Bioeng 2006; 94: Vinarov A, Robysheva Z, Sokolov D, Smirnov V. Examination of the long-term process for purifying gaseous discharges in industrial biofilter. Commun Agric Appl Biol Sci 2003; 68: Valenzuela L, Chi A, Beard S, Orell A, Guiliani N, Shabanovitz J, Hunt DF, Jerez CA. Genomics, metagenomics and proteomics in biominimg microorganisms. Biotechnol Adv 2006; 24: Olson ME, Ceri H, Morck DW, Buret AG, Read RR. Biofilm bacteria: formation and comparative susceptibility to antibiotics. Can J Vet Res 2002; 66: Coetser SE, Cloete TE. Biofouling and biocorrosion in industrial water systems. Crit Rev Microbiol 2005; 31: Neria-Gonzales I, Wang ET, Ramirez F, Romero JM, Hernandez-Rodriguez C. Characterization of bacterial community associated to biofilms of corroded oil pipelines from the southeast of Mexico. Anaerobe 2006; 12: Smith JL, Fratamico PM, Novak JS. Quorum sensing: a primer for food microbiologists. J Food Prot 2004; 67: Ekman J, Kosonen M, Jokela S, Kolari M, Korhonen P, Salkinoja-Salonen M. Detection and quantification of colored deposit-forming Meiothermus spp. in paper industry processes and end products. J Ind Microbiol Biotechnol 2007; 34: Beech IB, Sunner JA, Arciola CR, Cristiani P. Microbially-influenced corrosion: damage to prostheses, delight for bacteria. Int J Artif Organs 2004; 29: Mah TF, O Toole GA. Mechanisms of biofilm resistance to antimicrobial agents. Trends Microbiol 2001; 9:34-9. Lewis K. Persister cells and the riddle of biofilm survival. Biochemistry (Mosc) 2005; 70: Stewart PS, Costerton JW. Antibiotic resistance of bacteria in biofilms. Lancet 2001; 358: Lewis K. Riddle of biofilm resistance. Antimicrob Agents Chemother 2001; 45: Xu KD, McFeters GA, Stewart PS. Biofilm resistance to antimicrobial agents. Microbiology 2000; 146: Ehrlich GD, Hu FZ, Shen K, Stoodey P, Post JC. Bacterial plurality as a general mechanism driving persistance in chronic infections. Clin Orthop Relat Res 2005; 437:

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25 Vraneš et al Biofilm i kronične infekcije Significance of microbial biofilm occurence in the pathogenesis and treatment of chronic infections Jasmina Vraneš 1, 2, Vladimira Leskovar 2 1 Zagreb University Medical School, 2 Institute of Public Health Dr. Andrija Štampar, Zagreb; Zagreb, Croatia ABSTRACT The ability of bacterial cells to adhere to biotic and abiotic surfaces, to function as a group and to mutually communicate is crucial in the development of chronic infectious diseases. Sessile communities of microorganisms today known as biofilm are involved in a number of chronic bacterial infections. Key characteristics of biofilm-infections are the persistence of infection and resistance to antimicrobial agents and host defenses. Advances in the technology and the application of new microscopic and molecular methods while studing ultrastructure and functional relationships inside the biofilm give hope that a new therapeutic way to control biofilm-infections, one of the greatest challenges of 21 st century, will be found. Key words: biofilm, chronic infections, pathogenesis, therapy Original submission: 06 April 2009; Revised submission: 08 April 2009; Accepted: 03 May

26 ORIGINAL ARTICLE Effect of inoculum size of Enterobacteriaceae producing SHV and CTX-M extended-spectrum ß-lactamases on the susceptibility to ß-lactam combinations with inhibitors and carbapenems Branka Bedenić 1,2, Jasmina Vraneš 1,3, Nataša Beader 1,2, Ines Jajić-Benčić 4, Vanda Plečko 1,2, Selma Uzunović-Kamberović 5, Smilja Kalenić 1,2 1 Department of Microbiology, School of Medicine, University of Zagreb; 2 Department of Clinical and Molecular Microbiology, Clinical Hospital Center Zagreb; 3 Department of Microbiology, Zagreb Institute of Public Health; 4 Department of Microbiology, Sisters of Mercy Hospital; Zagreb, Croatia; 5 Laboratory for Diagnostics, Cantonal Public Health Institution Zenica, Bosnia and Herzegovina ABSTRACT Corresponding author: Branka Bedenić Department of Microbiology, School of Medicine, University of Zagreb, Department of Clinical and Molecular Microbiology, Clinical Hospital Center Zagreb, Kišpatić Street 12, Zagreb, Croatia Phone: ; Fax: ; branka.bedenic@zg.htnet.hr Original submission: 03 June 2008; Revised submission: 14 August 2008; Accepted: 15 September Med Glas 2009; 6(2): Aim Many extended-spectrum β-lactamases (ESBL) producing isolates of E. coli and K. pneumoniae are susceptible in vitro to amoxycillin-clavulanate (AMC), ceftazidime-clavulanate (CAZ/ cl), and piperacillin-tazobactam (TZP), but MICs increase substantially when higher inoculum is applied. The aim of this study was to determine the effect of inoculum size on the susceptibility of E. coli and K. pneumoniae isolates with well characterized ES- BLs, to amoxycillin (AMX), AMC - amoxycilin + clavulanate, ceftazidime (CAZ), CAZ/cl - ceftazidime + clavulanate, piperacillin (PIP), TZP - tazobactam + piperacilin, imipenem (IMI) and meropenem (MEM). Methods Minimum inhibitory concentrations (MIC) were determined by broth microdilution method using inocula that differed 100 fold in density. Results Inoculum effect for CAZ/cl was detected in 52% of SHV-2 producing K. pneumoniae strains followed by AMC (43%) and TZP (38%). SHV-5 producing K. pneumoniae strains showed the most pronounced inoculum effect with CAZ/cl and AMC and to lesser extent with TZP. Inoculum effect was observed for AMC, CAZ/cl and TZP in 71% of SHV-12 producers. E. coli producing SHV-5 β-lactamase showed the most pronounced inoculum effect with AMC, followed by CAZ/cl and TZP. Strains producing CTX-M β-lactamases had a marked inoculum effect with CAZ/cl, AMC and TZP. Carbapenems did not show inoculum effect with any type of ESBLs. Conclusion According to the results of this study, carbapenems remain the antibiotics of choice for the treatment of infections caused by ESBL-producing Enterobacteriaceae. Key words: inoculum effect, extended spectrum β-lactamases, carbapenems, β-lactam/inhibitor combinations 166

27 Bedenić et al Inoculum effect of β-lactam antibiotics INTRODUCTION Extended-spectrum β-lactamases (ESBL) are enzymes capable of hydrolyzing oxyiminocephalosporins and aztreonam. They are produced by a variety of Gram-negative bacilli (1). A major problem with ESBLs is their capacity to cause therapeutic failures with cephalosporins and monobactams even when the causative agent appears susceptible in the laboratory tests (2-4). In response to this problem CLSI (Clinical and Laboratory Standard Institution, former NCCLS) recommends that laboratories should report ESBL producing isolates of K. pneumoniae and E. coli as resistant to penicillins, cephalosporins and monobactams regardless of the results of in vitro testing (5). There are also questions whether β-lactamase inhibitor combinations should be used for the therapy of infections caused by ESBL pathogens. Many ESBL producing isolates of E. coli and K. pneumoniae are susceptible in vitro to amoxycillin-clavulanate (AMC), ceftazidime-clavulanate (CAZ/cl) and piperacillin-tazobactam (TZP), but MICs (minimum inhibitory concentration) increases substantially when higher inoculum is applied (6-7). Efficacy has been reported in animal models but clinical failures were reported in patients. Previous studies have shown moderate inoculum effect of β-lactamase/ inhibitor combinations against Enterobacteriaceae in general (6), but there are only few reports so far for ESBL positive enteric bacteria with well defined resistance mechanisms (7-9). The aim of this study was to determine the effect of inoculum size on the susceptibility of E. coli and K. pneumoniae isolates with well characterized ESBLs to amoxycillin (AMX), amoxycillin/clavulanate (AMC), ceftazidime (CAZ), ceftazidime/clavulante (CAZ/cl), piperacillin (PIP), piperacillin/tazobactam (TZP) and carbapenems in comparison with ESBL negative strains. Inoculum effect was determined according to the type of ESBL in order to determine if there are differences in its magnitude between different types of ESBLs. MATERIAL AND METHODS The experiments were performed on the set of K. pneumoniae and E. coli isolates with well defined resistance mehanisms: 52 K. pneumoniae strains producing SHV-5 β-lactamase, 21 K. pneumoniae with SHV-2 β-lactamase, seven K. pneumonia strains possessing SHV-12 β-lactamase, 41 E. coli strains producing SHV-5 β-lactamase (10-12) and fourteen E. coli strains positive for CTX-M β-lactamases (nine CTX-M-3 and five- CTX-M-15) (Bedenić B, unpublished data). Twenty six ESBL negative isolates of K. pneumoniae were used as the control group. The β-lactamases were characterized by isoelectric focusing, PCR and sequencing of bla ESBL genes. Minimum inhibitory concentrations (MIC) of amoxycillin (AMX), amoxycillin/clavulanate (AMC), ceftazidime (CAZ), ceftazidime/clavulanic acid (CAZ/cl), piperacillin (PIP), piperacillin/tazobactam (TZP), imipenem (IMI) and meropenem (MEM) were determined by broth microdilution method using inocula that differed 100 fold in density according to CLSI (5). The inocula contained 10 5 CFU/ml and 10 7 CFU/ ml approximately in Mueller-Hinton broth. The MIC (minimum inhibitory concentration) was defined as the lowest antibiotic concentration that prevented the visible growth of bacteria after incubation at 37 C for 18 h. Inoculum effect was defined as at least eightfold increase in antibiotic MIC in the presence of high inoculum compared to standard inoculum size (7). RESULTS Inoculum effect for CAZ/cl was detected in 52% of SHV-2 producing K. pneumoniae strains followed by AMC (43%) and TZP (38%). Two strains showed inoculum effect with imipenem. SHV-5 producing K. pneumoniae strains showed the most pronounced inoculum effect with CAZ/ cl (57%) and AMC (55%) and to lesser extent with TZP (44%). Two strains showed inoculum effect with meropenem (Table 1). 167

28 Medicinski Glasnik, Volumen 6, Number 2, August 2009 Table 1. Percentage of strains showing inoculum effect with ß-lactam/inhibitor combinations and carbapenems, according to the type of ESBL. Antibiotic* Type of β-lactamase AMC CAZ/cl TZP IMI MEM K. pneumoniae ESBL-negative 1/26 (3.8%) 0/26 (0%) 1/26 (3.8%) 0/26 (0%) 0/26 (0%) SHV-2 9/21 (43%) 11/21 (52%) 8/21 (38%) 2/21 (9.5%) 0/21 (0%) SHV-5 29/52 (55%) 30/52 (57%) 23/52 (44%) 0/52 (0 %) 2/52 (3.8 %) SHV-12 5/7 (71%) 5/7 (71%) 5/7 (71%) 0/7 (0%) 0/7 (0%) E. coli SHV-5 25/41 (61%) 21/41 (51%) 9/41 (22%) 0/41 (0%) 0/41 (0%) CTX-M 8/14 (57%) 10/14 (71%) 7/14 (50) 0/14 (0%) 1/14 (7.1%) *AMC, amoxycillin/clavulanate, CAZ/cl, ceftazidime/clavulanate; TZP, piperacillin/tazobactam; IMI, imipenem; MEM, meropenem; Inoculum effect was observed for AMC, CAZ/cl and TZP in 71% of SHV-12 producers. None of the strains showed inoculum effect for the carbapenems (Table 1). E. coli producing SHV-5 β-lactamase showed the most pronounced inoculum effect with AMC (61%) followed by CAZ/cl (51%) (Table 1). TZP had the least inoculum effect (22%). Carbapenems were not affected. Strains producing CTX-M β-lactamases had a marked inoculum effect with CAZ/cl (71%), AMC (57%) and TZP (50%). One strain exhibited inoculum effect with meropenem (Table 1). The concentration necessary to inhibit 90% of the SHV-2 producing K. pneumoniae rose from 128 to 1024 mg/l for AMC and TZP, from 256 to 1024 mg/l for CAZ, and from 4 to 32 for CAZ/cl when high inoculum was applied as Table 2. MIC range, cumulative MIC values and percentage of resistant strains at standard and high inoculum testing, for Klebsiella pneumoniae strains producing SHV-ESBLs Standard inoculum High inoculum Antibiotic MIC range MIC 50 MIC 90 %R MIC range MIC 50 MIC 90 %R K. pneumoniae SHV-2 (n=21) amoxycillin amoxycillin/clavulanate 1-> > ceftazidime > ceftazidime/clavulanate piperacillin piperacillin/tazobactam imipenem meropenem K. pneumoniae SHV-5 (n=52) amoxycillin amoxycillin/clavulanate > ceftazidime > ceftazidime/clavulanate piperacillin piperacillin/tazobactam imipenem meropenem K. pneumoniae SHV-12 (n=7) amoxycillin amoxycillin/clavulanate 32-> ceftazidime ceftazidime/clavulanate piperacillin piperacillin/tazobactam imipenem meropenem

29 Bedenić et al Inoculum effect of β-lactam antibiotics Table 3. MIC range, cumulative MIC values and percentage of resistant strains at standard and high inoculum testing, for Escherihia coli strains producing SHV and CTX-M-ESBLs. Standard inoculum High inoculum Antibiotic MIC range MIC 50 MIC 90 %R MIC range MIC 50 MIC 90 %R E. coli - SHV-5 (n=41) amoxycillin amoxycillin/clavulanate > ceftazidime > ceftazidime/clavulanate piperacillin piperacillin/tazobactam imipenem meropenem E. coli - CTX-M (n=14) amoxycillin amoxycillin/clavulanate ceftazidime ceftazidime/clavulanate piperacillin piperacillin/tazobactam imipenem meropenem compared to the standard. Meropenem and imipenem were not affected by inoculum size with MIC 90 values of 0.25 and 1 mg/l respectively when standard inoculum was applied and MIC 90 of 0.5 and 4 mg/l in high inoculum testing respectively. With increased inoculum the percentage of SHV-2 producers resistant to AMC rose from 43 to 90%, to CAZ from 76% to 95% and to TZP from 19% to 57%. At the standard inoculum testing none of the SHV-2 producers were resistant to CAZ/cl whereas at high inoculum 9.5% of the strains became resistant (Table 2). With SHV-5 producing K. pneumoniae the highest increase in MIC 90 due to inoculum effect was observed for CAZ/cl (4 to 32 mg/l) and AMC (128 to >1024 mg/l) followed by TZP (256 to 1024 mg/l) whereas carbapenems showed only slight increase of the concentration necessary to inhibit 90% of the strains (0.5 to 1 mg/l for imipenem and 0.25 to 0.5 mg/l for meropenem). At the standard inoculum testing none of the SHV-5 producers was resistant to CAZ/cl while at high inoculum 38% of the strains showed resistance (Table 2). The percentage of resistant strains was also significantly increased due to inoculum effect for TZP (38% to 75%) and AMC (81 to 98%). MIC 90 for SHV-12 producers at standard inoculum size was 1024 for AMX, for TZP 128 mg/l, for CAZ/cl 4 mg/l, for IMI 1 mg/l and for MEM 0.25 mg/l whereas at high inoculum size it reached 1024 mg/l for AMX, AMC, CAZ, PIP and TZP, 32 mg/l for CAZ/cl, 2 mg/l for IMI and 1 mg/l for MEM. At the standard inoculum testing all strains were resistant to AMX, AMC, PIP and CAZ, whereas 57% were resistant to TZP. No resistance to CAZ/cl, IMI and MEM was observed. At high inoculum percentage of strains resistant to TZP rose to 100 % and to CAZ/cl to 14.1% but the susceptibility IMI and MEM was maintained in spite of slightly higher MIC values for particular strains (Table 2). The concentration necessary to inhibit 90% of the SHV-5 producing E. coli strains rose for two dilutions with increased inoculum for AMC ( mg/l), TZP ( mg/l), imipenem (0.5 2 mg/l) and meropenem ( mg/l) and for three dilutions in case of CAZ/cl (4 32 mg/l). When the inoculum was increased 100 fold, resistance increased from 14 to 90% for AMC, from 10 to 53% for TZP and from 0 to 7% for CAZ/cl (Table 3). The significant increase in the concentration that inhibited 90% of the CTX-M producers due to inoculum effect was obtained with AMC ( mg/l), CAZ/cl (1 8 mg/l) TZP ( mg/l), where MICs of carbapenems did not have a marked increase in MIC 90 at high inoculum testing (two dilutions). When the inoculum was increased 100 fold resistance of CTX-M positive E. coli strains was increased from 43 to 93% for AMC, from 35 to 57% for CAZ and from 43 to 64% for TZP. All CTX-M producers maintained susceptibility to CAZ/cl and carbapenems even with high inoculum testing (Table 3). 169

30 Medicinski Glasnik, Volumen 6, Number 2, August 2009 ESBL negative strains did not display inoculum effect with any antibiotic tested. MIC 90 of AMX, AMC, CAZ, CAZ/cl, PIP, TZP, IMI, and MEM was 8, 2, 0.5, 0.25, 8, 4, 0.12 and 0.06 mg/l respectively in the presence of the standard inoculum while the values in the presence of high inoculum were 32, 8, 2, 1, 16, 8, 0.5 and 0.12 mg/l respectively (Table 4). All ESBL negative strains were susceptible to all tested antibiotics at the standard inoculum testing. When testing was performed with high inoculum 19% of the ESBL negative strains were resistant to AMX, but no resistance to any other antibiotic was observed (Table 4). AMC and CAZ/cl were associated with inoculum effect against all type of ESBL producers: SHV-2, SHV-5, SHV-12 and CTX-M. TZP was less affected by the inoculum size then AMC, and CAZ/cl particularly with CTX-M producers. It was not possible to determine inoculum effect for AMX, PIP and CAZ alone because of the predominantly off- scale MIC values which exceeded 1024 mg/l even when tested with the standard inoculum size. DISCUSSION Clinicians rely on the results of in vitro susceptibility testing to choose appropriate antimicrobial agent for the therapy. Results of in vitro testing depend on many factors including inoculum effect (6). Inoculum effect was previously described for ceftazidime, cefotaxime, cefepime and other cephalosporins (13-15), but there are only few reports of inoculum effect with β-lactam/inhibitor combinations (6). Previous studies have shown small inoculum effect of β-lactamase/inhibitor combinations on Enterobacteriaceae in general (6), but in this research we studied the inoculum effect of these compounds in enteric bacteria with well defined resistance mechanisms. The studies on animal models have shown failures of ceftriaxone/sulbactam combination in experimental rabbit endocarditis due to the high density of K. pneumoniae producing TEM-3 β-lactamase (8) and E. coli producing SHV-2 β-lactamase in the cardiac vegetations (9). According to the results of this study, inoculum effect for all tested compounds was more pronounced for ESBL positive strains in comparison with ESBL negative. This is in concordance with previous reports which found the inoculum effect to be more significant if the antibiotic is susceptible to hydrolysis by a certain β-lactamase (7,13-14). It can lead to therapeutic failures if infections caused by ESBL producing microorganisms are treated with expanded-spectrum cephalosporins. Inoculum effect occurs when a bacterium produces enzyme capable of hydrolyzing an antibiotic (7). There are two explanations for the inoculum effect: antibiotic destruction by β-lactamases and filamentous transformations with continued growth (6). Susceptibility to AMC and CAZ/cl was more affected by inoculum size than TZP. There were slight differences observed in the magnitude of the inoculum effect with different types of ESBLs. The activity of TZP was mostly compromised in the presence of high density of SHV-5 producing K. pneumoniae. The fact that SHV-5 and SHV-12 producers showed the higher increase in the percentage of resistant strains for CAZ/cl in comparison with SHV-2 and CTX-M producers due to inoculum effect could be explained with higher hydrolysis rate of ceftazidime by SHV-5 and SHV-12 β-lactamase. Car- Table 4. MIC range, cumulative MIC values and percentage of resistant strains at standard and high inoculum testing, for ESBL negative Klebsiella pneumoniae strains. ESBL negative K. pneumoniae (n=26) Standard inoculum High inoculum Antibiotic MIC range MIC 50 MIC 90 %R MIC range MIC 50 MIC 90 %R amoxycillin amoxycillin/clavulanate ceftazidime ceftazidime/clavulanate piperacillin piperacillin/tazobactam imipenem < meropenem < <

31 Bedenić et al Inoculum effect of β-lactam antibiotics bapenems were the most stable to inoculum efect regardless of the type of ESBL. This observation is in concordance with previous reports (16-17). For that reason carbapenems which are stable in the presence of high inoculum should be antibiotics of choice for the treatment of infections caused by ESBL producing Enterobacteriaceae. β-lactamase/inhibitor combinations should be avoided in the therapy because of the inoculum effect and development of hyperproducing variants during treatment which are not sufficiently inhibited with therapeutic concentrations of clavulanic acid or sulbactam (18). AMC could be used for the treatment of urinary tract infections caused by ESBL producing Enterobacteriaceae due to its high concentrations in urine which overwhelm the inoculum effect and prevent development of hyperproducing mutants. Combinations of expanded-spectrum cephalosporins with β-lactamase inhibitors are not available at Croatian market but combination of cefoperazone with sulbactam is registered in France (18). The other important drawback of β-lactamase/inhibitor combinations is the selection of inhibitor resistant β-lactamases (18). ACKNOWLEDGEMENT/DISCLOSURE This research was supported by Croatian Ministry of Science, Education and Sport (Grant No ). Competing interests: none decleared. REFERENCES 1. Bradford PA. Extended-spectrum β-lactamases in the 21st century: characterization, epidemiology, and detection of this important resistance threat. Clin Microbiol Rev 2001; 14: Paterson DL. Recommendation for treatment of severe infections caused by Enterobacteriaceae producing extended-spectrum β-lactamases (ES- BLs). Clin Microbiol Infect 2000; 6: Paterson DL, Ko WC, von Gottberg A, Casellas JM, Mulazimoglu L, Klugman KP, Bonomo RA, Rice L, McCormack J, Yu V. Outcome of cephalosporin treatment for serious infections due to apparently susceptible organisms producing extended-spectrum β-lactamases: implication for the clinical microbiology laboratory. J Clin Microbiol 2001; 39: Karas JA, Pillay DG, Muckart D, Sturm W. Treatment failure due to extended-spectrum β-lactamase. J Antimicrob Chemother 1996; 37: CLSI. Performance Standards for Antimicrobial Susceptibility Testing. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically. Eighteen Informational Supplement. CLSI document M100-S18. Wayne, PA: Clinical and Laboratory Standards Institute; Goldstein EJ, Citron DM, Cherubin CE. Comparison of the inoculum effects of members of the family Enterobacteriaceae on cefoxitin and other cephalosporins, β-lactamase inhibitor combinations, and the penicillin-derived component of these combinations. Antimicrob Agents Chemother 1991; 35: Thomson KS, Smith-Molland E. Cefepime, piperacillin:tazobactam, and the inoculum effect in tests with extended-spectrum β-lactamase producing Enterobacteriaceae. Antimicrob Agents Chemother 2001; 45: Caron F, Gutmann L, Bure A, Pangon B, Vallois JM, Pechinot A, Carbon C. Ceftriaxonesulbactam combinations in rabbit endocarditis caused by a strain of Klebsiella pneumoniae producing extended-spectrum TEM-3 β-lactamase. Antimicrob Agents Chemother 1990; 34: Fantin B, Pangon B, Potel G, Caron F, Vallee E, Vallois JM, Mohler J, Bure A, Philippon A, Carbon C. Activity of sulbactam in combinations with ceftriaxone in vitro and in experimental endocarditis caused by Escherichia coli producing SHV-2 likw β-lactamase. Antimicrob Agents Chemother 1990;

32 Medicinski Glasnik, Volumen 6, Number 2, August Bedenić B, Žagar Ž. Extended-spectrum β-lactamases in clinical isolates of Klebsiella pneumoniae from Zagreb, Croatia. J Chemother 1998;10: Bedenić B, Randegger CC, Stobberingh E, Hachler H. Molecular epidemiology of extendedspectrum β-lactamases from Klebsiella pneumoniae strains isolated in Zagreb, Croatia. Eur J Clin Microbiol Infect Dis 2001; 20: Bedenić B, Schmidt H, Herold S, Monaco M, Plečko V, Kalenić S, Skrlin J. Spread of Klebsiella pneumoniae producing SHV-5 β-lactamase in Dubrava University Hospital, Zagreb. J Chemother 2005; 17: Queenan AM; Foleno B, Gownley C, Wira E, Bush K. Effects of inoculum and β-lactamase activity in AmpC and extended-spectrum β-lactamase (ESBL)-producing Escherichia coli and Klebsiella pneumoniae clinical isolates tested by using NCCLS ESBL methodology. J Clin Microbiol 2004; 42: Kang CI, Pai, H, Kim SB, Kim HB, Kim EC, Oh MD, Choe KW. Cefepime and the inoculum effect in tests with Klebsiella pneumoniae producing plasmid-mediated AmpC-type β-lactamase. J Antimicrob Chemother 2004; 54: Bedenić B, Beader N, Žagar Ž. Effect of inoculum size on the antibacterial activity of cefpirome and cefepime against Klebsiella pneumoniae strains producing SHV extended-spectrum β-lactamases. Clin Microbiol Infect 2001; 7: Wiseman LR, Wagstaff AJ, Brogden RN, Bryson HM: Meropenem: A Review of its antibacterial activity, pharmacokinetic properties and clinical efficacy. Drugs 1995; 50: Betriu C, Salso S, Sanchez A, Culebras E, Gomez M, Rodrigez-Avial I, Picazo JJ. Comparative in vitro activity and the inoculum effect of ertapenem against Enterobacteriaceae resistant to extended-spectrum cephalosporins. Int J Antimicrob Agents 2006; 28:1-5. Amyes SGB, Miles RS. Extended-spectrum β-lactamases: the role of inhibitors in the therapy 1998; 42:

33 ORIGINAL ARTICLE Comparison of the frequency and the occurrence of antimicrobial resistance among C. jejuni and C. coli isolated from human infections, retail poultry meat and poultry in Zenica-Doboj Canton, Bosnia and Herzegovina Selma Uzunović-Kamberović 1, Tina Zorman 2, Ingrid Berce 3, Lieve Herman 4, Sonja Smole Možina 2 1 Cantonal Public Health Institute Zenica, Laboratory for Clinical and Sanitary Microbiology, Zenica, Bosnia and Herzegovina; 2 University of Ljubljana, Biotechnical Faculty, Department for Food science and Technology, Ljubljana, Slovenia; 3 Institute of Public Health Nova Gorica, Nova Gorica, Slovenia; 4 Agricultural Research Center-Ghent, Department for Animal Product Quality and Transformation Technology, Melle, Belgium; ABSTRACT Aim To compare the frequency of isolation and occurrence of antimicrobial resistance among C. jejuni and C. coli isolated in humans, retail poultry meat and poultry. Methods Fifty-three human, 52 retail poultry meat and 15 poultry Campylobacter jejuni/coli isolates were investigated for antibiotic susceptibility to eight antimicrobials by disk-diffusion method. Erythromycin and ciprofloxacin susceptibility were further determined by E-test, and additionally the MICs of erythromycin and ciprofloxacin were determined using the broth microdilution method. Corresponding author: Selma Uzunović-Kamberović, Cantonal Public Health Institute Zenica, Laboratory for Clinical and Sanitary Microbiology, Zenica, Bosnia and Herzegovina Phone: ; Fax.: ; selma_kamb@yahoo.com Original submission: 06 January 2009; Revised submission: 17 March 2009; Accepted: 06 April Results Prevalence of C. coli in humans, retail poultry meat and poultry was 28.3%, 56.9% and 53.3%, respectively. No significant differences were found in the overall resistance rates between C. jejuni and C. coli isolated from all three sources (p>0.05). Erythromycin and ciprofloxacin resistance was high and similar in humans, retail poultry meat and poultry (26.4%, 35.3%, 26.7%, and 32.1%, 23.5%, 26.7%, respectively) (p>0.05). C. jejuni displayed higher prevalence of resistance to erythromycin than C. coli in all investigated sources (p>0.05). All ciprofloxacin and 94.4% of erythromycin positive isolates were highly resistant ( 32 μg/ml and 128 μg/ml, respectively). Conclusion The high prevalence of C. coli isolates from humans, poultry meat and poultry and higher both overall and erythromycin-resistance in C. jejuni than in C. coli isolates suggests that there may be a common source in the environment, which might be absent in other geographical regions. Further studies are required to determine the role of efflux mechanism in erythromycin- and ciprofloxacin-resistance related to the level of resistance. Key words: ciprofloxacin-resistance, erythromycin-resistance, Bosnia and Herzegovina, Campylobacter jejuni, Campylobacter coli Med Glas 2009; 6(2):

34 Medicinski Glasnik, Volumen 6, Number 2, August 2009 INTRODUCTION Campylobacter species are one of the most common inducers of bacterial diarrhoea in humans worldwide (1). Campylobacters is considered a zoonotic disease, and occur in the intestine of domestic, production and wild animals. Transmission to humans occurs via food, drinking water and pets (2,3). The major route of infection in humans is thought to be the consumption of contaminated poultry meat, probably because of the high prevalence of contamination of chicken carcasses with Campylobacter and the frequency of poultry consumption (2, 4-7). Although most reports based on molecular typing have shown a major contribution of poultry to human Campylobacter infections, its epidemiology is still not completely defined (8,9). Erythromycin and fluoroquinolones are considered the drugs of choice for the treatment of gastrointestinal infections. An increase of antibiotic resistance of human Campylobacter isolates, especially to fluoroquinolones has been reported in many countries, but resistance to erythromycin and other antimicrobials has also been observed (10-12). As Campylobacter may be transferred from animals to humans, the possible development of antimicrobial resistance in Campylobacter spp., due to the use of antimicrobial agents in food animals, is a matter of concern (10,13). C. jejuni isolated from clinical infections is generally susceptible to erythromycin, whereas a much higher level of erythromycin resistance among C. coli isolates has been reported (10,14). Until recently, the fact that the majority of C. coli isolates were usually obtained from pigs, suggested that they were the most probable food source of human infections with C. coli (14). Since 1991 in Zenica-Doboj Canton, Bosnia and Herzegovina region, a significant proportion of C. coli in human infections has been reported as compared to other countries in both asymptomatic carriers and diarrhoeic patients comprising 36% and 26.5% of thermo tolerant Camyplobacter isolates (15,16). Moreover, C. coli isolates showed no difference to erythromycin-resistance as compared to C. jejuni isolates in this region (11). The majority of C. coli isolates analyzed in previous studies was obtained from pigs, suggesting that pigs were the most probable source of human infections with C. coli, rather than chicken and cattle (17). If pigs were the source of human C. coli infections, it would be surprising given that the ethnic structure of the Zenica-Doboj Canton population has changed due to mass migration during the wartime, with Muslims accounting for 82.3% of the post-war population (Statistical yearbook of R/F B&H, Sarajevo, 2000, 2004). For Muslims the consumption of pork is almost nonexistent and for that reason in the Zenica city there was no pork available in the markets before This suggested that the primary source of C. coli infections might be other than pigs. The aim of this study was the comparison of antimicrobial resistance among C. jejuni and C. coli isolated from human infections, retail poultry meat and poultry in Zenica-Doboj Canton, Bosnia and Herzegovina. MATERIALS AND METHODS The Laboratory for Sanitary and Clinical Microbiology of the Cantonal Public Health Institute in Zenica serves a total population of 331,229 inhabitants in Zenica-Doboj Canton: 149,053 in the urban area and 182,176 in the rural area. During the entire year of 2002, stool specimens were received from 2,491 consecutive outpatients with sporadic diarrhoea. There were 1,557 specimens taken from children (0-6 years of age), 331 from elementary school students (7-14 years of age), 204 from high school students (15-19 years of age), 311 and 88 from adults (20-64 and >64 years of age, respectively). The samples were cultured on modified Preston medium (OXOID, Basingstoke, United Kingdom) and incubated in a microaerophilic atmosphere (CampyGen, OX- OID) at 42 C for 48 h. A total of 147 samples of retail poultry meat products (25 samples of chicken liver and

35 Uzunović-Kamberović et al Antimicrobial resistance C. jejuni and C. coli samples of poultry leg skin) from 53 different markets in the central zone of the Zenica city were examined for the presence of C. jejuni and C. coli during thirteen sampling visits between May and October Sampling was done by laboratory technicians with a permission of market managers. All poultry samples found in the markets which originated from different countries were sampled. Poultry meat samples came from 14 national (81 samples) and 7 international chicken meat producers (66 samples) imported from Croatia and Germany (20 samples from each country), Slovenia (22 samples), Turkey and Holland (2 samples from each country). For the isolation of Campylobacter from poultry meat, the standardized procedure recommended by ISO was followed (9,18). Chicken liver or skin from legs (5 g) were enriched in 45 ml of selective Preston broth (Oxoid), containing 5% of horse blood (SR 048C, Oxoid) and incubated in a microaerophilic atmosphere for 18 hours at 42 C (CampyGen, Oxoid). One loopful of enrichment broth was streaked on modified Preston medium (Oxoid) and incubated in a microaerophilic atmosphere at 42 C for 3 days. We also isolated 15 C. jejuni C. coli strains from 23 cloacal samples of chicken collected from the biggest local farm with conventional housing, and production of 750 kg/month. Sampling was done by farm staff with farm owner s permission. A questionnaire about the contents of food and antibiotics given to poultry (duration of breeding before slaughtering, indication for antibiotic distribution), monthly production, a place of one-day storage of chickens was filled by a farm owner. The single sampling took place in July Isolates were stored at -70 C in a medium consisting of nutrient broth No. 2 (CM0271 Oxoid) (32 g), agar (1.2 g), glycerol (150 ml) and distilled water (up to 1000 ml), supplemented with two vials of Campylobacter growth supplement (SR0232E, Oxoid, Basingstoke, United Kingdom). After original isolation, the isolates were long-term stored at -80 C in Biotechnical Faculty in Ljubljana (Slovenia) for further studies. C. jejuni and C. coli were identified using standard microbiological methods and multiplex PCR (5,19). The disc diffusion method was performed to test the resistance to eight antimicrobials used in human and veterinary medicine (Oxoid): ampicillin (10 µg); erythromycin (15 µg); gentamicin (10 µg), tetracycline (30 µg), chloramphenicol (30 µg) nalidixic acid (30 µg), ciprofloxacin (5 µg); nitrofurantoin (300 µg). The inocula were adjusted to turbidity of a 0.5 McFarland standard and plated on Mueller-Hinton agar (Oxoid, Basingstoke, UK) supplemented with 5% sheep blood. The plates were incubated at 37 C for 48 hrs in a microaerobic atmosphere. The applied susceptibility criteria were in accordance with CLSI (NCCLS) [20]. The following cutoff values were used: ampicillin, 13 mm, erythromycin, 13 mm, gentamicin, 12 mm, tetracycline, 14 mm, chloramphenicol, 12 mm, nalidixic acid, 13 mm, ciprofloxacin, 15 mm, nitrofurantoin, 14 mm. C. jejuni ATCC 3356, E. coli ATCC and Staphylococcus aureus ATCC control strains were used. Erythromycin and ciprofloxacin susceptibility were further determined by Etest (AB Biodisc, Solna, Sweden) as described previously (21). The MICs (minimal inhibitory concentration) of erythromycin and ciprofloxacin (Sigma Aldrich, Saint Louis, USA) in Campylobacter isolates were determined using the broth microdilution method as described previously (20). For erythromycin the method was slightly modified: two-fold serial dilutions of erythromycin were used at the concentrations from to 512 μg/ ml. The MICs lowest concentration where no growth was observed was determined on the base of fluorescent signal measured by Microplate Reader (Tecan, Mannedorf/Zurich, Switzerland) after adding CellTiter-Blue Reagent following manufacturer s instructions (Promega Corporation, Madison, USA) to culture the media. Isolates were considered resistant to erythromycin when MIC 32 mg/l, and resistant to cipro- 175

36 Medicinski Glasnik, Volumen 6, Number 2, August 2009 floxacin when MIC 4 mg/l. The strains that repeatedly presented the MIC of erythromycin and ciprofloxacin of <128 mg/l and <32 mg/l, respectively, and 128 mg/l and 32 mg/l, respectively, were termed low-level resistant (LLR) and high level of resistant (HLR) strains, respectively. One isolate per patient was included in the analysis of antibiotic susceptibility. The significance of differences in resistance was determined by means of the χ 2 test, Fisher s exact test for independence and ANOVA. A statistically significant difference was defined as a p value of <0.05 and 95% confidence interval was used. RESULTS During 2002, C. jejuni strains were isolated from 37 (69.8%), C. coli from 15 (28.3%) confirmed human stool samples, and in one sample the coexistence of both Campylobacter species was found (it was included only in the overall antibiotic susceptibility testing). The frequency of isolation of Campylobacter strains in retail poultry meat was 35.3% (52/147). Twenty nine strains (56.9%) were identified as C. coli, 21 strains (41.2%) as C. jejuni, and in one sample the coexistence of both Campylobacter species was found. Fifteen out of 23 farm chicken cloacal samples, obtained from biggest local farm, were positive for Campylobacter. Eight out of 15 positive samples (53.3%) contained C. coli and 7 out of 15 (46.7%) samples contained C. jejuni. The distribution and results of antibiotic susceptibility testing of C. jejuni and C. coli strains are shown in Table 1. Retail poultry meat and human isolates have shown significantly higher prevalence of resistance to one or more antibiotics tested, than the poultry isolates (p<0.05). Prevalence of multiresistant isolates, defined as resistant to four or more antibiotics tested, were similar in all three sources (p>0.05). No significant difference was found in the overall resistance rates between C. coli and C. jejuni isolated from all three sources (p>0.05). High and similar prevalence rates of resistance among human, retail poultry meat (overall) and poultry isolates to erythromycin and ciprofloxacin were observed (p>0.05). Among poultry meat isolates, the isolates from imported retail poultry meat have shown higher resistance rate for erythromycin than those from domestic poultry meat, while ciprofloxacin resistance was higher among isolates of domestic retail poultry meat, but there was no statistical significance Table 1. Antimicrobial susceptibility of Campylobacter jejuni and Campylobacter coli isolates from humans, poultry meat and farm chickens Origin and species No of isolates (%) resistance MDR* AMP ERY GEN TET C NA CIP NIT % of overall % of Percentage (%) of resistance to antimicrobial agents Human All C. jejuni 37 (69.8%) C. coli 15 (28.3%) Poultry meat overall All C. jejuni 21 (41.2%) C. coli 29 (56.9%) Poultry meat domestic All C. jejuni 11 (35.5%) C. coli 19 (61.3%) Poultry meat imported All C. jejuni 11 (52.3%) C. coli 10 (47.6%) Poultry All C. jejuni 7 (46.7) C. coli 8 (53.3%) *MDR, multidrug resistance, AMP, ampicillin (10 µg); ERY, erythromicyin (15 µg); GEN, gentamicin (10 µg); TET, tetracycline (30 µg); CHL, chloramphenicol; NAL, nalidixic acid; CIP, ciprofloxacin (5 µg); NIT, nitrofurantoin (300 µg); a sample with coexistence of both Campylobacter species was included only in overall antibiotic susceptibility testing 176

37 Uzunović-Kamberović et al Antimicrobial resistance C. jejuni and C. coli between them (p>0.05). The resistance rates to erythromycin and ciprofloxacin of clinical isolates were higher in C. jejuni, 29.7% and 35.1%, respectively, than in C. coli, 20.0% and 26.7%, respectively (p>0.05). It is true for retail poultry and poultry isolates too. All ciprofloxacin positive isolates had a high-level resistance ( 32 μg/ml), and 94.4 % of erythromycin positive isolates had high-level resistance ( 128 μg/ml) (Table 2). Significantly higher prevalence of resistance for both erythromycin and ciprofloxacin was observed in the isolates from the age group of (53.8% for both antibiotics) as compared to the age group of 0-6 (23.3% for ciprofloxacin and 22.3% for erythromycin) (data not shown). DISCUSSION Resistance rates to one or more antibiotics as found in this study for Campylobacter strains have surpassed the highest previously reported resistance rates for human and poultry meat isolates (4,23,24). Reportedly, multidrug resistant (MDR) isolates are usually present in humans to a much lesser extent (0.8% to 21%) compared to the results from this study (23-25). A significant increase in ciprofloxacin resistance of human isolates in this region between 1998 (14%) and 2002 (32.1%) was observed (1). Several studies have shown that food animals can be a substantial source of human infections and that the same serotypes and genotypes can be isolated from humans and food animals (10,14). The consumption of imported chickens was identified as a possible risk factor for the acquisition of fluoroquinolone-resistant strains (10,13). Widespread use of antimicrobials in veterinary medicine has resulted in the emergence of strains of Campylobacter displaying a MDR phenotype (26). These strains are transmitted to humans usually through the consumption of undercooked contaminated food, particularly poultry. Of concern to public health is the increase in strains resistant to fluoroquinolone and macrolide drugs, important antibiotics used in the front-line treatment of campylobacteriosis (27). The results of our study do not show a significant difference in ciprofloxacin-resistance between domestic and imported poultry meat samples. We have got a confirmation of the examined farm owner that no antibiotics were applied to animal food, but still there is a possibility for an increase in their use for therapeutic purposes instead (28). The fact that the decrease of fluoroquinolone-resistant campylobacters had been observed before and during the use of antimicrobial growth promoters suggests that other factors might be involved (29). The reports about the connection between individual fluoroquinolone used in humans and development of fluoroquinolone-resistance in Campylobacter isolates are still controversial (4,10,12). It is worth mentioning that 56.6% of isolates from this study and 67% isolates from the previous study (11), all from the Zenica- Doboj region, originated from children up to 6 years old. High ciprofloxacin-resistance in this age group (23.3%) was found and due to the fact that the use of quinolones is restricted for children, the high ciprofloxacin-resistance is probably not influenced by overuse of this drug. It is unlikely that the human use of fluoroquinolones alone could be responsible for high-resistance rates of human Campylobacters to fluoroquinolones observed in many countries (4,10,12). Our results indicate the resistance of an animal origin Table 2. Number of strains according to level of resistance to erythromycin and ciprofloxacin* Origin of isolates (No) Species (No) No of strains inhibited at MIC(µg/mL) of erythromycin: No of strains inhibited at MIC(µg/mL) of ciprofloxacin: < >512 < >32 Human (52) C. jejuni (37) C. coli (15) Retail poultry (51) C. jejuni (22) C. coli (29) Poultry (15) C. jejuni (7) C. coli (8) *cutoff values: erythromycin MIC 32 mg/l; ciprofloxacin MIC 4mg/L; 177

38 Medicinski Glasnik, Volumen 6, Number 2, August 2009 rather than the overuse of these drugs in humans as proposed previously (12). Antibiotic-sensitive bacteria that are gradually exposed to increasing concentrations of a given antibiotic develop increasing resistance to that antibiotic and that induces the resistance increase to other unrelated antibiotics. The development of a MDR phenotype suggests that the development of multidrug resistance in Gram-negative bacteria in patients treated with subinhibitory doses of the antibiotic occurs via the same mechanism (30). The prevalence of erythromycin-resistance for human isolates (32.1%) reported in this study has surpassed the usually reported rates (<2.2% to 12%) (4,12). Both species have shown high prevalence of erythromycin-resistance from all three sources. Moreover, C. jejuni have shown higher prevalence of resistance to erythromycin than C. coli which is a rare finding, and in contrast with other reports where C. jejuni isolates from both humans and broilers, have usually showen low prevalence, or no resistance to erythromycin (4,13). In case of C. coli the highest reported erythromycin-resistance was among pig isolates (67.2%) as compared to poultry isolates (34.78%) (13,14). The macrolide tylosin has been permitted as a growth promoter in pigs, but not in broilers, and this could explain the lower proportion of erythromycin-resistant strains observed in broilers as compared to pigs (13,31). These high levels of erythromycin-resistant human strains from this study are uncommon in comparison to other studies (4,14,32,33) especially due to the fact that campylobacter strains in this region probably do not originate from pigs; postwar population of the Zenica-Doboj Canton is mostly the Muslims (82.3%) (Statistical yearbook of R/F B&H, Sarajevo, 2000, 2004), in which consumption of pork is almost nonexistent (15,16). It has shown that food-independed reservoirs of antibiotic-resistant Campylobacter do exist (34) and human-to-human transmission of both macrolide-susceptible and macrolide-ressistant strains is known to occur (35). Previously reported prevalence of C. coli strains in humans, at chicken farm level and retail poultry meat were lower than in our study (4,32,36). Such high prevalence of C. coli in chicken carcasses of domestic origin has been reported in only one report (37). We are aware of the limitations of this study where only two possible sources of campylobacter infections were investigated and a low number of human, retail poultry meat and poultry isolates were analyzed. The PFGE-typing analysis of human and poultry meat isolates in our study, where only a minority of clinical and poultry meat isolates of Campylobacter shared identical PFGE types, showed that, sources other than poultry and pigs might be important (8,9). However, the high prevalence of C. coli isolates from humans, poultry meat and poultry and higher overall as well as erythromycin-resistance in C. jejuni than in C. coli isolates suggests that there may be a common source in the environment, which might be absent in other geographical regions. We have not yet been able to explain the high prevalence of C. coli human infections in this region. Further studies including larger number of isolates from different sources should be directed to detection of efflux mechanism role in erythromycin resistance regarding to level of resistance, which provides bacterial pathogens a rapid adaptation to environmental changes (38,39). Besides of restoring the efficacy of existing drugs, specific efflux pump inhibitors (EPIs) may be useful as feed additives designed to decrease colonization of the animal gastrointestinal tract and thus reduce transmission to humans via the food chain (26). Further prospective research is required to examine potential reservoirs of Campylobacter spp. in the environment. A formal surveillance system supported by the characterisation & typing of larger numbers of isolates from different sources may help to identify epidemiological relationship. ACKNOWLEDGEMENTS/DISCLOSURE The authors would like to thank to the Ministry of Education, Science, and Sport of Republic of Slovenia, the Federal Ministry of Education 178

39 Uzunović-Kamberović et al Antimicrobial resistance C. jejuni and C. coli and Science of Bosnia and Herzegovina, as well as the Ministry of the Flemish Community for the financial support of the project and authors bilateral cooperation. This study was presented as part of: Uzunovic-Kamberovic S, Zorman T, Herman L, Berce I, Smole-Mozina S. Campylobacter jejuni and C. coli resistance in humans, poultry products and farm chickens: an epidemiological and laboratory study. Proceeding of the 14 th European Congress of Clinical Microbiology and Infectious Diseases, Prague, Chek, May 1-4, Clin Microbiol Infect 2004; 10 (Suppl 3):195. Competing interests: none decleared REFERENCES 1. Tauxe RV. Emerging foodborne pathogens. Internat J Food Microbiol 2002; 78: Workman SN, Mathison GE, Lavoie MC. Pet dogs and chicken meat as reservoirs of Campylobacter spp. in Barbados. J Clin Microbiol 2005; 43: Sacks JJ, Lieb S, Baldy LM, Berta S, Patton CM, White MC, Bigler WJ, Witte JJ. Epidemic campylobacteriosis associated with a community water supply. Am J Public Health 1986; 76: Gallay A, Prouzet-Mauléon V, Kempf I, Lehours P, Labadi L, Camou C, Denis M, de Valk H, Desenclos JC, Mégraud F. Campylobacter antimicrobial drug resistance among humans, broiler chickens, and pigs, France. Emerg Infect Dis 2007; 13: Zorman T, Smole Mozina S. Classical and molecular identification of thermotolerant campylobacters from poultry meat. Food Technol Biotechnol 2002; 40: Corry JEL, Atabay HI. Poultry as a source of Campylobacter and related organisms. J Appl Microbiol 2001; 90:90S-114S. 7. Stafford RJ, Schluter PJ, Wilson AJ, Kirk MD, Hall G, Unicomb L; OzFoodNet Working Group. Population-attributable risk estimates for risk factors associated with Campylobacter infection, Australia. Emerg Infect Dis 2008; 14: Hopkins KL, Desai M, Frost JA, Stanley J, Logan JM. Fluorescent amplified fragment length polymorphism genotyping of Campylobacter jejuni and Campylobacter coli strains and its relationship with host specificity, serotyping, and phage typing. J Clin Microbiol 2004; 42: Uzunovic-Kamberovic S, Zorman T, Heyndrickx M, Smole Mozina S. Role of poultry meat in sporadic Campylobacter infections in Bosnia and Herzegovina: laboratory-based study. Croat Med J 2007; 48: Smith KE, Besser JM, Hedberg CW, Leano FT, Bender JB, Wicklund JH, Johnson BP, Moore KA, Osterholm MT. Quinolone-resistant Campylobacter jejuni infections in Minnesota, New Engl J Medicine 1999; 340: Uzunovic-Kamberovic S: Antibiotic susceptibility of Campylobacter jejuni and Campylobacter coli human isolates from Bosnia and Herzegovina. J Antimicrob Chemother 2003; 51: Gupta A, Nelson JM, Barrett TJ, Tauxe RV, Rossiter SP, Friedman CR, Joyce KW, Smith KE, Jones TF, Hawkins MA, Shiferaw B, Beebe JL, Vugia DJ, Rabatsky-Ehr T, Benson JA, Root TP, Angulo FJ; NARMS Working Group. Antimicrobial resistance among Campylobacter strains, United States, Emerg Infect Dis 2004; 10: Van Looveran M, Daube G, De Zutter L, Dumont J-M, Lammens C, Wijdooghe M, Vandamme P, Jouret M, Cornelis M, Goossens H. Antimicrobial susceptibilities of Campylobacter strains isolated from food animals in Belgium. J Antimicrob Chemother 2001; 48: Aarestrup MF, Nielsen EM, Madsen M, Engberg J. Antimicrobial susceptibility patterns of thermophilic Campylobacter spp. from humans, pigs, cattle and broilers in Denmark. Antimicrob Agents Chemother 1997; 41: Uzunovic-Kamberovic S. Changes in Campylobacter jejuni / Campylobacter coli carriage rates in the Zenica region of Bosnia and Herzegovina in the pre- and postwar periods. J Clin Microbiol 2001; 39: Uzunovic-Kamberovic S. Some epidemiologic features of Campylobacter jejuni/coli infections in Bosnia and Herzegovina after the war. Clin Microbiol Infect 2003; 9: Nielsen EM, Engberg J, Madsen M. Distribution of serotypes of Campylobacter jejuni and C. coli from Danish patients, poultry, cattle and swine. FEMS Immunol Med Microbiol 1997; 19: Zorman T, Heyndrickx M, Uzunovic-Kamberovic S, Smole Mozina S. Genotyping of Campylobacter coli and C. jejuni from retail chicken meat and humans with campylobacteriosis in Slovenia and Bosnia and Herzergovina. Intern J Food Microbiol 2006; 110: Smibert, RM: Genus Campylobacter Sebald and Véron In Bergey`s Manual of Systematic Bacteriology. Krieg NR, Holt JG, eds. Baltimore: Williams & Wilkins; 1984:

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41 ORIGINAL ARTICLE Atelektaza pluća i infekcije donjih dišnih puteva u djece na odjelu za intenzivno liječenje Nada Mladina¹, Devleta Hadžić¹, Amela Selimović² ¹Klinika za dječije bolesti, ²Klinika za ginekologiju i akušerstvo; Univerzitetski klinički centar Tuzla, Bosna i Hercegovina SAŽETAK Cilj rada Prikazati etiološke, kliničke i radiološke karakteristike atelektaze pluća kod djece na intenzivnom tretmanu u Odjeljenju intenzivne njege i terapije Klinike za dječije bolesti Tuzla u jednogodišnjem periodu. Metode Uzorak je obuhvatio 31 dijete sa infekcijom donjih dišnih puteva i atelektazom pluća. Prosječna dob djece iznosila je 3,6 ± 3,9 g. Analizirane su etiološke, kliničke i radiološke karakteristike atelektaze pluća kod djece sa infekcijom donjih dišnih puteva. Corresponding author: Nada Mladina, Klinika za dječije bolesti, Univerzitetski klinički centar Tuzla, Trnovac bb, Tuzla, Bosna i Hercegovina Phone: nada.m@bih.net.ba Originalna prijava: 22. juli 2008.; Korigirana verzija: 25. oktobar 2008.; Prihvaćeno: 08. novembar Rezultati U promatranom jednogodišnjem periodu zbog infekcija donjih dišnih puteva, bronhitisa i pneumonije, liječeno je ukupno 332 pacijenta, od čega 208 dječaka (62,7%) i 124 djevojčice (37,3%). Kod 224 (67,5%) pacijenta radiološki nalaz je pokazao pneumoniju, dok je kod 31 (9,3%), uz pneumoniju, opisana i atelektaza pluća. Najčešća je bila desnostrana atelektaza (20 ili 64,5%), dok je u 10 ili 32,3% registrovana lijevostrana, a kod jednog pacijenta (3,2%) obostrana. Općenito osnovno oboljenje bila je infekcija donjih dišnih puteva (30 ili 96,8%), dok je kod samo jednog pacijenta to bio medijastinalni ekspanzivni proces. Klinički znaci, nalazi gasnih analiza i pulsne oksimetrije, bili su u korelaciji i u smislu hipoksemijskog tipa respiratorne insuficijencije. Najčešći uzrok atelektazi pluća bila je staza gustog sekreta, koja je dovela do smetnji ventilacije. Kontinuiranu oksigenoterapiju zahtijevalo je svih 31 pacijenata, najmanje 24 sata, dok je monitoring vitalnih parametara kod 12 ili 38,7% pacijenata bio potreban duže od 24 sata. Prosječna dužina intenzivnog liječenja bila je 4,3 ± 2,7 dana (od 1 do 15 dana). Zaključak Atelektaze pluća kod djece sa infekcijama donjih dišnih puteva u odjeljenju intenzivne njege nisu rijetke. One predstavljaju dodatni faktor rizika za ozbiljne poremećaje plućne ventilacije, naročito u dojenačkoj dobi. Ključne riječi: atelektaza pluća, donji dišni putevi, dijete Med Glas 2009; 6(2):

42 Medicinski Glasnik, Volumen 6, Number 2, August 2009 UVOD Atelektaza pluća predstavlja poremećaj plućne ventilacije u kojem manji ili veći dio plućnog parenhima nije u stanju da se ispuni vazduhom. Ovaj poremećaj može da se manifestuje odmah poslije rođenja, kao urođena totalna ili parcijalna atelektaza, ili kasnije, tokom života, kao stečena atelektaza (1). Atelektaza nije bolest za sebe već posljedični poremećaj ventilacije pluća nastao zbog niza bolesti i poremećaja. Uzroci atelektaze su brojni. Najčešće je to opstrukcija bronha koja može biti intraluminalna, muralna i ekstramuralna (2). Neke bolesti, kao što su astma, cistična fibroza, neuromuskularne bolesti i dr., uključuju i razvoj atelektaze. (3, 4). Poremećaji plućne ventilacije naročito su česti kod djece mlađe od pet godina, s obzirom da i minimalne promjene u disajnim putevima u tom uzrastu mogu da dovedu do opstrukcije disajnog puta (1, 2). Atelektaza može da protiče asimptomatski i da se otkrije tokom rutinskog fizikalnog ili radiološkog pregleda, a može biti i vrlo dramatična, naročito kod aspiracije stranog tijela u disajnim putevima (1, 2). Klinička slika zavisi od veličine atelektaze, a fizikalni nalaz i od primarne bolesti koja je doprinijela njenom razvoju. Rentgenski snimak pluća je zlatni standard za dijagnozu atelektaze (5, 6). Fleksibilna bronhoskopija korisna je u dijagnostici i u liječenju atelektaze (7). Atelektaza može nastati i posredstvom refleksnog bronhospazma uslijed bola, prijeloma rebara, hirurških intervencija na abdomenu i toraksu, te dijagnostičkih i drugih procedura, kao što su bronhoskopija, bronhografija, anestezija i drugo (8-10). Prognoza i tretman zavise od uzroka atelektaze. Dijagnostika i liječenje moraju biti uzročno usmjereni (2, 6, 8). Cilj ovog istraživanja bila je analiza učestalosti, te etioloških, kliničkih i radioloških karakteristika atelektaza pluća kod djece na intenzivnom tretmanu radi infekcija donjih dišnih puteva u jednogodišnjem periodu. ISPITANICI I METODE Retrospektivnim istraživanjem analizirani su svi pacijenati liječeni na Odjeljenju intenzivne njege i terapije Klinike za dječije bolesti u Tuzli, sa radiološki i klinički potvrđenom atelektazom pluća, u periodu do godine. Izvor podataka bio je protokol Odjeljenja intenzivne njege Klinike za dječije bolesti, kao i istorije bolesti liječenih pacijenata. Analizirani su anamnestički podaci, klinički i laboratorijski nalazi, terapijski postupci, dužina boravka u Odjeljenju intenzivne njege i terapije, te ishod liječenja. Od anamnestičkih podataka analiza je obuhvatala mjesto stanovanja, broj hospitalizacijâ, sezonsku distribuciju hospitalizacije, te vodeće kliničke simptome. Od kliničkog nalaza analizirani su uzrast, spol djeteta, opći pedijatrijski nalaz, lokalni auskultatorni nalaz, vitalni parametri (frekvenca pulsa, broj respiracija, tjelesna temperatura, saturacija krvi kisikom, mjerena pulsnim oksimetrom). Od laboratorijskih parametara analizirani su biohemijski nalazi: sedimentacija eritrocita, kompletna krvna slika i hematokrit, C-reaktivni protein, gasne analize, radiološki nalazi i mikrobiološke analize. Sve navedene pretrage rađene su na Poliklinici za laboratorijsku dijagnostiku, Zavodu za radiologiju i Zavodu za mikrobiologiju Univerzitetskog kliničkog centra Tuzla. Od terapijskih postupaka posebno je analizirana primjena i vremenski period oksigenoterapije sa visokim protokom kisika, inhalatorne terapije bronhodilatatorima, antibiotske terapije, sistemske primjene kortikosteroida, potrebe za primjenom kardiotonika i diuretika, rehidratacije kristaloidima, otvaranja dišnog puta, upotrebe balona sa maskom, primjene mehaničke ventilacije, dužine liječenja u Odjeljenju intenzivne njege i terapije, kao i ishodi liječenja. U statističkoj obradi rezultata korištene su standardne metode deskriptivne statistike. REZULTATI U periodu od do u Odjeljenju intenzivne njege i terapije Klinike za dječije bolesti Tuzla liječeno je ukupno 767 djece. Zbog respiratornih bolesti liječeno je 332 djece, od čega 208 dječaka (62,7% ) i 124 djevojčice (37,3%). Radiološki nalaz bio je uredan kod 108 pacijenata (32,5%). Kod 224 pacijenta (67,5%) 182

43 Mladina et al Atelektaza pluća i infekcije donjih dišnih puteva u djece Tabela 1. Dob pacijenata liječenih zbog atelektaze pluća Ukupno Dječaci Djevojčice Dob (godine) ( n = 31) ( n = 18) ( n = 13) (41,9) (16,1) (19,4) (22,6) 6 1 radiološki je opisana pneumonija, od toga kod 100 pacijenata (44,6%) desnostrana, kod 22 (9,8 %) lijevostrana, a kod 102 pacijenta (45,5%) obostrana pneumonija. Atelektaza pluća radiološki je verificirana kod 31 pacijenta, uzrasta od 2,5 mjeseca do 12,9 godina. Prosječna dob te djece iznosila je 3,60 ± 3,96 godine. Odnos dječaka i djevojčica iznosio je 1,4 : 1. Najveći broj pacijenata bio je dojenačke dobi u kojoj je distribucija po spolu bila ujednačena, dok su u starijim dobnim skupinama dominirali dječaci (Tabela 1). Prvu hospitalizaciju zbog respiratornog oboljenja imalo je 18 pacijenata (58,1%), a njih 13 (41,9%) sa radiološki verificiranom atelektazom pluća imalo je više rehospitalizacija zbog respiratorne bolesti. Najveći broj pacijenata sa radiološki verificiranom atelektazom pluća zabilježen je u mjesecu decembru (12; 38,7%), potom u mjesecima maju i junu (po 5 pacijenata; 16,1%). Najviše liječenih pacijenata bilo je iz Tuzle, (12; 38,7%), potom iz Lukavca i Banovića (po 5 pacijenata; 16,1%). U Tabeli 2. prikazane su prateće bolesti i stanja koja su dijagnosticirana kod pacijenata liječenih zbog atelektaze pluća. Uz atelektazu pluća, najčešće su zabilježene infekcije donjih dišnih puteva, bronhitis i pneumonija (kod 30 pacijenata), dok je jedan pacijent imao ekspanzivni medijastinalni proces. Pacijenti sa ponavljanim hospitalizacijama zbog respiratornih bolesti imali su obično prateća hronična oboljenja, najčešće imunodeficijenciju, anemiju, urođene srčane greške, neuromišićne i neurorazvojne bolesti. Lokalizacija atelektaze bila je Tabela 3. Vitalni parametri kod djece sa atelektazom pluća Parametar Mean ± SD* Minimum Maksimum Median Puls 134 ± Broj respiracija 42 ± Tjelesna temperatura ( C) 37,6 ± 0,74 36,4 39,4 37,5 Pulsna 93 ± oksimetrija (%) *aritmetička sredina ± standardna devijacija Tabela 2. Prateće bolesti i stanja dijagnosticirana kod pacijenata liječenih zbog atelektaze pluća Prateće bolesti i stanja Broj pacijenata* (%) Bronhitis 20 (43,5) Pneumonija 10 (21,7) Imunodeficijencija 5 (10,9) Anemija 4 (8,7) Urođene srčane greške 2 (4,3) Neuromišićne bolesti 2 (4,3) Neurorazvojne bolesti i poremećaji 2 (4,3) Limfom 1 (2,2) *neki pacijenti imali su više od jednog favorizirajućeg faktora za nastanak atelektaze pluća najčešće u desnom plućnom krilu (kod 20 pacijenata; 65%), u lijevom (kod 10 pacijenata; 32%), a samo kod jednog pacijenta bila je prisutna obostrana atelektaza pluća. Klinički atelektaza pluća kod naših ispitanika karakterizirala se znacima dispneje i povećanog rada pluća (tahipneja, tahikardija), hipoksemijom potvrđenom sniženim vrijednostima saturacije krvi kisikom, te znacima prijeteće respiratorne insuficijencije. Vitalni parametri pacijenata sa atelektazom u našem uzorku prikazani su u Tabeli 3. Vitalni parametri (puls i broj respiracija) pacijenata sa atelektazom, u našem uzorku, pokazali su odstupanje i bili većih vrijednosti u odnosu na normalne vrijednosti za dječiju dob. U Tabeli 4 prikazane su vrijednosti pulsa i broja respiracija pacijenata s atelektazom pluća po pojedinim dobnim skupinama i u poređenju sa normalnim vrijednostima za dob. Tabela 4. Vitalni parametri pacijenata s atelektazom pluća po pojedinim dobnim skupinama Dob (godine) Puls (u min.) normalno Respiracije (u min.) mean 146, ,3 120,6 minimalno maksimalno mean 49,3 47, ,4 minimalno maksimalno normalno

44 Medicinski Glasnik, Volumen 6, Number 2, August 2009 U Tabeli 5 prikazani su parametri acidobaznog statusa i gasnih analiza pacijenata s atelektazom pluća. Rezultati osnovnih laboratorijskih parametara krvne slike, sedimentacije eritrocita i nalaza C-reaktivnog proteina prikazani su u Tabeli 6. U 20 pacijenata (64,5%) vrijednost C- reaktivnog proteina kod prijema bila je iznad 10 mg/l. Broj leukocita kod prijema bio je u 14 pacijenata (45,2%) iznad 15 x 10 9 /L, a 13 je pacijenata (41,9%) bilo febrilno kod prijema, sa izmjerenom tjelesnom temperaturom iznad 38 C. Mikrobiološka analiza pokazala je pozitivne kulture brisa ždrijela i nosa kod 9 (29,0%), odnosno kod 3 (9,7%) pacijenta. Najčešće su bili izolirani Staphylococcus aureus (5 izolata), Klebsiella spp. (4 izolata), te Pseudomonas aeruginosa, Candida albicans i Mycobacterium tuberculosis (po 1 izolat). Nije bilo pozitivnih nalaza hemokulture. Oksigenoterapija tokom najmanje 24 sata, primijenjena je kod svih (31) pacijenata. Monitoring vitalnih parametara duži od 24 sata, zahtijevalo je 12 pacijenata (38,7%). Antibiotska parenteralna terapija, terapija bronhodilatatorima i intenzivna respiratorna fizikalna terapija s ciljem drenaže bronhalnog sekreta, primijenjena je kod svih (31) pacijenata. Kardiotonike, u okviru terapijskog tretmana, zahtijevalo je 13 pacijenata (41,9%). Najveći broj pacijenata, koji su imali manifestne znake srčanog popuštanja i zahtijevali u terapiji kardiotonike i diuretike, bila su dojenčad; osam pacijenata (61,5%). U jednog pacijenta bila je indicirana terapijska bronhoskopija i drenaža. Pacijent sa dijagnosticiranim malignim procesom u medijastinumu podvrgnut Tabela 5. Parametri acidobaznog statusa pacijenata s atelektazom Parametar Mean ± SD* ph 7,35 ± 0,07 7,08 7,47 7,35 pco2 (kpa) 4,99 ± 0,96 2,64 7,49 4,9 po2 (kpa) 6,62 ± 1,27 3,59 8,6 6,85 Bazni višak -2,4 ± ,5-1,4 Bikarbonati (mmol/l) 21 ± 3, ,25 Saturacija kisikom (%) 78 ± 12 45, *aritmetička sredina ± standardna devijacija je odgovarajućoj terapiji prema usvojenim protokolima. Prosječna dužina liječenja u Odjeljenju intenzivne njege bila je 4,3 ± 2,7 dana (od 1 do 15 dana), a prosječna dužina ukupnog bolničkog tretmana bila je 14,6 ± 11,4 dana (od 5 do 57 dana). DISKUSIJA Dobna distribucija pacijenata sa atelektazom pluća u našem istraživanju pratila je dobnu distribuciju ukupnog uzorka pacijenata sa respiratornim bolestima, što se uglavnom slaže sa podatkom da su, uz atelektazu pluća, najčešće zabilježene infekcije donjih dišnih puteva, bronhitis i pneumonija, kao i sa podacima iz literature (1-3). Distribucija po spolu pacijenata sa atelektazom u našem istraživanju razlikovala se u odnosu na ukupni uzorak pacijenata sa respiratornim bolestima. Nasuprot prednosti dječaka u ukupnom uzorku respiratornih bolesti, koja je u dojenačkom periodu najizrazitija, u našem uzorku pacijenata sa atelektazom pluća ustanovljena je minimalna prednost djevojčica u dojenačkom periodu. Ovu razliku teško je objasniti, čak i kad se uzmu u obzir i prateće bolesti i stanja koja su zabilježena kod naših pacijenata sa atelektazom. Mogući razlog mogao bi biti i relativno kratak period istraživanja. Pacijenti sa ponavljanim hospitalizacijama zbog respiratornih bolesti, u našem istraživanju imali su obično prateća hronična oboljenja, najčešće imunodeficijenciju, anemiju, urođene srčane greške, neuromišićne i neurorazvojne Tabela 6. Laboratorijski pokazatelji pacijenata sa atelektazom Parametar Sedimentacija eritrocita C-reaktivni prot. (mg/l) Hemoglobin (g/l) Broj eritrocita (x1012/l) Broj leukocita (x109/l) Hematokrit (L/L) Mean ± SD* Minimum Maksimum Median Minimum Maksimum Median 33 ± ± 56 0, ,5 116 ± ,23 ± 0,52 3,2 5,4 4,2 14,4 ± 5,4 5, ,7 0,34 ± 0,04 0,27 0,4 0,34 184

45 Mladina et al Atelektaza pluća i infekcije donjih dišnih puteva u djece bolesti. Sva ova stanja obično podrazumijevaju teškoće sa disanjem i hranjenjem, što donekle može objasniti češću zastupljenost pacijenata sa ovim hroničnim bolestima i poremećajima među pacijentima sa atelektazom pluća. Ovo se uglavnom slaže sa rezultatima sličnih istraživanja (3, 4, 6). Sezonska distribucija pacijenata sa atelektazom pratila je sezonsku distribuciju ukupnog uzorka pacijenata sa respiratornim bolestima. Klinička slika zavisi od veličine područja zahvaćenog atelektazom (1-3). U našem istraživanju, kliničke karakteristike pacijenata sa atelektazom pluća uglavnom su bili znaci dispneje i povećanog rada pluća (tahipneja, tahikardija), što se slaže sa činjenicom da je u našem uzorku najčešći uzrok atelektazi pluća (kod 96,8%) najvjerovatnije bila staza sekreta u pacijenata sa bronhitisom i pneumonijom, dok je kod jednog pacijenta (3,2%) atelektaza bila uzrokovana kompresijom tumorskih masa iz medijastinuma. Podaci iz literature značajno se razlikuju od ustanove do ustanove (2-7) i uglavnom su ovisni o patologiji koju određeni zdravstveni centar zbrinjava. Našim istraživanjem nisu obuhvaćeni pacijenti sa stranim tijelom u bronhima jer se oni, po organizaciji posla, liječe u Klinici za otorinolaringologiju. Fizikalni nalaz, također, zavisi od primarne bolesti koja je doprinijela razvoju atelektaze (1-3). Mada je atelektaza uglavnom mehaničkog porijekla, vrlo često postoje znaci bakterijske infekcije, što najviše zavisi od primarnog plućnog poremećaja (9, 15-19). Atelektaze su vrlo podložne i sekundarnim bakterijskim infekcijama (9, 14). Nespecifični markeri upale (sedimentacija eritrocita, C-reaktivni protein i leukociti) imaju ograničeno značenje pri dijagnosticiranju bakterijskih infekcija kod djece s atelektazom. Ipak, značajno je istaći da se vrlo visoke vrijednosti pojedinih upalnih markera rijetko viđaju pri virusnim infekcijama (15-17). Lala i saradnici (18) ustanovili su kako se kod djece sa pneumonijom nalaz CRP-a veći od 10 mg/l signifikantno češće javlja u grupi djece s bakterijskim pneumonijama u odnosu na ostale uzročnike. Slične rezultate objavili su Bircan i saradnici (19). U našem istraživanju našli smo umjereno povišene biohemijske parametre bakterijske infekcije. Najčešće izolovani bakterijski uzročnici u našem istraživanju bili su Staphylococcus aureus i Klebsiella spp. što govori o populaciji našeg uzorka, s obzirom da ovi uzročnici više odgovaraju etiologiji bolničkih pneumonija i pneumonija kod imunodeficijentnih pacijenata. Podaci iz literature su različiti, a rezultati slični našima, objavljeni su u nekoliko studija (14, 20). Liječenje pacijenata sa atelektazom pluća zavisi od njezinog uzroka (6). U slučaju aspiracije i stranog tijela u disajnim putevima, terapija izbora je bronhoskopska evaluacija, evakuacija stranog sadržaja i drenaža. Fizikalna terapija općenito je od posebnog značaja i koristi. Drenaža sekreta je osnovna mjera i cilj terapije bilo koje endobronhalne opstrukcije (20). Smatra se da je kod atelektaze, koja traje duže od godinu dana, proces na plućima ireverzibilan i da je u tom slučaju indicirano hirurško liječenje (22). U našem istraživanju, tokom jednogodišnjeg perioda, u jednog pacijenta bila je indicirana terapijska bronhoskopija i drenaža, dok mehaničku potporu disanju i hirurško liječenje nije bilo neophodno primijeniti ni kod jednog djeteta sa atelektazom pluća. Analiza učestalosti atelektaze pluća kod djece sa infekcijama donjih dišnih puteva, koja su zahtijevala intenzivni nadzor i tretman, pokazala je da je učešće atelektaza kao precipitirajućeg faktora poremećaja plućne ventilacije značajno, naročito u dojenačkoj populaciji. Atelektaza pluća kod ovih pacijenata klinički je odgovarala slici osnovnog oboljenja. Blagovremena dijagnostika, kao i energične mjere liječenja infekcije donjih dišnih puteva, uz adekvatan fizikalni tretman, kod većine pacijenata u našem istraživanju bile su dovoljne za uspješno terapijsko rješavanje nastalog poremećaja plućne ventilacije. Kod tumačenja ovih rezultata svakako treba uzeti u obzir i kratak period istraživanja, te su potrebna daljnja prospektivna istraživanja koja će obuhvatiti veći broj pacijenata. ZAHVALE / IZJAVE Komercijalni ili potencijalni dvostruki interes ne postoji. 185

46 Medicinski Glasnik, Volumen 6, Number 2, August 2009 LITERATURA Šićević S. Atelektaza pluća. U: Plućne bolesti u dece. Savremena administracija. Beograd 1990; Raos M, Klancir SB, Dodig S, Kovač K. Atelektaze pluća u dječjoj dobi. Paediatr Croat 1999; 43: Peroni DG, Boner AL. Atelectasis: mechanisms, diagnosis and management. Pediatr Respir Rev 2000; 1: Birnkrant DJ. The assessment and management of the respiratory complications of pediatric neuromuscular diseases. Clin Pediatr (Phila) 2002; 41: Ashizawa K, Hayashi K, Aso N, Minami K. Br J Radiol 2001; 74: Schindler MB. Treatment of atelectasis: where is the evidence? Crit Care 2005; 9: Holmgren NL, Cordova M, Ortuzar P, Sanchez I. Role of flexible bronchoscopy in the re-expansion of persistent atelectasis in children. Arch Bronconeumol 2002; 38: Hendriks T, de Hoog M, Lequin MH, Devos AS, Merkus PJ. Crit Care 2005; 9: Riccetto AG, Zambom MP, Pereira IC, Morcillo AM. Influence of socioeconomic and nutritional factors on the evolution to complications in children hospitalized with pneumonia. Rev Assoc Med Bras 2003; 49: Krause MF, von Bismarck P, Oppermann HC, Ankerman T. Bronchoscopic surfactant administration in pediatric patients with persistent lobar atelectasis. Respiration 2008; 75: Toyoshima M, Maeoka Y, Kawahare H, Maegaki Y, Ohno K. Pulmonary atelectasis in patients with neurological or muscular disease; gravity-related lung compression by the heart and intra-abdominal organs on persistent supine position. No To Hattatsu 2006; 38: Lutterbey G, Wattjes MP, Doerr D, Fischer NJ, Gieseke J Jr, Schild HH. Atelectasis in children undergoing either propofol infusion or positive pressure ventilation anesthesis for magnetic resonance imaging. Pediatr Anesth 2007; 17: Blitman NM, Lee HK, Jain VR, Vicencio AG, Girshin M, Haramati LB. Pulmonary atelectasis in children anesthetized for cardiothoracic MR: evaluation of risk factors. J Comput Assist Tomogr 2007; 31: Wu KH, Lin CF, Huang CJ, Chen CC. Rigid ventilation bronchoscopy under general anesthesia for treatment of pediatric pulmonary atelectasis caused by pneumonia: A review of 33 cases. In Surgery 2006; 91: Tumgor G, Celik U, Alabaz D, Cetiner S, Yaman A, Yildizdas D, Alhan E. Aetiological agenst, interleukin-6, interleukin-8 and CRP concentrations in children with communitiy-acquired pneumonia. Ann Trop Pediatr 2006; 26: Almirall J, Bolibar I, Toran P, Pera G, Boquet X, Balanzo X, Sauca G. Contribution of C-reactive protein to the diagnosis and assesment of severity of community-acquired pneumonia. Chest 2004; 125: Lagerstrom F, Engfeldt P, Holmberg H. C-reactive protein in diagnosis of comunity-acquired pneumonia in patient in primary care. Scand Infect Dis 2006; 38: Lala SG, Madhi SA, Pettifor JM. The discriminative value of C-reactive protein levels in distinguishing betwen community-acquired bacteriaemic and respiratory virus-associated lower respiratory tract infections in HIV-1-infected and uninfected children. Ann Trop Pediatr 2002; 22: Bircan A, Kaya O, Gokirmak M, Ozturk O, Sahin U, Akkaya A. C-reactive protein, leukocyte count and ESR in the assesment of severity of community-acquuired pneumonia. Tuberk Toraks 2006; 54:22-9. Schechter MS. Airway clearence applications in infants and children. Respir Care 2007; 52: Bar-Zohar D, Sivan Y. The yield of flexible fiberoptic bronchoscopy in pediatric intensive care patients. Chest 2004; 126: Choudhury SR, Chadha R, Mishra A, Kumar V, Singh V, Dubey NK. Lung resections in children for congenital and acquired lesions. Pediatr Surg 2007; 23:

47 Mladina et al Atelektaza pluća i infekcije donjih dišnih puteva u djece Lung atelectasis and lower respiratory tract infections in children in the intensive care unit Nada Mladina¹, Devleta Hadžić¹, Amela Selimović² ¹Pediatrics Clinic, ²Gynecology and Obstetrition Clinic; University and Clinical Center Tuzla, Bosnia and Herzegovina ABSTRACT Aim To determine etiologic, clinical and radiological findings of lung atelectasis in children undergoing intensive treatment in the Department of Intensive Care Unit at the Pediatric Clinic Tuzla in one-year period. Methods We analyzed a group of 31 children with lower respiratory tract infections and pulmonary atelectasis. Their average age was 3,6 ± 3,9 years. We analyzed etiologic, clinical and radiological findings of pulmonary atelectasis among children with lower respiratory tract infections. Results In one year period we treated 332 patients due to lower respiratory tract infections, bronchitis and pneumonia, 208 boys (62, 7%) and 124 girls (37, 3%). In 224 (67,5%) patients radiologic findings showed pneumonia, while in 31(9,3%) of patients radiological findings showed pneumonia and pulmonary atelectasis, as well. The most frequent was the right-sided atelectasis (20 or 64,5%), while in 10 or 32,3% left sided atelectasis was noticed. In only one patient (3,2%) bilateral atelectasis was found. Generally etiologic base was the infection of lower respiratory tract (30 or 96, 8%). It was only one patient with mediastinal expansive process. Clinical signs, gas analyses and plus oximetries were in correlation and showing hypoxemic type of respiratory insufficiency. The most frequent cause of lung atelectasis was mucus stasis. All 31 patients demanded oxygen therapy and monitoring of vital parameters at least for 24 hours, while in 12 or 38,7% of them, monitoring of vital parameters was needed longer than 24 hours. Average duration of intensive treatment was 4, 3± 2, 7 days (1-15 days). Conclusion Pulmonary atelectasis are not rare in children with lower respiratory tract infections in Intensive Care Unit. They are an additional risk factor for serious disturbances of pulmonary ventilation, especially in infancy. Key words: pulmonary atelectasis, lower respiratory tract, child Original submission: 22 July 2008.; Revised submission: 25 October 2008; Accepted: 08 November

48 ORIGINAL ARTICLE Evaluacija dijagnostičke vrijednosti interleukina-6 i C-reaktivnog proteina iz krvi pupčanika u prepoznavanju rane infekcije terminske novorođenčadi male porođajne mase Almira Ćosićkić 1, Fahrija Skokić 2, Selmira Brkić 3 1 Klinika za dječije bolesti, 2 Ginekološko-akušerska klinika, Odjel za neonatologiju, Univerzitetski klinički centar Tuzla; 3 Zavod za patološku fiziologiju, Medicinski fakultet Univerziteta u Tuzli; Tuzla, Bosna i Hercegovina SAŽETAK Cilj Evaluirati dijagnostičku vrijednost interleukina-6 (IL-6) i C-reaktivnog proteina (CRP) iz krvi pupčanika u prepoznavanju rane novorođenačke infekcije (RNI) prema kliničkoj slici, hematološkim parametrima i mikrobiološkim nalazima. Corresponding author: Almira Ćosićkić, Klinika za dječije bolesti, Univerzitetski klinički centar Tuzla, Trnovac bb, Tuzla, Bosna i Hercegovina Phone/fax.: ; almira_cosickic@yahoo.com Originalna prijava: 12. august 2008.; Korigirana verzija: 16. septembar 2008.; Prihvaćeno: 07. novembar Metode Provedeno je retrospektivno-prospektivno istraživanje u Klinici za ginekologiju i akušerstvo u Tuzli. Uključujući kriteriji bili su porođajna masa < grama, dob od 37. do 42. gestacijske nedjelje, novorođenčad oba spola, iz jednoplodne trudnoće i bez vidljivih anomalija na rođenju. Isključujući kriteriji bili su porođajna masa > grama, gestacijska dob < 37 nedjelja, višeplodna trudnoća, novorođenčad sa kongenitalnim anomalijama. Kriterije je zadovoljilo 120 novorođenčadi, a formirane su dvije grupe: ispitivana, novorođenčad s RNI (n = 28) i kontrolna grupa, novorođenčad bez RNI (n = 92). Analizirane su vrijednosti IL-6 i CRP-a iz krvi pupčanika, klinička slika, hematološki parametri i mikrobiološki nalazi. Rezultati Vrijednosti IL-6 < 10 pg/ml smatrane su normalnim za novorođenačku dob. Medijan vrijednosti IL-6 u ispitivanoj grupi bio je 49 pg/ml, a u kontrolnoj 9.7 pg/ml, uz značajnu razliku među grupama (p < ). Senzitivnost testa IL-6 bila je 82% i specifičnost 89%. Vrijednosti CRP-a < 5.0 mg/l smatrane su normalnim za novorođenačku dob. Medijan CRP-a u ispitivanoj grupi iznosio je 3.5 mg/l, a u kontrolnoj 2.8 mg/l bez značajne razlike među grupama (p = 0.997). Senzitivnost testa CRP-a bila je 25% i specifičnost 86%. Niska je bila i senzitivnost kombinacije oba testa IL-6 i CRP-a 21% prema dijagnostičkim parametrima RNI, ali je specifičnost bila značajna (87%). Zaključak Dijagnostička vrijednost IL-6 iz krvi pupčanika u prepoznavanju RNI je značajna, dok je vrijednost CRP-a iz krvi pupčanika ograničena. Ključne riječi: interleukin-6, C-reaktivni protein, rana novorođenačka infekcija. Med Glas 2009; 6(2):

49 Ćosićkić et al IL-6 i CRP u ranoj novorođenačkoj infekciji UVOD Rana novorođenačka infekcija (RNI) značajan je uzrok morbiditeta i mortaliteta novorođenčadi, a njeno prepoznavanje često se temelji na procjeni znakova i simptoma koji su nespecifični i mogu podsjećati na mnoga druga neinfektivna stanja, koja su zapravo odraz ekstrauterine adaptacije novorođenčeta (1). Novorođenčad male porođajne mase (MPM) predstavljaju rizičnu grupu jer imaju visoku perinatalnu smrtnost, češće komplikacije u novorođenačkom periodu, veću učestalost kongenitalnih anomalija i sklonija su infekcijama (2). Imunološki sistem novorođenčeta razvija se postepeno, te osim što neki njegovi dijelovi nisu potpuno razvijeni, oni su i bez iskustva jer u zaštićenoj intrauterinoj sredini nisu morali reagirati na strane antigene. Komponente nespecifične, a posebno specifične imunosti, ispoljavaju funkcijske i kvantitativne nedostatke što rezultira neadekvatnim upalnim odgovorom i povećanom sklonosti novorođenčadi ka infekciji, naročito novorođenčadi MPM (3). Interleukin-6 (IL-6), citokin prisutan u najranijoj fazi upale, značajan je medijator upalnog odgovora, stimulira stanice jetre na stvaranje reaktanata akutne faze upale, a među njima i C reaktivnog proteina (CRP) (4). Određivanje citokina iz krvi pupčanika može predstavljati put kojim se prepoznaju novorođenčad ugrožena infekcijom (5). Procjena pouzdanosti povišenih vrijednosti reaktanata akutne faze upale u novorođenčadi za otkrivanje infekcije sve je potrebnija u svakodnevnoj kliničkoj praksi. Uz hipotezu da bi se određivanje citokina iz krvi pupčanika moglo koristiti za pravovremeno prepoznavanje novorođenčadi sa RNI, cilj ovoga rada jeste da se evaluira dijagnostička vrijednost IL-6 i CRP-a iz krvi pupčanika u pravovremenom prepoznavanju RNI u odnosu na kliničku sliku, hematološke parametre i mikrobiološke nalaze. ISPITANICI I METODE Provedeno je retrospektivno-prospektivno istraživanjem u Klinici za ginekologiju i akušerstvo Univerzitetskog kliničkog centra u Tuzli (UKC Tuzla) u vremenskom periodu od marta do septembra godine. Uključujući kriteriji bili su porođajna masa novorođenčadi manja od grama, gestacijska dob novorođenčadi od 37. do 42. gestacijske nedjelje, novorođenčad iz jednoplodne trudnoće, oba spola, bez vidljivih anomalija na rođenju. Isključujući kriteriji bili su porođajna masa novorođenčadi iznad grama, gestacijska dob novorođenčadi < 37 nedjelja, višeplodna trudnoća majke, novorođenčad sa kongenitalnim anomalijama. Uslove istraživanja zadovoljilo je 120 novorođenčadi od kojih su formirane dvije grupe: ispitivana grupa novorođenčadi (ispitanici) sa RNI (novorođenčad sa pozitivnim dijagnostičkim parametrima: klinička slika, hematološki parametri i mikrobiološki nalazi) (n = 28) i kontrolna grupa (kontrolni) novorođenčadi bez infekcije (n = 92). U prvih 48 sati po rođenju praćeni su klinički znaci od strane respiratornog i kardiovaskularnog sistema, poremećaj regulacije tjelesne temperature i patološke promjene u krvnoj slici (6, 7). Zahvaćenost respiratornog sistema bila je prisutna ako je postojao bar jedan od slijedećih poremećaja: tahipneja > 60 udisaja u minuti, dispneja, apneja ili postojanje potrebe za ventilacijom novorođenčeta (8). Pozitivni znaci od strane kardiovaskularnog sistema uključivali su prisutnost jednog ili više slijedećih parametara: srčana akcija u mirovanju iznad 160 otkucaja u minuti, slab periferni puls, krvni pritisak ispod petog percentila za dob, nivo bikarbonata ispod 15 meq/ litar, ph krvi ispod 7.30 i potreba za nadoknadom volumena (9). Tjelesna temperatura, mjerena rektalno, manja od 36,5 C ili veća od 38,0 C smatrana je patološkom. Hematološki parametri obuhvaćali su patološke promjene u krvnoj slici i smatrani su pozitivnim kada je bilo prisutno tri i više od sedam parametara (ukupan broj leukocita < ili > , ukupan broj segmentiranih leukocita iznad 5.000, ukupan broj nesegmentiranih leukocita veći od 500, odnos nesegmentiranih i segmentiranih leukocita veći od 0.2, prisutnost toksičnih granulacija, prisutne mlade forme leukocita i trombocitopenija) hematološkog skor si- 189

50 Medicinski Glasnik, Volumen 6, Number 2, August 2009 stema za ranu dijagnozu neonatalne sepse prema Rodwellu i suradnicima (10). Analizirane su vrijednosti IL-6 i CRP-a iz krvi pupčanika novorođenčadi, te dijagnostički parametri za RNI: klinička slika, hematološki parametri i mikrobiološki nalazi (kultura krvi, urina i likvora). Za identifikaciju uzročnika infekcije korišten je nalaz kulture krvi, te je za svu novorođenčad, uključenu u istraživanje, uzet 1mL krvi iz pupčanika u bočice za uzimanje hemokulture (BD Bactec PEDS PLUS/F, Dickinson and Company, SparksLab Supplies Ltd, Shannon, Ireland). Kultura likvora i standardni pregled likvora načinjeni su samo za onu novorođenčad koja su imala simptome koji su upućivali na meningitis. Urin za bakteriološku kulturu, za svu novorođenčad uključenu u istraživanje, prikupljen je urinarnim vrećicama koje su zamijenjivane svakih 30 minuta do prikupljanja uzorka. Uzorci su analizirani u Poliklinici za laboratorijsku dijagnostiku, Odjeljenje mikrobiologije UKC Tuzla. Izolacija bakterijskog uzročnika u značajnom broju (>10 5 ) za određenu kulturu smatrana je pozitivnim nalazom. Krv iz pupčanika za određivanje vrijednosti IL-6 i CRP-a uzeta je neposredno po rođenju, standardnom procedurom i analizirana je u Poliklinici za laboratorijsku dijagnostiku, Odjeljenje imunologije UKC Tuzla. Uzorci krvi za analizu IL-6 centrifugirani su brzinom okretaja u minuti tokom 10 minuta. Nakon centrifugiranja, odvojeni serum čuvao se u flakonima na temperaturi -80 C, do postupka određivanja na svim uzorcima. IL-6 određivan je metodom ELISA (Metertech 960, Quantikine Human IL-6, R&D systems, Metertech Inc, Taipei, Taiwan). Vrijednosti IL-6 od 0 do 10 pg/ml smatrane su normalnim. Uzorci krvi za određivanje CRP-a analizirani su u toku 24 sata po uzimanju uzorka pomoću nefelometra (BN2, Behring,GmBH, Marburg, Germany). Vrijednosti CRP-a od 0 do 5.0 mg/l smatrane su normalnim (11). Prethodno je dobiven pristanak Etičkog komiteta i svake majke da želi sudjelovati u navedenom istraživanju. U statističkoj obradi podataka za testiranje statističkog značaja razlike među uzorcima korišten je neparametrijski Mann-Whitneyev test za numeričke varijable, χ 2 test korišten je za uspoređivanje frekvencija, te omjer izgleda (OR) sa 95%-tnim rasponom pouzdanosti (CI). Razlika među uzorcima smatrana je značajnom ako je vrijednost p iznosila < Validnosti analiza IL-6 i CRP-a iz krvi pupčanika procijenjene su izračunavanjem senzitivnosti, specifičnosti, njihovih pozitivnih i negativnih prediktivnih vrijednosti, te dijagnostičke tačnosti. Za obradu podataka korišten je statistički program Arcus QuickStat (12). REZULTATI U periodu od 1. marta godine do 30. septembra godine u Klinici za ginekologiju i akušerstvo u Tuzli od ukupno živorođene novorođenčadi porođajnu masu manju od grama imalo je 171 (6,98%) novorođenče. Iz istraživanja je isključeno 51 novorođenče male porođajne mase (MPM), od kojih je 41 rođeno prije 37. gestacijske nedjelje, 9 novorođenčadi je bilo iz višeplodne trudnoće, a jedno je rođeno s vidljivim anomalijama. Porođajna masa novorođenčadi kretala se u rasponu od grama do grama (medijan grama). Gestacijska dob novorođenčadi bila je u rasponu od 37. do 42. (medijan 38) gestacijske nedjelje. Rođena su u relativno dobroj kondiciji s Apgar-skorom u prvoj minuti u rasponu vrijednosti od 6 8 (medijan 6.78), te su bila češće ženskog spola. Kliničke karakteristike novorođenčadi prikazane su u Tablici 1. Pozitivne mikrobiološke nalaze imalo je 38/120 (31,7%) terminske novorođenčadi MPM, od toga pozitivnu kulturu krvi imalo je 22/38 (57,9%) novorođenčadi (u najvećem broju izolovan je Streptococcus β-haemoliticus, 11/22), pozitivna urinokultura bila je kod 12/38 (31,6%) novorođenčadi (u najvećem broju izolovana je E. coli, 7/12) i kod 4/38 novorođenčadi bila je pozi- 190

51 Ćosićkić et al IL-6 i CRP u ranoj novorođenačkoj infekciji Tablica 1. Kliničke karakteristike ispitivane djece Kliničke karakteristike novorođenčadi (N = 120) Gestacijska dob (nedjelje), raspon (medijan) (38) Porođajna masa (g) raspon (medijan) (2.230) Porođajna dužina (cm) raspon (medijan) (49.8) Apgar-skor 1. minut, raspon (medijan) 6-8 (6.78) Apgar-skor 5. minut, raspon (medijan) 7-9 (8.2) Perinatalna asfiksija (n; %) 4 (3,3%) Muški spol (n; %) 38 (31,7%) tivna kultura likvora, gdje je Staphylococcus aureus izolovan u tri slučaja, dok je iz jednog uzorka izolovan Streptococcus β haemoliticus. Kliničku sliku za RNI imalo je 36/120 (30%) terminske novorođenčadi MPM, a najčešći su bili: odbijanje i/ili loše podnošenje hrane (82%), povišena tjelesna temperatura (60%), letargija (58%), povraćanje (44%), hiperbilirubinemija (42%). Pozitivne hematološke parametre za RNI imalo je 32/120 (26,7%) terminske novorođenčadi MPM, a najčešći su bili: odnos nesegmentiranih i segmentiranih leukocita > 0.2 (68%), leukocitoza (66%), trombocitopenija (52%) i leukopenija (32%). Pozitivne sve dijagnostičke parametre (klinička slika, hematološki parametri i mikrobiološki nalazi) za RNI imalo je 28/120 (23,3%) terminske novorođenčadi MPM koja su i činila ispitivanu grupu. Povišene vrijednosti IL-6 zabilježene su kod 33/120 (27,5%) terminske novorođenčadi MPM. Vrijednosti IL-6 uzetog na rođenju iz krvi pučanika u ispitivanoj grupi kretale su se u rasponu od 9.5 pg/ml do 60 pg/ml, uz medijan od 49 pg/ml. U kontrolnoj grupi vrijednosti IL-6 uzetog iz krvi pupčanika kretale su se u rasponu 3.1 pg/ml do 52 pg/ml, uz medijan 9.7 pg/ml. Mann-Whitneyevim testom uočena je statistički značajna razlika između medijana vrijednosti IL-6 u ispitivanoj i kontrolnoj grupi (P < ). Tablica 2. Brojčani odnos vrijednosti interleukina-6 i dijagnostičkih parametara za ranu novorođenačku infekciju u terminske novorođenčadi, omjer izgleda pozitivnog i negativnog nalaza IL-6 (pg/ml) Dijagnostički parametri za RNI* Pozitivan Negativan Ukupno > Ukupno * rana novorođenačka infekcija; χ2 = 54.69; P < 0.001; OR = (95%CI: ) Povišene vrijednosti CRP-a imalo je 20/120 (16,7%) terminske novorođenčadi MPM. Vrijednosti CRP-a uzete iz krvi pupčanika, u ispitivanoj grupi kretale su se u rasponu 0.1 mg/l do 12.5 mg/l, uz medijan 3.5 mg/l, dok su u kontrolnoj grupi bile u rasponu 0.1 mg/l do 9.4 mg/l, a medijan je iznosio 2.8 mg/l. Ustanovljena je statistički značajna razlika između medijana vrijednosti CRP-a u ispitivanoj i kontrolnoj grupi (P = 0.997). Vrijednosti IL-6 iznad 10 pg/ml i pozitivne dijagnostičke parametre za RNI (klinička slika, hematološki parametri i mikrobiološki nalazi) imalo je 23/120 (19,1%) terminske novorođenčadi MPM (Tablica 2). Uočena je statistički značajna povezanost povišenih vrijednosti IL-6 i pozitivnih dijagnostičkih parametara za RNI (χ 2 = 54.69, P <0.001). Vjerovatnost pojave povišenih vrijednosti IL-6 je bila 37,72 puta veća (95%CI: ) kod terminske novorođenčadi MPM s pozitivnim dijagnostičkim parametrima za RNI, nego kod onih s negativnim dijagnostičkim parametrima. Vjerovatnost pojave povišenih vrijednosti IL-6 bila je barem 11,72 puta veća ako su dijagnostički parametri za RNI bili pozitivni, nego kada su bili negativni. Validnost povišene vrijednosti IL-6 u odnosu na analizirane dijagnostičke parametre praćena je putem senzitivnosti, specifičnosti, pozitivne i negativne prediktivne vrijednosti, te dijagnostičke tačnosti (slika 1). Uočena je značajno visoka senzitivnost, specifičnost, pozitivna prediktivna vrijed- * PPV, pozitivna prediktivna vrijednost; ** NPV, negativna prediktivna vrijednost Slika 1. Validnosti testa interleukina-6 u odnosu na dijagnostičke parametre rane novorođenačke infekcije terminske novorođenčadi male porođajne mase* 191

52 Medicinski Glasnik, Volumen 6, Number 2, August 2009 Tablica 3. Brojčani odnos vrijednosti C-reaktivnog proteina i dijagnostičkih parametara za ranu novorođenačku infekciju u terminske novorođenčadi male porođajne mase, omjer izgleda pozitivnog i negativnog nalaza CRP (mg/l) Dijagnostički parametri za RNI* Pozitivan Negativan Ukupno > Ukupno/Total *rana novorođenačka infekcija; χ2 = 1.82; P =0.176; OR = (95%CI: ) nost i dijagnostička tačnost vrijednosti IL-6, dok je negativna prediktivna vrijednost bila niska. Povišene vrijednosti CRP-a i pozitivne dijagnostičke parametre RNI imalo je 7/120 (5,8%) terminske novorođenčadi MPM, dok je 21/120 (17,5%) novorođenčadi sa prisutnim dijagnostičkim parametrima za RNI imalo normalne vrijednosti CRP-a za novorođenačku dob (Tablica 3). Nije uočena statistički značajna povezanost povišene vrijednosti CRP-a s dijagnostičkim parametrima RNI u terminske novorođenčadi MPM (p = 0.17). Vjerovatnost pojave visokih vrijednosti CRP-a bila je samo 2,02 puta veća (95%CI: ) kod novorođenčadi s pozitivnim dijagnostičkim parametrima, nego kod novorođenčadi s negativnim. Vjerovatnost pojave povišenog CRP-a bila je samo 0,71 puta veća ako su i dijagnostički parametri za RNI bili pozitivni. Validnost povišene vrijednosti CRP-a u prepoznavanju RNI praćena putem senzitivnosti, specifičnosti, pozitivne i negativne prediktivne vrijednosti, te dijagnostičke tačnosti prikazana je na Slici 2. Uočena je niska senzitivnost i negativna prediktivna vrijednost povišene vrijednosti Tablica 4. Brojčani odnos vrijednosti interleukina-6, C-reaktivnog proteina i dijagnostičkih parametara za ranu novorođenačku infekciju u terminske novorođenčadi male porođajne mase, omjer izgleda pozitivnog i negativnog nalaza IL-6 (granična vrijednost 10 pg/ml) CRP (granična vrijednost 5 mg/l) > < Dijagnostički parametri RNI* Pozitivan 6 22 Negativan Ukupno Ukupno/ Total * rana novorođenačka infekcija; χ2 = 1.183, P = 0.276; OR = (95%CI: ) CRP-a u odnosu na analizirane dijagnostičke parametre, ali je specifičnost i dijagnostička tačnost bila značajna. Povišene vrijednosti IL-6 i CRP-a, kao i pozitivne dijagnostičke parametre RNI, imalo je 6/120 (5%) terminske novorođenčadi MPM, dok je 22/120 (18,3%) terminske novorođenčadi MPM s pozitivnim dijagnostičkim parametrima imalo IL-6 i CRP normalnih vrijednosti za novorođenačku dob (Tablica 4). Nije bilo statistički značajne povezanosti povišenih vrijednosti IL-6, CRP-a sa analiziranim dijagnostičkim parametrima (p = 0.276). Vjerovatnost pojave visokih vrijednosti IL-6 i CRP-a zajedno bila je samo 1,81 puta veća (95%CI: ) kod novorođenčadi s pozitivnim dijagnostičkim parametrima za RNI nego kod novorođenčadi s negativnim. Validnost povišene vrijednosti IL-6, uz povišene vrijednosti CRP-a u prepoznavanju RNI, a prema analiziranim dijagnostičkim parametrima, praćena putem senzitivnosti, specifičnosti, pozitivne i negativne prediktivne vrijednosti, te dijagnostičke tačnosti, prikazana je na Slici 3. * PPV, pozitivna prediktivna vrijednost; ** NPV, negativna prediktivna vrijednost Slika 2. Validnost testa C-reaktivnog proteina u odnosu na dijagnostičke parametre rane novorođenačke infekcije terminske novorođenčadi male porođajne mase* * PPV, pozitivna prediktivna vrijednost; ** NPV, negativna prediktivna vrijednost Slika 3. Validnost testova interleukina-6 i C-reaktivnog proteina u odnosu na dijagnostičke parametre rane novorođenačke infekcije terminske novorođenčadi male porođajne mase* 192

53 Ćosićkić et al IL-6 i CRP u ranoj novorođenačkoj infekciji Uočena je niska senzitivnost, ali je specifičnost i dijagnostička tačnost bila značajna. DISKUSIJA U ovom istraživanju istražena je dijagnostička vrijednost interleukina-6 (IL-6) i C-reaktivnog proteina (CRP-a) iz krvi pupčanika prema dijagnostičkim parametrima za ranu novorođenačku infekciju (RNI) (klinička slika, hematološki parametri i mikrobiološki nalazi) s namjerom da se utvrdi njihova pouzdanost u pravovremenom prepoznavanju RNI-a u terminske novorođenčadi male porođajne mase (MPM). U strukturi morbiditeta i mortaliteta novorođenčadi, RNI ima značajan udio. Do teških oštećenja ili smrti, obično dolazi u prva 24 sata po rođenju, dok je klinička slika još nejasna, laboratorijski nalazi nespecifični, a mikrobiološki nalazi tek uzeti (13). Pristupanje inicijalnom antibiotskom tretmanu zasniva se upravo na kliničkim znacima bolesti, hematološkim parametrima i podacima o epidemiološkim faktorima vezanim za RNI. Ove su procjene subjektivne, tako da terapija često bude primijenjena nepotrebno. Od trideset novorođenčadi kod kojih se terapija započne na osnovu ovakvih procjena, potreba za antibiotskom terapijom dokaže se samo u jednog novorođenčeta (14, 15). U našem istraživanju uočili smo značajnu povezanost vrijednosti IL-6 iz krvi pupčanika s pozitivnim dijagnostičkim parametrima za RNI, kao i značajno visoku senzitivnost i specifičnost IL-6 iz krvi pupčanika. Visoka pozitivna prediktivna vrijednost, te značajna povezanost vrijednosti IL-6 i pozitivnih dijagnostičkih parametara za RNI, potvrđuju i u našem istraživanju pouzdanost ovog dijagnostičkog parametra u prepoznavanju RNI. Naši rezultati o validnosti testa IL-6 iz krvi pupčanika donekle su slični rezultatima drugih istraživanja. Yoon i suradnici (16) dokazali su značajnu povezanost povišenih vrijednosti IL-6 iz krvi pupčanika s nastankom RNI. Reyes i suradnici (17) istakli su senzitivnost IL-6 za prepoznavanje RNI od 80%. Rezultati nekoliko studija potvrdili su da je IL-6 iz krvi pupčanika senzitivni marker za dijagnozu novorođenačke infekcije koja nastaje u prva 72 sata života, uz senzitivnost % i negativnu prediktivnu vrijednost % (18-20). Drugi istraživači ističu pouzdanost IL-6 i u prepoznavanju kasne novorođenačke infekcije, uz senzitivnost 89% i negativnu prediktivnu vrijednost 91%, te kako je značajno senzitivniji od drugih markera, a među njiima i CRP-a (21). Hadžijaki i suradnici (22) analizirali su vrijednosti IL-6 majčine krvi, iz krvi pupčanika i periferne krvi novorođenčadi. Došli su do rezultata da su vrijednosti IL-6 iz krvi pupčanika značajno veće u novorođenčadi s infekcijom, uz senzitivnost 95%, specifičnost 100%, pozitivnu prediktivnu vrijednost 100% i negativnu prediktivnu vrijednost 97,4%, te su zaključili da je IL-6 iz krvi pupčanika jedan od najsenzitivnijih markera za prepoznavanje RNI. S druge pak strane, Janota i suradnici (23) su istakli da vrijednosti IL-6 iz krvi pupčanika ili iz krvi novorođenčeta, uzete u prva dva sata života, nema dovoljnu senzitivnost, ni specifičnost za prepoznavanje RNI. Dooy i suradnici (24) analizirali su povezanost stvaranja inflamatornih medijatora i perinatalne kolonizacije respiratornog trakta. Njihovi rezultati govore o tome da su proinflamatorni citokini, među kojima je i IL-6, bili značajno povećani u novorođenčadi koja je bila inficirana gram-negativnim patogenima koji su izolirani mikrobiološkim nalazima. U našem istraživanju, povišene vrijednosti CRP-a i pozitivne dijagnostičke parametre za RNI imalo je 5,8% terminske novorođenčadi MPM i nismo pronašli ovisnost povišene vrijednosti CRP-a iz krvi pupčanika i dijagnostičkih parametara za RNI. Procjenom validnosti povišene vrijednosti CRP-a iz krvi pupčanika u prepoznavanju RNI, našli smo nisku senzitivnost, ali značajnu specifičnost i dijagnostičku tačnost. Ovako značajna specifičnost testa CRP-a potvrda je da negativan nalaz CRP-a sa značajnom sigurnošću znači i odsutnost infekcije. Mali broj novorođenčadi ispitivane grupe s povišenim CRP-om tumačimo njegovom biološkom ulogom u organizmu i dinamikom njegove sinteze. Odgađanje njegovog porasta 193

54 Medicinski Glasnik, Volumen 6, Number 2, August 2009 za nekoliko sati počiva na kaskadi zbivanja koja dovode do porasta razine CRP-a, uključujući aktivaciju neutrofila, poticaja od strane IL-6 i same hepatičke sinteze CRP-a (25). Benitz i suradnici (25) u svojoj studiji ukazali su kako je serijsko mjerenje CRP-a iz krvi pupčanika, a potom sata nakon rođenja, značajno za otkrivanje novorođenčadi s infekcijom. Nameće se zaključak da je senzitivnost testa CRP-a iz krvi pupčanika, u našem istraživanju, očekivano snižena, s obzirom na potrebu praćenja vrijednosti CRP-a u određenim vremenskim intervalima (12 do 24 sata). Arshad i suradnici (26) ustanovili su da povišen CRP, 12 do 24 sata nakon pojave znakova infekcije, ima pozitivnu prediktivnu vrijednost od samo 7-43%, ali negativnu prediktivnu vrijednost od 97-99,5%, te se CRP može smatrati vrlo korisnim u procjeni odsutnosti infekcije. Isti autori ističu kako je senzitivnost CRP-a iznosila 86,7% za slučajeve RNI s pozitivnom hemokulturom i 80,6% za slučajeve RNI s negativnom hemokulturom. Zeeshan i suradnici (27) izvijestili su o senzitivnosti, specifičnosti, pozitivnoj i negativnoj prediktivnoj vrijednosti CRP-a od 85,7%, 95%, 82,7% i 95,9% u grupi novorođenčadi s pozitivnim nalazom hemokulture. Jedan od razloga širokog raspona senzitivnosti CRP-a, koji se kreće od % i specifičnosti od 6-97% u detekciji RNI, donekle je i u činjenici o još uvijek neusuglašenom stavu u literaturi o dopuštenoj gornjoj vrijednosti CRP-a u novorođenačkom periodu. Mogući razlog diskrepance u senzitivnosti CRP-a u različitim studijama može biti i različitost dijagnostičkih kriterija, ali i različitih metoda kojima se određuje vrijednost CRP-a (28). U našem istraživanju nismo uočili značajnu povezanost povišene vrijednosti oba testa, i IL-6 i CRP-a, sa pozitivnim dijagnostičkim parametrima RNI. Evaluacijom validnosti kombinacije oba testa, i IL-6 i CRP-a, našli smo nisku senzitivnost, ali je specifičnost bila značajna. Naši rezultati o validnosti kombinacije testova IL-6 i CRP-a nešto se razlikuju od rezultata drugih istraživanja. Khassawnwh i suradnici (29) izvještavaju o značajnoj prednosti CRP-a prema vrijednostima IL-6 i IgM u prepoznavanju infekcije novorođenčadi sa senzitivnošću CRP-a 95% i negativnom prediktivnom vrijednosti 98%. Zaključuju da su CRP, IL-6 i IgM u kombinaciji veoma korisni za ranu dijagnozu gram-negativne neonatalne sepse. Døllner i suradnici (30) ističu pouzdanost kombinovanog testa CRP-a i IL-6 u prepoznavanju novorođenčadi s infekcijom i vjerovatnom infekcijom i njihove senzitivnosti 85% i specifičnosti 62%. Dijagnostička vrijednost IL-6 iz krvi pupčanika u prepoznavanju RNI značajna je zbog visoke senzitivnosti, specifičnosti i prediktivne pozitivne vrijednosti, dok je dijagnostička vrijednost CRP-a iz krvi pupčanika niske senzitivnosti, ali visoke specifičnosti, te može poslužiti kao brza i danas lako dostupna pretraga kojom se mogu, s velikom sigurnošću, probrati novorođenčad bez infekcije. ZAHVALE / IZJAVE Komercijalni ili potencijalni dvostruki interes ne postoji. LITERATURA Stoll BJ. Infections of the neonatal infant. U: Behrman RE, Kliegman RM, Jenson HB ur. Nelson textbook of pediatrics. 17. izd. Philadelphia: WB Saunders, 2004: Levene M, Tudehope D, Thearle J. Neonatal transport and organition of perinatal services.u: Levene M, Tudehope D, Thearle M, ur. Essentials of neonatal medicine. London: Blackwell Science Ltd, 2000: Prober CG. Clinical approach to the infected neonate. U: Long SS, Pickerin LK, Prober CG ur. Principles and practice of pediatric Infectious diseases. New York: Churchill Livingstone, 1997: Naccasha N, Hinson R, Montag A, Ismail M, Bentz L, Mittendorf R. Association between funisitis and elevated interleukin-6 and C-reactive protein in cord blood. Obstet Gynecol 2001; 97:

55 Ćosićkić et al IL-6 i CRP u ranoj novorođenačkoj infekciji Heep A, Behrendt D, Nitsch P, Fimmers B, Bartmann P, Dembinski J. Increased serum levels of interleukin 6 are associated with severe intraventricular haemorrhage in extremely premature infants. Dis Child Fetal Neonatal Ed 2003; 88: Bromberger P, Lawrence JM, Braun D, Saunders B, Contreras R, Petitti DB. The Influence of intrapartum antibiotics on the clinical spectrum of early onset group B streptococcal infection in term infants. J Pediatr 2000; 106: Saez-Llorens X, McCracken GH. Sepsis syndrome and septic shock In pediatrics: current concepts of terminology, patophysology and management. J Pediatr 1993; 123: D Harlingue AE, Durand DJ. Recognition, stabilization and transport of the high risk newborn. U : Klaus MH, Fanaroff AA, ur. Care of the high risk neonate. 4.izd. Philadelphia: WB Saunders Co, 1993; Graves GR, Rhodes PG. Tachycardia as a sign of early onset neonatal sepsis. Pediatr Infect Dis 1984; 3: Rodwell RL, Leslie AL, Tudhope DL. Early diagnosis of neonatal sepsis using a haematologic scoring system. J Pediatr 1993; 12: Mathers NJ, Pohlandt F. Diagnostic audit of C- reactive protein in neonatal infekctions. Eur J Pediatr. 1987; 146: Buchan IE. Arcus QuickStat Biomedical version 1.izd. Cambridge: Adisson Wesley Longman Ltd; Hengst JM. The role of C- reactive protein in the evaluation and management of infants with suspected sepsis. Adv Neonatal Care 2003; 3:3-13. Hammerschlag MR, Klein JO, Herschel M, Chen FC, Fermin R. Patterns of use of antibiotics in two newborn nurseries. N Engl J Med 1997; 296: Philip AG, Hewitt JR.Early diagnosis of neonatal sepsis. Pediatrics 1980; 65: Yoon BH, Roberto RP, Shin J. The relationship among inflammatory lesions of the umbilical cord (funisitis), umbilical cord plasma interleukin 6 concentration, amniotic fluid infection, and neonatal sepsis. Am J Obstet & Gynecol 2000; 183: Reyes SC, Garcia-Munoz F, Reyes D, Gonzalez G, Dominguez C, Domenech E. Role of cytokines (interleukin-1 beta, 6, 8, tumor necrosis factor alpha and soluble receptor of interleukin-2) and C- reactive protein in the diagnosis of neonatal sepsis. Acta Paediatr 2003; 92: Messer J, Eyer D, Donato L. Evaluation of interleukin-6 and soluble receptors of tumor necrosis factor for early diagnosis of neonatal infection. J Pediatr 1996; 129: Brener R, Niemeyer CM, Leititis JU. Plasma levels and gene expression of granulocyte colonystimulating factor, tumor necrosis factor-alpha, interleukin (IL)-1-beta and soluble intercellular adhesion molecule-1 in neonatal early onset sepsis. Pediatr Res 1998; 44: Sulian JC, Vintzileos AM, Lai YL. Maternal chorioamnionitis and umbilical vein interleukin-6 levels for identifying early neonatal sepsis. J Matern Fetal Med 1999; 8: Ng PC. Diagnostic markers of infection in neonates. Arch Dis Child Fetal Neonatal Ed 2004; 89:229. Hatzidaki E, Gourgiotis D, Manoura A, Korakaki E, Bossios A, Galanakis E. Interleukin- 6 in preterm premature rupture of membranes as an indicator of neonatal outcome. Am J Perinatol 2001; 18: Janota J, Stranák Z, Bĕlohlávková S, Jirásek JE. Chorioamnionitis and early-onset neonatal sepsis do not significantly affect levels of interleukin-6 in very low birth weight infants. Sb Lek 2001; 102: Dooy JD, Ieven M, Stevens W. Endotracheal colonization at birth is associated with a pathogendependent aro-and antiinflammatory cytokine response in ventilated preterm infants: a prospective cohort study. Pediatr Res 2004; 54: Benitz WE, Gould JB, Druzin ML. Preventing early onset group B streptococcal sepsis, strategy development using decision analysis. Pediatrics 1999; 103:76. Arshad A, Asghar I, Tariq MA. Role of serum C- reactive protein in the rapid diagnosis of neonatal sepsis. Pak Armed Forces Med J 2003; 53: Zeeshan A, Ghafoor T, Waqar T, Ali S, Aziz S, Mahmud S. Diagnostic value ff C-reactive protein and haematological parameters in neonatal sespsis. JCPSP 2005; 15: Chiesa C, Panero A, OsbornJF, Simonetti AF, Pacifico L. Diagnosis of neonatal sepsis: a clinical and laboratory challenge. Clin Chem 2004; 50: Khassawneh M, Hayajneh WA, Kofahi H, Khader Y, Amarin Z, Daoud A. Diagnostic markers for neonatal sepsis: comparing C-reactive protein, interleukin-6 and immunoglobulin M. Scand J Immunol 2007; 65: Døllner H, Vatten L, Austgulen R. Early diagnostic markers for neonatal sepsis: comparing C-reactive protein, interleukin-6, soluble tumour necrosis factor receptors and soluble adhesion molecules. J Clin Epidemiol 2001; 54:

56 Medicinski Glasnik, Volumen 6, Number 2, August 2009 Diagnostic value of Interleukin 6 and C-reactive protein from umbilical cord blood in recognition of early infection in full-term newborns with low birth weight Almira Ćosićkić 1, Fahrija Skokić 2, Selmira Brkić 3 1 Clinic for children s diseases, 2 Clinic for gynecology and obstetrics, Department for neonates; University Clinical Center in Tuzla, 3 Depatment of Pathophysiology; Medical Faculty intuzla, ABSTRACT Aim Diagnostic value evaluation of Interleukin-6 (IL-6) and C-reactive protein (CRP) from the umbilical cord blood in diagnosis of early newborn infection (ENI) on the basis of clinical picture, hematological parameters and microbiological tests. Methods A retrospective-prospective study conducted in the Department of Gynecology and Obstetrics. One hundred and twenty newborns were included in the study. Inclusion criteria were: newborns with birth weight less than 2500 grams, gestational age from 37 to 42 weeks, both genders, no visible anomalies at birth, from a single pregnancy. Newborns were divided into two groups: the examined group, newborns with ENI (n=28), and second, control group of newborns without ENI (n=92). IL-6 and CRP were determined from the umbilical cord blood and clinical picture, hematological parameters and microbiological test results. Results IL-6 values of from 0 to 10 pg/ml were considered normal. Median IL-6 value of in the first group was 49 pg/ml and in the second group 9,7 pg/ml with significant difference between these two groups (P<0.0001). Sensitivity of IL-6 test was 82% and specificity 89%. CRP values from 0 to 5 mg/l were considered normal. Median value of CRP in first group was 3,5 mg/l, and in the control group 2,8 mg/l with no significant difference between the groups (P=0,997). Sensitivity of CRP test was 25% and specificity was 86%. Sensitivity of both IL-6 and CRP tests combined was low 21%, whereas specificity was 87%. Conclusion Diagnostic value of IL-6 from umbilical cord blood in recognition of ENI is remarkable, while CPR value from umbilical cord blood has limited importance. Keywords: Interleukin-6, C-reactive protein, early newborn infection Original submission: 22 September 2008.; Revised submission: 13 November 2008; Accepted: 12 January

57 ORIGINAL ARTICLE Alterations in body weight and biochemistry in patient treated with different psychotropic drugs in a clinic in Istanbul Aliye Ozenoglu 1, Serdal Ugurlu 2, Huriye Balci 3, Gunay Can 4, Funda Elmacıoglu 1, Yeltekin Demirel 5, Engin Eker 6 1 Ondokuz Mayis University, Samsun Health School, Samsun; 2 Division of Rheumatology, Department of Medicine, Fatih sultan Mehmet Education and Research Hospital, Istanbul; 3 Central Laboratory, Cerrahpasa Medical Faculty, University of Istanbul; 4 Department of Public Health, Cerrahpasa Medical Faculty, University of Istanbul,Department of Family Medicine, Medical faculty, University of Cumhuriyet, Sivas; 6 Cerrahpasa Medical Faculty, University of Istanbul, Istanbul; Turkey. ABSTRACT Aim Was to compare adult female patients receiving psychiatric drugs with obese adult females who didn t receive any drug treatment with respect to the alterations in body weight and biochemistry, and find out the contrubution of a team approach for the management of these alterations. Corresponding author: Aliye Özenoğlu, Ondokuz Mayis University, Samsun Health School Samsun, Turkey Phone: Fax: aozenoglu@omu.edu.tr; Original submission: 17 November 2008; Revised submission: 30 April 2009; Accepted: 18 May Methods A total of 102 female patients aged mean 40.9±12.4 years who had been followed up and treated in the Psychiatry Outpatient Clinics in Istanbul University for their psychiatric disorders and were complaining about increased body weight in the treatment period were included. The controls were composed of 261 females aged mean 39.8±13.0 years who had been referred by various departments to dietitians due to exogenous obesity but had no endocrine-metabolic or psychiatric disorders or history of drug use. Initially, antropometric measurements and biochemical tests were performed for all patients. Results In the group receiving psychiatric treatment, the mean body weight, BMI, waist and hip circumferences, body fat percentage (p<0.001); blood insulin, triglyceride, TSH, fibrinogen and homocysteine levels, and HOMA-IR were found to be higher than those of the controls (p<0.05), whereas the total protein, albumin, zinc and folate levels were significantly lower (p<0.001). Conclusion The results of this study showed that patients who need psychopharmacotherapies were also more susceptible to several metabolic and cardiovascular disorders. Therefore, it would be useful if psychiatric patients are treated with a multidisciplinary team approach consisting of an endocrinologist, psychiatrist and a dietitian specialized in this area to prevent or delay the metabolic disorders caused by psychiatric disorders and treatments. Key words: body composition, obesity, psyhcotropic drugs Med Glas 2009; 6(2):

58 Medicinski Glasnik, Volumen 6, Number 2, August 2009 INTRODUCTION During psychiatric treatment, body weight usually increases and this is frequently accompanied by an increase in appetite (1). This side effect, which is difficult to foresee of its development and timing, finally causes obesity and results in the cessation of an effective treatment in some of the patients. Obesity not only affects the psychological state of the patient, but also increases the risk of development of several chronic diseases such as coronary artery disease, hypertension, hyperlipidaemia, diabetes, some types of cancer, cerebrovascular diseases, osteoarthritis, pulmonary diseases and sleep apnoea (2-4). Therefore, reduction of body weight plays a pivotal role in the prevention of several chronic diseases and improvement of the prognosis of an ongoing disease. In this research, we aimed to compare the changes in anthropometric and biochemical parameters in patients treated with psychiatric drugs with ones who did not, and to find out the contrubution of a dietitian specialized in this area for the treatment of nutrition related metabolic problems arising out of psychiatric pharmacotherapies. PATIENTS AND METHODS In order to determine patients group for this research the records of all patients who addmitted to outpatient clinic of Psychiatric Department in Cerrahpasa Medical Faculty University of Istanbul in the 2004 to 2008 period retrospectively analyzed. It has chosen two groups among 363 patients who suffer from obesity and were followed by a dietician. All patients are informed and accepted their inclusion in the research. The study group included 102 (28.1%) adult female patients taking psychiatric pharmacotherapies and complained about increased body weight within this period. The mean ±SD age of study group was 40.9±12.44 years. Medications that patients in the study group have taken were antipsychotics 34 (38,2%), mood stabilisers 36 (40,4%), antidepressants 72 (80,9%) or anxiolytic 9 (10,1%) which were mostly taken as combined pharmacotherapy. The controls included 261 (71.9%) adult females who had no endocrine, metabolic or psychiatric disorders or history of drug use and had been referred to dietitians due to exogenous obesity. The mean ±SD age of control group was 39.8±13.0 years. As body composition depends on age, sex and severity of obesity (5, 6) both groups included adult females with body-mass index (BMI) 25. All patients underwent antropometric assessment, body composition analysis using a Bioelectrical Impedance Analyzer (Bodystat Quadscan 4000, England). Biochemical parameters of patients were studied with overnight fasting blood samples taken from the antecubital vein. Biochemical parameters, serum fasting blood glucose (FBG), total protein, albumin, uric acid, triglyceride, and total, HDL and LDL cholesterol were measured using an Olympus AU 800 autoanalyzer (Olympus, Japan). FBG was analysed using the hexokinase method. The methods were biuret for total protein, BCG for albumin and the uricase / PAP method for uric acid. Levels of total, HDL cholesterols and triglycerides were measured using enzymatic methods in all samples. Serum hscrp concentrations were determined with immunonephelometry using the BN II Systems Analyzer (Dade Behring, Malburg, Germany). FT3, FT4, third-generation thyroid sitimulating hormone (TSH) were measured on Immulite 2000 (DPC; LosAngeles,USA). FT3 and FT4 were measured by a competitive analog-based immunoassay. TSH levels were determined by two-side chemiluminescent immunometric assay. Insulin and cortisol were measured with Immulite 2000 analyzer (DPC,USA) by chemiluminescent immunometric assay. Insulin resistance (IR) was determined by HOMA- IR index, e.g. serum insulin (mg/dl) x plasma glucose (mg/dl) / 405. Serum B12, folic acid levels were measured by radioimmunoassay (RIA) (DPC, USA). Plasma level of homocysteine was determined by high-performance liquid chromatography (HPLC Agilent 1100 Series), coupled with fluorescence detector. Plasma fibrinogen levels were measured by BCT (Dade Behring, Malburg, Germany) analyser. Serum zinc, and copper concentrations were determined using the standard atomic absorption spectrophotometry. 198

59 Ozenoglu et al Body weight and psychotropic drugs treatment The data have been analysed with the Student s t-test and correlations were calculated using Pearson correlation. All data are expressed as mean ± standard deviation (SD). RESULTS The results of anthropometric measurements and body composition analyses of study and control groups are shown in Table 1, while the biochemical parameters are shown in Table 2. In the study group, the mean body weight, BMI, waist and hip circumferences, the waist/ hip ratio, body fat percentage, and serum insulin, triglyceride, TSH, fibrinogen and homocysteine levels were found to be significantly greater while the percent of body water and lean body mass, total protein, albumin, zinc and folate levels were significantly lower than those of the controls. In the study group, there was a positive correlation between BMI, waist and hip circumferences, body fat percentage, basal metabolic rate, insulin, HOMA-IR and ferritin levels (r=0.779, p<0.001; r=0.895, p<0.001; r=0.438, p<0.001; r=0.593, p<0.001; r=0.379, p<0.001; r=0.340, p<0.001; r=0.470, p<0.001; respectively). However, there was a negative correlation between BMI and the percentage of body water and lean body mass (r= , p<0.001; r=-0.423, p=0.006; respectively). Table 1. Comparison of the anthropometric measurements and body analyses of study group and control group Group Anthropometric Study Control measures Mean ± SD Mean ± SD p* Height (cm) ± ± Weight (kg) ± ± <0.001 Body Mass Index (BMI) ± ± 6.40 <0.001 Waist circumference (cm) ± ± <0.001 Hip circumference (cm) ± ± <0.001 Waist/hip ratio ± ± % of body water ± ± 4.41 <0.001 % of body fat ± ± 5.75 <0.001 % of lean body mass ± ± 6.03 <0.001 Basal metabolic rate ± ± (kcal) * p-values below 0.05 were considered significant In the control group, there was a positive correlation between BMI and waist and hip circumferences, the waist/hip ratio, body fat percentage, basal metabolic rate, fasting blood glucose, insulin, HOMA-IR, triglycerides, fibrinogen levels (r=0.869, p<0.001; r=0,917, p<0.001; r=0.195, p=0.009; r=0.370, p<0.001; r=0.671, p<0.001; r=0.273, p<0.001; r=0.368, p<0.001; r=0.416, p<0.001; r=0.196, p=0.003; r=0.467, p=0.001; respectively). But BMI showed negative correlation with the percentage of body water and lean body mass, and HDL-C level (r=-0.554, p<0.001; r= , p<0.001; r=-0.210, p=0.002; respectively). Mean duration for being treated with psychotropic drugs of patients in the study group was found median 2 years (Interquartile range, IQR: 1-4) and the increase in body weight was 11.9 ± 5.0 kg in this period. In study group there was a positive correlation between the duration of drug treatment and body weight, BMI, waist and hip circumferences, body fat percentage, insulin, triglycerides, TSH and homocystein (r=0.293, p=0.005; r=0.272, p=0.010; r=0.339, p=0.002; r=0.280, p=0.016; r=0.310, p=0.009; r=0.262, p=0.019; r=0.241, p=0.025; Table 2. Comparison of the biochemical parameters of the groups* Group Biochemical Study Control parameters Mean ± SD Mean ± SD p* FBG (mg/dl) ± ± Insulin (miciu/ml) ± ± Triglycerides (mg/dl) ± ± Total cholesterol (mg/dl) ± ± HDL (mg/dl) ± ± LDL (mg/dl) ± ± Total protein (g/dl) 7.17 ± ± Albumin (g/dl) ± ± 0.35 <0.001 Hemoglobin (g/dl) ± ± Haematocrit (%) ± ± TSH (miu/ml) 2,2864 ±2,1395 1,83 ± 1, FT3 (pg/ml) 2,93 ± 0,598 3,229 ± 0, FT4 (ng/dl) 1,532 ± 2,67 2,058 ± 3, HOMA-IR 4.16 ± ± Fibrinogen (mg/dl) ± ± <0.001 CRP (mg/l) 7.69 ± ± Cortisol (µg/dl) ± ± Zinc (µg/dl) ± ± Copper (µg/dl) ± ± Uric acid (mg/dl) 4.94 ± ± Vitamin B12 (pg/ml) ± ± Folic acid (ng/ml) 6.99 ± ±3.98 <0.001 Homocysteine (umol/l) ± ± *FBG, fasting blood glucose; TSH, thyroid sitimulating hormone; FT3, free T3; FT4, free T4; HOMA-IR, homeostasis model of assesment-insulin resistance; CRP, C-reactive protein; *p-values below 0.05 were considered significant 199

60 Medicinski Glasnik, Volumen 6, Number 2, August 2009 r=0.394, p=0.000; r=0.344, p=0.030; respectively), but there was a negative correlation between the duration of drug treatment and percentage of body water, percentage of lean body mass, and albumin (r=-0.278, p=0.020; r=0.293, p=0.013; r=-0.415, p=0.006; respectively). It has also found a positive correlation in the study group between weight gain and length of drug treatment, BMI, waist and hip circumferences, body fat percentage, insulin, TSH and HOMA-IR (r=0.646, p=0.000; r=0.534, p=0.000; r=0.504, p=0.000; r=0.511, p=0.000; r=0.412, p=0.000; r=0.382, p=0.000; r=0.419, p=0.000; r=0.319, p=0.004; respectively), but there was a negative correlation between weight gain and percentage of body water, percentage of lean body mass, LDL-C and folat levels (r=-0,534, p=0.000; r=-0.388, p=0.001; r=-0.283, p=0.010; r=-0.247, p=0.041; respectively). DISCUSSION Abdominal obesity is strongly associated with disorders of glucose, insulin and lipid metabolism (7-11). Waist circumference is among the fixtures of the diagnostic criteria of metabolic syndrome (12). In people with abdominal obesity, the presence of at least the two of the diagnostic criteria is regarded as indicative of metabolic syndrome (12). In these people, high serum uric acid, leptin, insulin, and CRP levels and high plasma fibrinogen as well as clinical depression, non-alcoholic steatosis, and policystic ovary syndrome are more commonly encountered (12, 13). Recent studies have also indicated the association of insulin resistance and impaired glucose tolerance with decreased cortical functions and Alzheimer s disease (AD) (14-20). In our study, higher levels of FBG, serum insulin, triglycerides and homocysteine levels in the study group (Table 2) may indicate that these patients have greater risk of not only cardiovascular and metabolic diseases, but also of AD. In our study, the group receiving pharmacotherapy had a mean HOMA-IR value which was significantly greater than that of the controls. Besides, the extremely significantly greater BMI, waist and hip circumferences, waist/hip ratio, body fat percentage and fasting blood insulin values in the study group as compared with the controls also are findings compatible with the diagnostic criteria of metabolic syndrome (Tables 1 and 2). Metabolic syndrome has been reported to be considerably prevalent among patients treated for schizophrenia (21). This situation indicates significantly increased risks of cardiovascular and metabolic disorders. Therefore, evaluation and follow-up of the risks associated with metabolic syndrome should be part of the clinical treatment in patients treated with antipsychotics. The influence of psychopharmacological treatment on body weight does not only vary with the drug classes, but also among patients receiving the same treatment (22). Clinical parameters that may explain the increased body weight include the duration and dosage of treatment, duration of the disease, clinical response, age, sex, smoking, BMI, environmental factors, prominent alteration of appetite, deviation from the normal body weight at the beginning of treatment, and drug-induced activation of the tumor necrosis factor (TNF) system (23-25). Many authors have reported that increased weight gain is correlated with improved clinical symptoms (26-28). In the present study, majority of patients on psychiatric pharmacotherapy use more than one drug. In addition, the proportion of antidepressant users was the highest (80.9%). It is possible that individual factors may have contributed to significant weight gain as well as the effect of antidepressants on appetite and body weight. When the factors such as premobid body weight, tendency to metabolic diseases, nutritional and physical activity habits, and coping levels with modification in appetite and sedation caused by psychotropic drugs are considered together, it may be possible to explain the development of weight gain and its relation with risk factors (26). Therefore, our suggestion is that patient should be evaluated in terms of other hormonal metabolic disorders that may develop during treatment, in addition to psychiatric disorders, and should be followed during the course of treatment. Chronic lithium treatment has been shown in several studies to cause increased body weight (29-30). The extent of this increment varies among reports. It has been reported that weight gain occurs within the first 2 years, and body weight does 200

61 Ozenoglu et al Body weight and psychotropic drugs treatment not increase significantly despite continuation of lithium intake (31, 32). Not all of the patients underwent lithium treatment in our study; however, thyroid stimulating hormone (TSH) levels were significantly higher in the study group than the controls. Because slowing down the functions of thyroid gland would contribute to obesity and other related risk factors by decreasing the basal metabolic rate, patients should be followed up regarding this issue. Recent studies have proposed that clozapine, an atypical antipsychotic, is associated with insulin resistance (22, 26). It has been suggested that hyperleptinemia may form an important link between the development of obesity and insulin resistance syndrome, particularly in patients using atypical antipsychotics such as clozapine (22-24, 33). The majority of patients examined in our study used polypharmacy, which makes it difficult to ascribe insulin resistance established in the study group to solely antipsychotic use. In one study, a positive correlation was found between body fat and fibrinogen, while plasma leptin concentrations were shown to be correlated with fibrinogen and CRP (33). In our study, the mean fibrinogen level in the group receiving drugs was significantly higher from those of the controls (Table 2). While CRP was high in both groups (normal values; 0 to 5 mg/l), cortisol levels were higher in the study group. It is thought that higher cortisol levels in psychiatric patients are associated with their endogenous stress, and increased preference of sweet foods may be a mechanism developed for coping with this stress (8). In this study, we have found that while the body weight, waist circumference and body fat ratio increase in patients undergoing pharmacotherapy for psychiatric disorders, increased circulating glucose, insulin, triglyceride and homocysteine levels accompanied by increased proinflammatory cytokines render these patients more susceptible to various metabolic and cardiovascular problems. Therefore, we conclude that it would be useful if candidates for psychiatric pharmacotherapy treated with multidiscipliner team approach consisting of an endocrinologist, psychiatrist and a dietitian to prevent or delay the metabolic disorders developing along with the psychiatric disorders and treatments. Although the importance of team approach in the management of complex diseases has been stressed adequately in the literature, in clinical practice, while the dominant problem of the patient is given priority, other problems that may develop in time may be neglected. If a dietitian experienced in and informed on particularly disorders associated with endocrine metabolism and psychiatry fields and who is skilled in communicating with patientsis included in the team, he/ she may serve as a bridge in cooperating with other units and in maintaining this cooperation. In the present study, it was established that a dietitian experienced in and informed on this field may assume an important role in the treatment of nutritional, metabolic and cardiovascular diseases, which may develop in addition to psyhiatric disorders in patients receiving psyschiatric treatment. However, it will not always be posssible to consult endocrinologists and dietitians for each patients receiving psychiatric treatment in the clinic. Therefore, family physicians and young psychiatrists should also be aware of the impact of various diseases and drug treatments on appetite and body weight and cardiometabolic diseases that may develop during treatment. ACKNOWLEDGEMENT/DISCLOSURE Competing interests: none decleared. REFERENCES Ozenoğlu A. Changes in appetite and body weight during tretament of psychiatric diseases and approaches to overcome them. Diabet Bilimi 2006; 4: Aronne LJ. Epidemiology, morbidity and treatment of overweight and obesity. J Clin Psychiatry 2001; 62 (Suppl): Rosmond R, Björntorp P. The role of antidepressants in the treatment of abdominal obesity. Med Hypotheses 2000; 4: Ozenoglu A. Medical nutrition treatment of obesity. Istanbul: Dilek Basımevi, Ozenoglu A, Can G, Hatemi H. Reference parameters of body composition among man and women with normal weight, overweight, obesity and morbid obesity acoording to their BMI. Endokrinolojide Yonelisler 2001;10:

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63 ORIGINAL ARTICLE Klinička revizija lipidnog statusa kod tipa 2 dijabetesa na nivou timova porodične/obiteljske medicine u općini Zenica, Bosna i Hercegovina Larisa Gavran 1, Selmira Brkić 2 1 Edukativni centar porodične medicine Travnička, Dom zdravlja Zenica, 2 Medicinski fakultet Univerziteta u Tuzli; Bosna i Hercegovina SAŽETAK Cilj Cilj ove studije jeste istražiti da li su timovi porodične/obiteljske medicine (TOM) u općini Zenica, nakon uvođenja posebnog kartona za dijabetes (PKD), mogli dovesti do poboljšanja kontrole nivoa lipida za pacijente oboljele od dijabetes melitusa (DM) tipa 2, prema preporučenim smjernicama. Metode Klinička revizija prakse bila je izvedena pregledom kartona pacijenata oboljelih od DM-a tipa 2, starijih od 18 godina, za 19 timova porodične/obiteljske medicine u Zenici, dvije godine prije ( ) i dvije godine poslije ( ) implementacije vodiča za DM. Podijelili smo sve zabilježene vrijednosti lipida i sve TOM na one koji su dostigli optimalni nivo (UK < 4.5 mmol/l; LDL- kolesterol < 2.5 mmol/l; TG < 1.7 mmol/l) i one koji to nisu (neoptimalni nivo UK > 4.5 mmol/l; LDL- kolesterol > 2.5 mmol/l; trigliceride > 1.7 mmol/l). Corresponding author: Larisa Gavran, Edukativni centar porodične medicine Travnička, Dom zdravlja Zenica, Fra Ivana Jukića 2, Zenica, Bosna i Hercegovina Phone: ; gavranlarisa@yahoo.com Originalna prijava: 22. septembar 2008.; Korigirana verzija: 14. decembar 2008.; Prihvaćeno: 31. mart Rezultati Pregledana su 853 kartona pacijenata oboljelih od DM tipa 2, 46 po jednom TOM-u. Od 19 voditelja TOM-a, četiri (21%) su bili muškog i 15 (79%) ženskog spola. Prosječna starosna dob iznosila je 46,6 godina. Ustanovljen je statistički značajan napredak za optimalni nivo za LDL - kolesterol (19 u odnosu na 531; p < ), kolesterol (67 u odnosu na 212; p < ) i trigliceride (227 u odnosu na 463; p < ) u periodu prije implementacije PKD-a u odnosu na period poslije. Statistički značajan napredak optimalnog nivoa za trigliceride po timovima nađen je za 10 od 19 TOM (P < ). Zaključak Nakon implementacije vodiča za kontrolu lipida kod DM tip 2 pacijenata, većina TOM-a unaprijedila je optimalni nivo lipida. Ključne riječi: lipidi, diabetes mellitus, vodiči, timovi porodične/ obiteljske medicine Med Glas 2009; 6(2):

64 Medicinski Glasnik, Volumen 6, Number 2, August 2009 UVOD Diabetes mellitus (DM) tip 2 je heterogeni poremećaj kompleksne etiologije koji se javlja kao odgovor na genetske utjecaje i utjecaje spoljne sredine. Centralni događaj u razvoju DM-a jesu insulinska rezistencija i poremećena sekrecija insulina, mada još uvijek ima oprečnih stavova šta je primarni poremećaj (1, 2). Širom svijeta suočeni smo sa povećanom prevalencijom obolijevanja od dijabetes melitusa tipa 2 zbog povećanja gojaznosti i smanjenog nivoa aktivnosti (1, 3). Morbiditet i mortalitet od komplikacija DM-a može se uveliko smanjiti pravovremenim i stalnim procedurama kontrole. Ovi metodi skrininga indikovani su za sve osobe sa DM-om, mada su brojne studije pokazale kako najveći broj osoba sa dijabetesom ovu bolest sveobuhvatno ne liječi (4-7). Skrining za dislipidemiju i hipertenziju treba da se izvodi svake godine (1). DM tipa 2 prisutan je u oko 90% svih slučajeva dijabetesa. Najučestalija dislipidemija u tipu 2 dijabetesa sastoji se od hipertrigliceridemije, niskog HDL-kolesterola, tzv. dobri kolesterol i normalnog LDL-kolesterola, tzv. loši kolesterol (8). Klinički vodiči prakse (KVP) sistematski su razvijane preporuke koje pomažu liječniku i pacijentu u donošenju odluke u specifičnim kliničkim okolnostima odgovarajuće zdravstvene njege (9). Koristi od vodiča u kliničkoj praksi su višestruke. Za naše istraživanje bilo je značajno iskoristiti upute zasnovane na dokazima i omogućavati postizanje ocjene i osiguranje kvalitetne zaštite pregledom kliničkog kvaliteta (auditom) (10). Naprimjer, Američka asocijacija za dijabetes (American Diabetic Association, ADA) objavi četiri vodiča godišnje za nekoliko promjenjivih faktora rizika za kardiovaskularne bolesti, uključujući kontrolu glikemije, krvnog tlaka i koncentracije lipida u krvi (4, 11). Prema preporukama Kanadske dijabetološke asocijacije (Canadian Diabetic Association, CDA) jedna od komponenti prvog i tzv. follow up posjeta dijabetičara liječniku jeste metabolička kontrola i evaluacija faktora rizika: glukoza natašte, HbA1c, profil lipida natašte, mikroalbuminurija, serum kreatinin, izračunavanje kreatinin klirensa, elektrokardiogram, pregled stopala (monofilamentom ili vibracijom velikog prsta stopala), indeks tjelesne mase (BMI), krvni tlak, thyroid-stimulirajući hormon (kod svih pacijenata sa tipom 1 dijabetesa; kod pacijenata sa tipom 2 samo ako je klinički indicirano) (8). Dijabetes je sedmi vodeći razlog posjeti liječniku primarne zdravstvene zaštite, PZZ (12, 13). Dugotrajna socijalna i ekonomska korist, koja se postiže dobrom kontrolom nivoa glukoze i unapređenjem kvalitete njege, trebala bi stimulirati liječnike PZZ-a da svakodnevno rade po kliničkim vodičima prakse (14). Studije u Europi i svijetu, koje su uključivale liječničke prikaze, revizije prakse i pregled administrativnih podataka, pokazale su da je kvalitet kontrole DM-a od strane liječnika PZZ-a suboptimalan. Zapravo iste studije ukazuju na korisne intervencije na nivou PZZ-a koje mogu dovesti do signifikantnog poboljšanje kvalitete njege za dijabetes (4, 6, 12, 15, 16-18). Većina razvijenih zemalja ima program za nadzor kroničnih bolesti kao što je DM (19). Bosna i Hercegovina i dalje nema takvog nacionalnog programa. Donešene su određene zakonske odredbe kojima se afirmira izrada takvih programa i objavljeni akreditacijski standardi za timove porodične/obiteljske medicine (Agencije za kvalitet i akreditaciju u zdravstvu Federacije Bosne i Hercegovine, AKAZ FBiH) koji očekuju od tima da tretira pacijente s hroničnim oboljenjima u skladu sa savremenim saznanjima i vodiljama za kliničku praksu (20). Svi stanovnici koji mjestom stanovanja gravitiraju najbližoj ambulanti porodične/obiteljske medicine u općini Zenica, u kojima je rađena studija, registrirani su kod određenog liječnika koji je odgovoran u prosjeku za oko pacijenta. Upravni odbor Zavoda zdravstvenog osiguranja Zeničko-dobojskog kantona, u maju godine, donio je Odluku za usvajanje osnovnih uslova potrebnih za priznavanje porodične/ obiteljske medicine na području Zeničkodobojskog kantona (OUPPM-ZDK), a jedan od uslova je i dokument pod nazivom Uspostavljanje sistema aktivnog nadzora nad dijabetesom i hipertenzijom (broj: /05). 204

65 Gavran et al Klinička revizija lipidnog statusa Posebni karton za dijabetes (PKD) sadrži 13 parametara (8 brojčanih i 5 opisnih) za praćenje, od kojih se za DM tip 2 svaka tri mjeseca prati vrijednost šećera u krvi natašte, ishrana, aktivnost, tjelesna težina i krvni tlak; svakih šest mjeseci kolesterol, trigliceridi, proteini u urinu, te pregled stopala; a kreatinin, EKG, te pregled fundusa oka jedanput godišnje. PKD su bili distribuirani svim ordinacijama porodične/obiteljske medicine u ZDK, u periodu januar-februar godine. Cilj kliničke revizije (audita) lipidnog statusa kod tipa 2 dijabetesa bio je istražiti da li su timovi porodične/obiteljske medicine (TOM) u Zenici, nakon uvođenja posebnog kartona za dijabetes (PKD), mogli dovesti do poboljšanja kontrole nivoa lipida za svoje pacijente sa DM-om tipa 2 prema preporučenim smjernicama. PACIJENTI I METODE Provedena je audit-studija u Domu zdravlja u Zenici, u deset ambulanti porodične/obiteljske medicine. U istraživanju su analizirani podaci iz perioda maj maj godine. Ovim istraživanjem kompariran je nivo evidentiranih parametara prije i poslije implementacije posebnog kartona za dijabetes (PKD). Podaci iz PKD-a pregledani su u periodu dvije godine prije ( ) i dvije godine poslije ( ) implementacije posebnog kartona za dijabetes. Studijom su obuhvaćeni pacijenti oboljeli od dijabetes melitusa tipa 2, oba spola, stariji od 18 godina. Analizirani su podaci za ukupno 843 pacijenta. Devetnaest liječnika porodične/obiteljske medicine u Zenici, odnosno 19 timova porodične/ obiteljske medicine (TOM), učestvovalo je u studiji i omogućilo uvid u medicinske kartone oboljelih pacijenata od dijabetes melitusa. Jedan liječnik specijalista porodične/obiteljske medicine odbio je sudjelovati u istraživanju. Za svakog od ovih liječnika uzorak pacijenata sa DM-om bio je izabran na osnovu slijedećih kriterija: dob pacijenta od 18 godina i više (u toku izvođenja istraživanja); dijagnosticiran DM prema međunarodnoj kvalifikaciji bolesti - 9 revizija (E11, E10 ili DM tip 2); redovitost dolazaka pacijenta u periodu istraživanja; najmanje jedan dolazak pacijenta prije i jedan dolazak poslije implementacije PKD-a; pacijenti koje je obiteljski liječnik češće sam pregledao i uputio na pretrage bez prethodne konsultacije liječnika specijaliste. Slučajnim odabirom pregledani su kartoni pacijenata praćenih od strane jednog liječnika, a koji su zadovoljili kriterije. Za svakog pacijenta iz kartona su, osim rednog broja, dobi i spola, uzeti podaci za slijedeće parametre, prije i poslije upotrebe PKD-a: zadnji nalaz lipoproteina niskog denziteta (LDL-kolesterol), ukupni kolesterol (UK-kolesterol) i trigliceridi (TG). Podaci iz kartona dokumentirani su na posebnim obrascima revizije DM prakse, koji su kreirani prema PKD-u. Nivoi vrijednosnih parametara preuzeti su iz europskog vodiča u prevenciji kardiovaskularnih oboljenja, kojeg je objavio Komitet Europskog udruženja kardiologa za kliničke vodiče, (21). Vrijednosti evidentiranih parametara označene su na slijedeći način: LDL-kolesterol > 2.5 mmol/l, kao nedovoljan nivo; LDL-kolesterol < 2.5 mmol/l, kao optimalan nivo; UK-kolesterol > 4.5 mmol/l, kao nedovoljan nivo; UK-kolesterol < 4.5 mmol/l, kao optimalni nivo; TG > 1.7 mmol/l, kao nedovoljan nivo; TG < 1.7 mmol/l, kao optimalni nivo. S obzirom da je etički komitet ustanove bio tek u osnivanju u času početka ove studije, o sprovedbi revizije prakse na kartonima pacijenata sa dijabetesom tipa 2, saglasnost smo dobili od direktora i kolega koji su učestvovali u izvedbi studije. U statističkoj obradi podataka korištene su standardne metode deskriptivne i inferentne statistike. Kako su podaci sa kojima se radilo vezani isključivo za učestalost, odgovarajuće statističke hipoteze testirane su χ2 testom ili testom proporcija (z-test proporcija) koji je analogan Studentovom t-testu za brojčane (kvantitativne) podatke. Statističke hipoteze testirane su sa nivoom signifikantnosti p=0.05, tj. nulta hipoteza jednakosti dviju proporcija odbacivana je u korist alternativne kod vrijednosti p <

66 Medicinski Glasnik, Volumen 6, Number 2, August 2009 REZULTATI Ukupno su analizirana 853 kartona bolesnika sa šećernom bolesti. U uzorku pacijenata dominirao je ženski spol, 538 (63.1%), u odnosu na muški, 315 (36.9%) (P < ). Najviše bolesnika bilo je u dobnoj skupini od preko 60 godina, 603 (70.7%) u odnosu na mlađe bolesnike, 250 (29.3%). U 19 timova porodične/obiteljske medicine ukupno je registrirano pacijenata, od toga sa DM-om. U prosjeku svaki od timova imao je registrovanih pacijenata, 83 DM po timu, a revizija prakse po timu odrađena je u prosjeku na 45 kartona pacijenata oboljelih od DM-a tipa 2. S obzirom na stručnost timova najviše su bili zastupljeni specijalisti porodične/obiteljske medicine (spec. P/OM, 13, 74%), dok je 5 (21%) liječnika bilo dodatno educirano iz oblasti porodične/obiteljske medicine (engl. programm additional training, PAT), a 1 (5%) liječnik bio je na specijalizaciji iz porodične/obiteljske medicine. Prosječna starosna dob timova iznosila je 46.6 godina, 4 (21%) uzorka timova bili su muškog, a 15 (79%) ženskog spola. Procent nivoa lipida za nedovoljan i optimalan nivo, ukupno za sve timove, statistički značajno je promijenjen poslije upotrebe obrasca (P < ) (Tabela 1). U Tabeli 2 prikazane su vrijednosti optimalnog nivoa lipoproteina niskog denziteta po Tabela 1. Vrijednosti lipida, prije i poslije upotrebe posebnog kartona za dijabetes, po nivoima evidentiranosti za sve timove ukupno* LDL Prije upotrebe posebnog kartona N 283 O 53 Vrijednost lipida (mmol/l) Poslije upotrebe posebnog kartona N 234 O 119 Z 5.4 N p < Z 5.4 O p < UK < < TG < < Ukupno *LDL, lipoprotein niskog denziteta ( loši kolesterol ); UK, ukupni kolesterol; TG, trigliceridi; N, nedovoljan nivo (LDL > 2.5 mmol/l, UK > 4.5 mmol/l i TG > 1.7 mmol/l); O, optimalan nivo (LDL < 2.5 mmol/l, UK < 4.5 mmol/l i TG < 1.7 mmol/l ); p, nivo statističkog značaja ; Z, test proporcija; timovima (LDL < 2.5 mmol/l). Ustanovljene su statistički značajne razlike proporcija optimalnog nivoa LDL-a poslije primjene posebnog kartona za dijabetes melitus tipa 2 za timove 17 i 19 (p < 0.01) u odnosu na zabilježene vrijednosti optimalnog nivoa LDL-a u periodu prije uvođenja posebnog kartona za dijabetes melitus tipa 2. U Tabeli 3 prikazane su vrijednosti optimalnog nivoa ukupnog kolesterola po timovima (UK < 4.5 mmol/l). Nađena je statistički značajna razlika proporcija optimalnog nivoa UK poslije primjene posebnog kartona za dijabetes melitus tipa 2 za timove 3 (p < 0.01), 13 i 14 (p < ) u odnosu na zabilježene vrijednosti optimalnog nivoa UK-a u periodu prije uvođenja posebnog kartona za dijabetes melitus tipa 2. Tabela 4 pokazuje vrijednosti optimalnog nivoa triglicerida po timovima (TG < 1.7 mmol/l). Nađene su statistički značajne razlike proporcija optimalnog nivoa TG-a poslije primjene posebnog Tabela 2. Optimalni nivoi vrijednosti lipoproteina niskog denziteta (LDL), prije i poslije upotrebe posebnog kartona za dijabetes, po timovima porodične/obiteljske medicine* Prije upotrebe posebnog kartona Poslije upotrebe posebnog kartona Tim N1 N2 (%) N2 (%) (2.4) 5 (12.2) (0.0) 0 (0) (8.0) 11 (22.0) (2.4) 1 (2.4) (7.5) 1 (2.5) (2.2) 0 (0) (6.5) 10 (21.7) (2.4) 5 (12.5) (9.1) 4 (9.1) (4.8) 11 (26.8) (10.0) 6 (15.0) (4.2) 9 (18.8) (6.0) 0 (0.0) (13.0) 9 (19.6) (8.2) 4 (8.2) (4.3) 0 (0.0) (2.1) 11 (23.4) 3.1 < (11.6) 14 (32.6) (2.2) 18 (39.1) 3.1 <0.01 * Z, test proporcija; p, nivo statističkog značaja; N1, broj obrađenih kartona po timu porodične/obiteljske medicine (P/OM); N2, broj kartona u kojima je nađena optimalna vrijednost lipoproteina niskog denziteta ( loši kolesterol - LDL < 2,5 mmol/l); program dodatne edukacije iz P/OM i/ili druga specijalizacija (opće medicine ili medicine rada); specijalista P/OM; specijalizant P/ OM; Z p 206

67 Gavran et al Klinička revizija lipidnog statusa kartona za dijabetes melitus tipa 2 za timove 1, 2, 7, 12 i 13 (p < ), timove 9, 14, 18 i 19 (p < 0.001), a za tim 6 (p < 0.01) u odnosu na zabilježene vrijednosti optimalnog nivoa TG-a u periodu prije uvođenja posebnog kartona za dijabetes tipa 2. DISKUSIJA U istraživanju je sudjelovalo 19 timova porodične/obiteljske medicine u općini Zenica, tj. 19 liječnika (kao vođa timova) koji su dobrovoljno pristali na reviziju prakse kartona njihovih pacijenata s DM-om tipa 2. U pomenutih 19 timova ukupno je bilo registrirano pacijenta, a od toga pacijenata sa dijabetes melitusom. Ova zastupljenost vjerojatno je i veća jer u Domu zdravlja Zenica ne postoji registar sa kontinuiranim uvođenjem oboljelih od dijabetesa koji bi se mogao unaprijediti sa budućom kompjuterizacijom ambulanti porodične/obiteljske medicine. U prosjeku, svi timovi zajedno imali su pacijenata. Za razliku od naših, holandski liječnici opće prakse po jednom liječniku imaju registriranih pacijenata (22). Tako su timovi 2, 5, 12, 14 i 16 imali najveći broj registriranih pacijenata (oko 3.000), dok su timovi 8 i 9 imali najmanji broj (oko 1.580). Prosječan broj registriranih dijabetičara po timovima kretao se od 50 (kod tima 11) do 135 (kod tima 13), što govori o različitoj svakodnevnoj angažiranosti timova u menadžmentu dijabetičara. Tokom našeg istraživanja željeli smo utvrditi da li poboljšana evidentiranost ključnih, vremenom zadatih parametara, može rezultirati poboljšanom kontrolom bolesti, tj. da li može dovesti do povećanja nivoa nekih vrijednosnih parametara. Primijetili smo da su vrijednosti optimalnog nivoa za parametre LDL, UK i TG statistički značajno povećane nakon uvođenja u odnosu prije uvođenja PKD-a za sve timove zajedno. Tabela 3. Optimalni nivoi vrijednosti ukupnog kolesterola, prije i poslije upotrebe posebnog kartona za dijabetes, po timovima porodične/obiteljske medicine* Prije upotrebe posebnog kartona Poslije upotrebe posebnog kartona Tim N1 N2 (%) N2 (%) (2.4) (6.1) (6.0) < (9.8) (12.5) (2.2) (6.5) (2.5) (9.1) (7.3) (10.0) (4.2) (2.0) < (15.2) < (40.8) ) * Z, test proporcija; p, nivo statističkog značaja; N1, broj obrađenih kartona po timu porodične/obiteljske medicine (P/OM); N2, broj kartona u kojima je nađena optimalna vrijednost ukupnog kolesterola (UK < 4,5 mmol/l); program dodatne edukacije iz porodične/obiteljske medicine (P/ OM) i/ili druga specijalizacija (opće medicine ili medicine rada); specijalista P/OM; specijalizant P/OM; Z p Tabela 4. Optimalni nivo vrijednosti triglicerida, prije i poslije upotrebe posebnog kartona za dijabetes, po timovima porodične/obiteljske medicine* Prije upotrebe posebnog kartona Poslije upotrebe posebnog kartona Tim N1 N2 (%) N2 (%) (26.8) 24 (58.5) 2.9 < (16.3) 35 (71.4) 5.5 < (40.0) 24 (48.0) (24.4) 21 (51.2) (22.5) 20 (50.0) (26.1) 27 (58.7) 2.9 < (26.1) 31 (67.4) 4.0 < (10.0) 14 (35.0) (29.5) 27 (61.4) 3.0 < (43.9) 27 (65.9) (27.5) 18 (45.0) (16.7) 30 (62.5) 4.9 < (22.0) 31 (62.0) 4.1 < (32.6) 32 (69.6) 3.5 < (24.5) 19 (38.8) (32.6) 14 (30.4) (25.5) 13 (27.7) (25.6) 27 (62.8) 3.5 < (30.4) 29 (63.0) 3.1 <0.001 * Z, test proporcija; p, nivo statističkog značaja; N1, broj obrađenih kartona po timu porodične/obiteljske medicine (P/OM); N2, broj kartona u kojima je nađena optimalna vrijednost triglicerida (TG < 1,7 mmol/l); program dodatne edukacije iz porodične/obiteljske medicine (P/ OM) i/ili druga specijalizacija (opće medicine ili medicine rada); specijalista P/OM; specijalizant P/OM; Z p 207

68 Medicinski Glasnik, Volumen 6, Number 2, August 2009 Naprotiv, zajedničkom timskom unapređenju optimalnog nivoa LDL-a, optimalan nivo vrijednosti LDL-a, po timovima pojedinačno, našli smo da je statistički značajnije zabilježen poslije u odnosu na prije uvođenja PKD-a jedino kod dva tima kojeg vode specijalisti P/OM. Tri tima uspjelo je statistički značajno dostići optimalan nivo vrijednosti ukupnog kolesterola poslije u odnosu na prije uvođenja PKD-a. Najbolje statistički značajno unapređenje optimalnog nivoa dostignuto je za vrijednosti triglicerida i zabilježeno je kod najvećeg broja timova (10/19) od kojih je 7/10 specijalista P/ OM, 5/10 doeduciranih liječnika iz P/OM i jedan specijalizant P/OM. Slične rezultate pokazali su i Kirk i sur. revizijom kartona 86 slučajno odabranih pacijenata sa DM-om u ambulantama obiteljske medicine u SAD-u ustanovljeno je da unatoč statistički značajnom poboljšanju vrijednosti promjenjivih parametara faktora rizika, proporcija pacijenata koji su imali po vodičima preporučene vrijednosti HbA1c, krvnog tlaka i LDL kolesterola, nije bila značajno promijenjena (4). Gill i Di Prinzio u istraživanju, sprovedenom godine, o utjecaju upotrebe unificiranih vodiča (UKV) za dijabetes na kvalitet njege (258 kartona dijabetičara u 28 ambulanti obiteljske medicine) ustanovili su da nije bilo statistički značajne promjene kod većine kvalitativnih indikatora godinu prije i godinu poslije implementacije UKV. U drugoj analizi istog istraživanja liječnici koji su koristili obrasce za praćenje imali su bolju kvalitetu njege za većinu mjerenja premda nisu bile za sva mjerenja ujednačeno bolja (23). Naša studija je pokazala kako je upotreba PKD-a pomogla da se unaprijedi praćenje za većinu zadatih parametara, zbirno za sve timove, kao i da je za veliki broj timova pojedinačno (10/19), a koje su većinom činili specijalisti POM, statistički značajno bio dostignut optimalni nivo parametra triglicerida nakon uvođenja upotrebe PKD-a u odnosu na period prije uvođenja. Za ostale vrijednosne parametre, LDL i UK, samo su neki timovi dosegli optimalne nivoe i to kod LDL-a 2/19, UK-a 3/19, koji također nisu bili ujednačeni i samim tim ne možemo biti zadovoljni. U istraživanju primjene preporučenih smjernica za unapređenje kontrole DM pacijenata sprovedenom u SAD-u, ustanovljeno je da su razlozi za nepridržavanje smjernica bile razlike u znanju liječnika, nepovjerenje u preporučeni vodič ili problem vezan za nepridržavanje pacijenata datim preporukama od liječnika (11). Istraživanjem utjecaja multikomponentnih intervencija koje mogu dovesti do unapređenja kvaliteta njege za dijabetičare mjerenjem 13 parametara, ustanovljeno je kako uključivanje pojedinih ambulanti primarne zdravstvene zaštite u povremene obilaske i godišnje sastanke, može unaprijediti praksu tako da pacijenti dobivaju praćenje i tretman, kao i dostizanje ključnih ciljeva za HbA1c, lipidni status i krvni tlak (17). Istraživanjem nivoa metaboličke kontrole kod tipa 2 dijabetičara, vođenih od strane 26 liječnika na programu dodatne edukacije iz porodične/obiteljske medicine u Tuzli, tokom godine, ustanovljena je loša metabolička kontrola ukupnog kolesterola kod pacijenata tipa 2 dijabetesa (24). Pokazatelji prezentirani u ovoj studiji sugeriraju da je primjena posebnog kartona za dijabetes na nivou porodične/obiteljske medicine u Zenici dovela do unapređenja optimalnog nivoa lipida kod većine pacijenata timova porodične/ obiteljske medicine i na taj način njihovi su pacijenti sa dijabetesom tipa 2 dostigli kliničke ciljeve za mjerenje lipida preporučene u europskim vodičima (21). Ovim istraživanjem ustanovljeno je kako je postojeći posebni karton za dijabetes (PKD) mogao stimulirati liječnike porodične/obiteljske medicine da poboljšaju svoje vještine i znanje, te implementiraju klinički vodič prakse u svom svakodnevnom radu. S druge strane, potrebno je napraviti novo istraživanje koje bi moglo ispitati razloge uslijed kojih liječnici porodične/obiteljske medicine u općini Zenica nisu dostigli optimalni nivo vrijednosti parametara (ukupni kolesterol, LDL) po trenutno preporučenim smjernicama. ZAHVALE / IZJAVE Komercijalni ili potencijalni dvostruki interes ne postoji. 208

69 Gavran et al Klinička revizija lipidnog statusa LITERATURA 1. Powers AC. Diabetes mellitus. U: Harrison TR, Braunwald E, Fauci AS, Kasper DL, Hauser SL, Longo DL i sur. Načela interne medicine. Knjiga 2. (15.izd.). Beograd: Bard-fin d.o.o., 2004: Metelko Ž, Granić M, Škrabalo Z. Šećerna bolest. U: Vrhovac B, ur. Interna medicina (drugo promijenjeno i dopunjeno izdanje). Zagreb: Medicinska Naklada, 1997: Bryant W, Greenfield JR, Chisholm DJ, Campbell LV. Diabetes guidelines: easier to preach than to practice? MJA 2006; 185: Kirk JK, Huber KR, Clinch CR. Attainment of goals from national guidelines among persons with type 2 diabetes: a cohort study in an academic family medicine setting. NC Med J 2005; 66: Lawler F, Viviani N. Patient and Physician Perspectives Regarding Treatment of Diabetes: Compliance with Practice Guidelines. J Fam Pract 1997; 44: Kirkman MS, Williams SR, Caffrey HH, David GM. Impact of a program to Improve adherence to diabetes guidelines by primary care physicians. Diabetes Care 2002; 25: Ornstein S, Nietert PJ, Jenkins GR, Wessell AM, Nemeth LS, Feifer C, Corley ST. Improving diabetes care through a multicomponent quality improvement model in a practice-based research network. Am J Med Qual 2007; 1: Anonymous. Canadian Diabetes Association Clinical Practice Guidelines Expert Committee. Canadian Diabetes Association. Clinical practice guidelines for the prevention and management of diabetes in Canada. Can J Diabetes 2003; 27 (Suppl 2): S Ratsep A, Kalda R, Oja I, Lember M Family doctors knowledge and self-reported care of type 2 diabetes patients in comparison to the clinical practice guideline: cross-sectional study. BMC Fam Pract 2006; 16: Mašić I. Porodična /obiteljska medicina - principi i praksa. Medicinski vodiči u praksi. Sarajevo: Avicena 2007; Anonymous. American Diabetes Association: Standard of medical care in diabetes. Diabetes Care 2006; 29 (suppl 1):S4-S Worrall G, Freake D, Kelland J, Pickle A, Keenan T. Care of patients with type II diabetes: a study of family physicians compliance with clinical practice guidelines. J Fam Pract 1997;44: Bodenheimer T, Wagner EH, Grumbach K. Improving primary care for patients with chronic illness. JAMA 2002; 288: Harris SB, Meltzer SJ, Zinman B. New guidelines for the of diabetes: a physician s guide. Canadian Medical Association 1998; 159: Harris SB, Stewart M, Brown JB, Wetmore S, Faulds C, Webster-Bogaert S, Porter S. Type 2 diabetes in family practice. Room for improvement. Can Fam Physician 2003; 49: Ziemer DC, Miller CD, Rhee MK. Clinical inertia con- tributes to poor diabetes control in a primary care setting. Diabetes Educ 2005; 31: Anonymous. Agency for Healthcare Research and Quality. National Healthcare Quality Rothman AA, Wagner EH. Chronic illness management: what is role of primary care? Ann Intern Med 2003; 138: Anonymous. Službene novine Federacije BiH. Zakon o sustavu poboljšanja kvalitete, sigurnosti i o akreditaciji u zdravstvu 2005; 59: Šabanović F, Selimbašić I, Pavlović J, Leovac Lj, Ćepo M, Mercvajer M, Čavkunović M, Trninić S, Falak V, Hodžić V, Jatić Z. U: Akreditacijski standardi za timove porodične/obiteljske medicine- verzija 3.3 (ur.). Riđanović Z, Nakaš B, Cerić K. Sarajevo: AVICENA 2005:27-9. Anonymous. Europski vodič za prevenciju kardiovaskularnih bolesti u kliničkoj praksi. Eur Heart J 2003; 24: Valk GD, Renders CM, Kriegsman DMW, Newton KM, Twisk KMN, Eijk van JThM, Wal van der G, Wagner EH. Quality of care for patients with Type 2. Diabetes Mellitus Netherlands and the United States: a comparison of improvement programs. Health Serv Res 2004; 39 (4 Pt 1): Gill JM, DiPrinzio MJ. The Medical Society of Delaware s Uniform Clinical Guidelines for diabetes: did they have a positive impact on quality of diabetes care? Del Med J 2004; 76: Herenda S, Tulumovic A, Omanović S, Jakupović B. Metabolic control among type 2 diabetic patients in the northeast part of Bosnia and Hercegovina. In: Abstract Book of the 14 th Wonca Europe Conference of ESGP/FM, Wonca Europe 2008, Istanbul, Turkey, September, 2008:

70 Medicinski Glasnik, Volumen 6, Number 2, August 2009 Clinical review of lipids control level for Diabetes Mellitus type 2 patients done by Family Medicine Teams in Zenica, Bosnia and Hercegovina Larisa Gavran 1, Selmira Brkić 2 1 Family Medicine Teaching Centre Travnička, Health Centre Zenica, 2 Medical Faculty University of Tuzla; Bosnia and Herzegovina ABSTRACT Aim To assess the effect of the implementation of the guidelines for Diabetes Mellitus (DM) in the Family Medicine Teams (FMT) in Zenica municipality using a special flowchart designed for diabetes (SFD) to control lipids level improvement in DM type 2 patients. Methods The review was conducted among 853 DM Type 2 patients older than 18. In 19 FMTs in Zenica we checked patients charts made two years before ( ) and two years after ( ) the implementation of guidelines for DM. We divided all the stablsihed values of lipids and all the FMT on the basis of the acievement of an optimal level (cholesterol < 4.5 mmol/l; LDL- cholesterol < 2.5; triglyceride < 1.7 mmol/l) and failure to achieve the optimal level (cholesterol > 4.5 mmol/l; LDLcholesterol > 2.5 mmol/l; TG > 1.7 mmol/l). Results A total number of 853 DM Type 2 patients records were analysed, 46 per one FMT. Four out of 19 FMT leaders (21%) were men and 15 (79%) were women. The average age was years. A statistically significant improvement for optimal level of LDL - cholesterol (19 vs. 531; p<0.0001), cholesterol (67 vs. 212; p <0.0001) and triglyceride (227 vs. 463; p <0.0001) before and after implementation of SFD, respectively, has been found. The statistically significant improvement (per teams) for optimal triglyceride level (p<0.0001) for 10/19 FMT has been found too. Conclusion After the implementation of lipids guidelines for DM, most of the FMT in Zenica town improved the optimal level of lipids. Key words: lipids, Diabetes Mellitus, guidelines, Family Medicine Teams Original submission: 22 September 2008; Revised submission: 14 December 2008; Accepted: 31 March Med Glas 2009; 6(2):

71 ORIGINAL ARTICLE Učestalost pušenja i nikotinska ovisnost kod medicinskih radnika Željko Martinović 1, Cvita Martinović 2, Mladen Čuturić 1 1 Kirurški odjel; 2 Interni odjel, Hrvatska bolnica Dr. fra Mato Nikolić, Nova Bila, Bosna i Hercegovina SAŽETAK Cilj Cilj istraživanja bio je utvrditi učestalost pušenja i stupanj nikotinske ovisnosti kod aktivnih pušača među medicinskim radnicima. Metode Istraživanje je provedeno na uzorku od 66 medicinskih radnika Hrvatske bolnice Dr. fra Mato Nikolić u Novoj Bili, metodom ankete i Fagerstromovog modificiranog upitnika za procjenu nikotinske ovisnosti. Rezultati Od ukupno 66 ispitanika, evidentirali smo 37 (56,1%) pušača, 19 (51,4%) žena i 18 (48,6%) muškaraca. Prosječna vrijednost skora nikotinske ovisnosti iznosila je 8 bodova što inače predstavlja visok stupanj nikotinske ovisnosti. Corresponding addres: Željko Martinović, Hrvatska bolnica Dr. fra Mato Nikolić, Dubrave bb, Nova Bila, Bosna i Hercegovina Phone: ; zeljko.martinovic3@gmail.com Zaključak Pušenje, kao preventabilni zdravstveni faktor rizika, predstavlja veliki problem i samim medicinskim radnicima. Oni, kao aktivni pušači, imaju visok stupanj nikotinske ovisnosti koja zahtijeva stručni tretman. Ključne riječi: pušenje, medicinski radnici, stupanj nikotinske ovisnosti, Fagerstromov upitnik Originalna prijava: 19. novembar 2008.; Korigirana verzija: 30. mart 2009.; Prihvaćeno: 13. april Med Glas 2009; 6(2):

72 Medicinski Glasnik, Volumen 6, Number 2, August 2009 UVOD U prvim desetljećima dvadesetog stoljeća, pušenje je postalo društveno prihvatljiva navika. Danas, prema procjeni Svjetske zdravstvene organizacije, tu naviku ima 1,3 milijarde ljudi i približno 5 milijuna godišnje umire od njenih posljedica. Pušenje je tako postala najsmrtonosnija pandemija današnjice (1, 2). Prvi znanstveni dokazi o štetnim učincima pušenja pojavljuju se sredinom prošlog stoljeća. Engleski liječnici R. Doll i A. B. Hill godine dokazali su uzročnu povezanost pušenja i karcinoma bronha i pluća, infarkta miokarda i kronične opstruktivne bolesti pluća (3, 4). Američka zdravstvena služba (Surgeon General s Report on Smoking and Health) objavila je godine izvješće o pušenju kao faktoru rizika koji znatno pridonosi morbiditetu i mortalitetu niza bolesti, a godine nikotinska ovisnost unesena je u Međunarodnu klasifikaciju bolesti, ozljeda i uzroka smrti (5). Iako predstavlja preventabilan čimbenik rizika, prema Svjetskoj zdravstvenoj organizaciji, pušenje duhana je drugi vodeći uzrok smrtnosti i četvrti zajednički zdravstveni čimbenik rizika u svijetu. Ako se nastave sadašnji trendovi pušenja, do godine broj umrlih od bolesti vezanih za pušenje duhana mogao bi doseći brojku od 650 milijuna (6). Danas se smatra kako je moguće reducirati značajan broj ovih smrtnih ishoda mjerama sprječavanja, suzbijanja i prestanka pušenja (7). Važnu ulogu u prevenciji i strategiji edukacije o prestanku pušenja imaju zdravstveni radnici, koji bi inače trebali biti primjer zdravog načina života. Međutim, značajan broj zdravstvenih radnika nisu uvijek dobar primjer svojim pacijentima. Štoviše, mnogi od njih ne smatraju prekid pušenja kao visoko prioritetan cilj za svoje pacijente, te savjet o potrebi prekida pušenja ne vide kao sastavni dio medicinske skrbi. Prema objavljenim podacima u različitim studijama, učestalost navike pušenja među zdravstvenim radnicima je visoka, posebice u državama u razvoju, u kojima antipušačke kampanje još nisu pokrenute u značajnijoj mjeri, pa tako ni u Bosni i Hercegovini (8, 9). Cilj ovog istraživanja bio je utvrditi učestalost pušenja i stupanj nikotinske ovisnosti kod aktivnih pušača medicinskih radnika primjenom modificiranog Fagerstromovog upitnika. Ako pušite, molimo Vas, da odgovorite na slijedeća Odgovor pitanja na način da križićem označite Vaš odgovor Koliko cigareta dnevno pušite? i više slabe (do 0,9 mg nikotina) 2. Kakve cigarete pušite? srednje (1,0-1,2 mg nikotina) jake (1,3 mg i više nikotina) nikad 3. Da li uvlačite dim cigarete? ponekad uvijek 4. Kada nakon buđenja zapalite svoju u prvih 5 minuta prvu cigaretu? unutar 6-30 minuta unutar minuta 5. Kad više pušite? u toku prijepodneva u toku ostalog dijela dana 6. Koje cigarete biste se najteže odrekli? prve jutarnje bilo koje druge 7. Da li Vam je teško suzdržati se od ne pušenja na mjestima gdje je to zabranjeno? da 8. Da li pušite i kada ste bolesni? ne da *Nikotinski skor ovisnosti: 0-2 vrlo niska; 3-4 niska; 5-6 umjerena; 7 i više bodova - visoka i vrlo visoka Bodovi* Slika 1. Modificirani Fagerstromov upitnik za procjenu nikotinske ovisnosti 212

73 Martinović et al Učestalost pušenja i nikotinska ovisnost ISPITANICI I METODE Istraživanje je provedeno na slučajnom uzorku od 66 ispitanika medicinskih radnika (liječnici, medicinske sestre i tehničari) Hrvatske bolnice Dr. fra Mato Nikolić u Novoj Bili, metodom ankete. Anketni listić sadržavao je podatke o pušenju, pušačkom stažu, godinama starosti i spolu, te Fagerstromov modificirani upitnik za procjenu nikotinske ovisnosti (10). Modificirani Fagerstromov upitnik (Slika 1) sastojao se od osam pitanja. Odgovori na pitanja bodovali su se od 0-3 boda, a zbrajanjem je dobiven skor nikotinske ovisnosti (raspon od 2 do 15 bodova). Skor od 2-6 bodova označavao je nisku do umjereno tešku nikotinsku ovisnost, a skor od 7 i više bodova označavao je teški stupanj nikotinske ovisnosti po Fagerstromu. REZULTATI Od ukupno 66 ispitanika, 36 (54,5%) je bilo ženskog, a 30 (45,5%) muškog spola. Prosječna starosna dob ispitanika iznosila je 30,34 ± 15,24 godina. Ispitanika pušača bilo je 37 (56,1%), a nepušača 29 (43,9%) (Tablica 1). Nije nađena statistički značajna razlika u spolnoj distribuciji ispitanika pušača i nepušača (p > 0,05, odnosno p > 0,01). Prosječna starosna dob ispitanika pušača iznosila je 32,05 ± 14,14 godina, a nepušača 28,17 ± 16,23 godina. Nije nađena statistički značajna razlika u starosnoj dobi ove dvije grupe (p > 0,05). Prosječan pušački staž iznosio je 13,16 ± 9,82 godine (raspon 1-34). U Tablici 2 prikazana je raspodjela ispitanika prema broju dnevno ispušenih cigareta (uglavnom niskog sadržaja nikotina). Većina ispitanika pušača više je pušila u popodnevnim satima (24, 64,9%). Prosječna vrijednost skora nikotinske ovisnosti iznosila je 8,08 ± 2,00 (raspon 3-12). Nije nađena Muškarci (%) Žene (%) Ukupno (%) Pušači 18 (27,3%) 19 (28,8%) 37 (56,1%) Nepušači 12 (18,2%) 17 (25,7%) 29 (43,9%) Ukupno 30 (45,5%) 36 (54,5%) 66 (100%) statistički značajna razlika u prosječnoj vrijednosti nikotinskog skora između ispitanika pušača muškog i ženskog spola (p > 0,05). Visok stupanj nikotinske ovisnosti nađen je kod 32 (48,5%) ispitanika, dok je pet (7,6%) ispitanika imalo nisku do umjerenu vrijednost nikotinske ovisnosti. Analizom međusobne povezanosti visine skora nikotinske ovisnosti i dužine pušačkog staža, te broja dnevno popušenih cigareta, utvrđena je pozitivna linearna povezanost ovih varijabla. Vrijednost koeficijenta korelacije između dužine pušačkog staža i skora nikotinske ovisnosti pokazivala je neznatnu povezanost između ovih dviju varijabla (r = 0,117; 95% CI = -0,2152-0,4249; p = 0,4903), a u obje varijable ustanovljeni su zajednički činioci (1,36%) (R 2 = 0,0136). Nađena je statistički značajna korelacija između broja dnevno popušenih cigareta i visine skora nikotinske ovisnosti (r = 0,594; 95% CI = 0,3350-0,7701; p < 0,0001). U obje varijable ustanovljeno je 35,3% zajedničkih činilaca (R 2 = 0,3534). DISKUSIJA Učestalost pušenja duhana u općoj populaciji u Bosni i Hercegovini izrazito je visoka. Neposredno nakon završetka rata svakodnevno je pušilo oko 48% odraslih osoba, dok je, prema podacima Svjetske zdravstvene organizacije iz godine, ta brojka bila osjetno niža, te je, u populaciji odraslih osoba, u dobi između 25. i 64. godine, iznosila 37,6% (11, 12). Drugim riječima, u Bosni i Hercegovini proširenost navike pušenja među najvećim je u državama Balkana (32,4%) i EU (29,3%) (11). Slična epidemiološka situacija utvrđena je i u populaciji medicinskih radnika. Prema podacima iz dostupne literature, učestalost navike pušenja u ovoj populaciji u Bosni i Hercegovini iznosi oko 48% (9, 13). U godini, prema Tablica 2. Ispitanici pušači prema broju dnevno popušenih Tablica 1. Spolna distribucija i distribucija navike pušenja ispitanika cigareta Broj dnevno popušenih cigareta Ispitanici pušači (%) (13,5%) (59,5%) (24,3%) 31 i više 1 (2,7%) 213

74 Medicinski Glasnik, Volumen 6, Number 2, August 2009 podacima Svjetske zdravstvene organizacije, u Bosni i Hercegovini svakodnevno je pušilo 55% liječnika i 50% liječnica (14). Zastupljenost navike pušenja bila je najveća kod medicinskih sestara i tehničara (58%) i liječnika opće prakse (oko 48%), dok je istu naviku imalo oko 43% liječnika specijalista (9). U usporedbi sa drugim državama Europe, učestalost navike pušenja od 48%, među medicinskim radnicima u Bosni i Hercegovini, ekstremno je visoka. Prema podacima iz literature, slična ili veća učestalost navike pušenja kod medicinskih radnika, zabilježena je u Grčkoj (39%), Rumuniji (42,3%) i Turskoj (oko 52%), dok je u većini država EU učestalost navike pušenja ispod 25%, s tendencijom stalnog opadanja (15, 16, 17). Izrazito niska učestalost ove navike dokumentirana je kod medicinskih radnika u SAD-u (2%), Australiji (3%) i Engleskoj (3%) (18). Učestalost pušenja kod naših ispitanika bila je 56,1% i veća je nego u općoj populaciji, i to većinom kod zdravstvenih radnika ženskog spola (54,5%), što je osobito zabrinjavajuće. Znanstvena istraživanja dokazala su negativan utjecaj pušenja na reproduktivno zdravlje žena, odnosno povećavanje rizika od neplodnosti, ekstrauterine trudnoće, spontanog pobačaja, prijevremenog poroda i menstrualnih poremećaja (7, 19, 20). Udisanje duhanskog dima kod dojenčadi i male djece dovodi do učestalije pojave različitih bolesti dišnog sustava i oštećenja plućne funkcije, a i sindrom iznenadne smrti dojenčeta učestaliji je kod dojenčadi koja su bila izložena duhanskom dimu (20, 21). Više je mogućih uzroka visoke učestalosti pušenja kod naših ispitanika. Jedan od najvažnijih jeste najvjerojatnije nedavno okončani ratni sukob u BiH i veliko breme odgovornosti pred zdravstvenim radnicima za život i zdravlje njegovih sudionika. Ovakvo razmišljanje potkrijepljuju demografski podaci naših ispitanika i podaci iz literature (9, 22). Drugi, ne manje važan uzrok, jeste općekulturalno prihvaćanje i odobravanje pušenja duhana kao društveno prihvatljive navike, kako u općoj populaciji, tako i u populaciji zdravstvenih radnika. Štoviše, zdravstveni radnici, posebice liječnici, predstavljaju jednu od društveno utjecajnih grupa kojoj je pušenje prihvatljiva navika u različitim socijalnim okolnostima kao što su stresne situacije na poslu i profesionalnom usavršavanju (9, 22). Treći uzrok leži u činjenici da, nažalost, ne mali broj zdravstvenih radnika još uvijek pušenje ne prihvaća kao bolest ovisnosti, te da ona, kao takva, zahtijeva i adekvatno liječenje, a ne samo mjere suzbijanja i sprječavanja. Ovakvo razmišljanje podupire i srednja vrijednost Fagerstrom nikotinskog skora, koja se, kod naših ispitanika, nalazila u rasponu vrlo visoke nikotinske ovisnosti. Od ukupnog broja pušača u ovom istraživanju, 86,5% odgovorili su da uvijek inhaliraju dim iz cigarete, a 84% da dnevno popuše cigareta, uglavnom niskog (0,9 mg) sadržaja nikotina, što odgovara prosječnoj dnevnoj dozi od oko 18,5 mg nikotina (raspon mg). Potrebnu dozu nikotina pušači mogu regulirati brojem dnevno popušenih cigareta, te brojem i dubinom inhalacija dima iz cigarete (7). Štetni učinci duhanskog dima rastu s dozom izloženosti. Zbog tih različitosti i stupanj nikotinske ovisnosti uveliko varira (7, 20). Sa povećanjem broja dnevno popušenih cigareta učestalije se javljaju i simptomi nikotinske ovisnosti, te raste vrijednost skora nikotinske ovisnosti što upućuje na njihovu pozitivnu linearnu povezanost (7, 8, 20, 23). I dobivena vrijednost koeficijenta korelacije, u našem istraživanju, pokazala je da postoji stvarna, statistički značajna povezanost između visine skora nikotinske ovisnosti i broja dnevno popušenih cigareta (p < 0,001). U obje varijable ustanovljeno je približno 35,3% zajedničkih čimbenika, što upućuje na zaključak o postojanju puno većeg broja čimbenika koji utiču na stupanj nikotinske ovisnosti kod medicinskih radnika. Možemo samo pretpostaviti da veliki udio čine psihološki i društveni čimbenici (7, 22). Vrijednost koeficijenta međusobne povezanosti skora nikotinske ovisnosti i dužine pušačkog staža pokazala je neznatnu povezanost ovih dvaju varijabla, uz približan udio zajedničkih čimbenika od 1,36%. Mnogi čimbenici određuju razlike i učinke pušenja među pušačima pojedinačno (8, 24). Jedan od njih jeste i dužina pušačkog staža, čiji se učinci izražavaju 214

75 Martinović et al Učestalost pušenja i nikotinska ovisnost kroz rizike nastanka i pojave različitih oboljenja, kvalitetu i dužinu života pušača, što je u literaturi dobro dokumentirano (25, 26). Usprkos tome, učestalost navike pušenja među zdravstvenim radnicima u mnogim državama Europe i svijeta i dalje je visoka. Danas postoji široko prihvaćen konsenzus o implementaciji strategije o prevenciji i suzbijanju pušenja (7, 8). Uloga medicinskih radnika u implementaciji ove strategije jeste od esencijalne važnosti. Jasno je da visoka učestalost navike pušenja među zdravstvenim radnicima može povećati neodlučnost i skepticizam prema prestanku pušenja i liječenju nikotinske ovisnosti kod njihovih pacijenata. To potvrđuju i podaci iz literature koji pokazuju da liječnici imaju bolje rezultate u uvjeravanju svojih pacijenata na prestanak pušenja, ako i oni sami nisu pušači (18). Osim izravnog utjecaja na prestanak pušenja među pacijentima, medicinski radnici svojim učešćem u kreiranju socijalne politike usmjerene na sprječavanje i suzbijanje pušenja, mogu imati značajan utjecaj na smanjenje stope pušenja u općoj populaciji i smanjenju mogućnosti prinudne izloženosti duhanskom dimu ( pasivnom pušenju ) kroz zakonsko osiguravanje radnih i javnih prostora bez duhanskog dima. Pasivno pušenje još uvijek je široko rasprostranjen čimbenik rizika u EU. Meta analize provedene u EU i SAD-u potvrdile su povezanost pasivnog pušenja sa rakom pluća odraslih i opstruktivnim plućnim bolestima kod djece (27). S druge strane, pušenje unutar bolničkih prostora nije rijetkost i ima snažan negativan edukacijski učinak na pacijente. Prema podacima iz literature, oko 75% medicinskog osoblja, aktivnih pušača, puši izvan svog radnog prostora u bolnici, a samo oko trećine bolničkog medicinskog osoblja vjeruje da je politika bolnicâ bez pušenja uopće provodiva (28). Prema našim podacima, više od trećine naših ispitanika (35,1%) više su pušili u prijepodnevnim satima, odnosno u vrijeme radnog vremena. Oko 14% ispitanika pušilo je i unutar prostora bolnice. Ova brojka je nešto manja od očekivane i rezultat je ranije uvedenih restrikcijskih mjera. U Bosni i Hercegovini tek se očekuje intenzivnija antipušačka kampanja, posebice na području zdravstvene prosvijećenosti i edukacije, te suzbijanju i zabrani pušenja u javnim i radnim prostorijama. Medicinski radnici bi svakako trebali imati vodeću ulogu u potpori takvoj socijalnoj politici, posebice u zdravstvenim ustanovama. Naravno, zbog visoke učestalosti navike pušenja i visokog stupnja nikotinske ovisnosti, i zdravstvene radnike trebalo bi uključiti u programe odvikavanja, a zatim dodatno educirati i uključiti u timove za pružanje savjetodavne i farmakološke pomoći u procesu odvikavanja. Kao i druge kronične bolesti, i nikotinska ovisnost zahtijeva različite modalitete tretmana, te neophodnu dodatnu edukaciju zdravstvenih radnika. ZAHVALE / IZJAVE Komercijalni ili potencijalni dvostruki interes ne postoji. LITERATURA WHO. Tobacco or health: a global status report WHO. Geneva: WHO, ( ) WHO. Guidelines for controlling and monitoring the tobacco epidemic. Geneve: WHO, ( ) Doll R, Hill AB. A study of the aetiology of carcinoma of the lung. Br Med J 1952; 2: Doll R, Hill AB. Lung cancer and other causes of death in relation to smoking: A second report on the mortality of British doctors. Br Med J 1956; 2: Doll R. Tobacco: a medical history. J Urban Health 1999; 76: European Commission. Tobacco or health in the EU: paste, present and future Luxembourg, Office of Official publications of the European Communities, health/ph determinants/life style/tobacco/documents/tobacco fr.eu.pdf ( ) NIDA Research report series: Tobacco addiction. U. S. Department of Health and Human Services. National Institute of Health, nida.nih.gov/pdf/rrttobacco.pdf. ( ) 215

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77 Martinović et al Učestalost pušenja i nikotinska ovisnost Incidence of smoking and nicotine depedence among medical workers Željko Martinović 1, Cvita Martinović 2, Mladen Čuturić 1 1 Department of Surgery; 2 Department of Internal Medicine, Croatian Hospital Dr. Fra Mato Nikolić, Nova Bila ABSTRACT Aim To determine the frequency of smoking and nicotine dependence level of active smokers among medical workers. Methods The research encompassed a sample of 66 medical workers of the Croatian Hospital Dr. Fra Mato Nikolić in Nova Bila using surveys and Fagerstrom s modified test for nicotine dependence. Results There were of 66 examinees,37 (56.1%) of them being smokers, 19 (51.4%) female and 18 (48.6%) male. The average value of nicotine dependence score was 8 points, which represented a high level of nicotine dependence. Conclusion Smoking as a preventable health risk factor is a big problem even for medical workers themselves. Medical workers, active smokers, have a high level of nicotine dependence requiring a professional treatment. Key words: smoking, medical workers, level of nicotine dependence, Fagerstorm s test Original submission: 19 November 2008; Revised submission: 30 March 2009; Accepted: 13 April

78 ORIGINAL ARTICLE Smoking is the most frequent risk factor for cardiovascular diseases in Croatian Western region: findings of the Croatian health survey 2003 \ulija Malatestinić 1, Nena Rončević 2, Henrietta Benčević-Striehl 1, Suzana Janković 1, Vladimir Mićović 1 1 Teaching Institute of Public Health of Primorsko-goranska County, University School of Medicine, 2 University School of Philosophy; Rijeka, Croatia ABSTRACT Aim To estimate the prevalence of selected behavioral risk factors for cardiovascular diseases in the western region of Croatia and to determine the differences based on age and gender. Methods A national survey on health status and health behavior of the adult population has been conducted. The representative sample of 10,766 households for six officially defined regions of Croatia has been determined, and Western region has been included with 1,562 inhabitants, aged 18 years and older. The overall response rate of administered face-to-face questionnaire was 85-6%. Prevalence rates per 100 inhabitants (smoking, eating habits, alcohol consumption, physical activity, socio-economic characteristics, chronic conditions) have been determined. Corresponding author: \ulija Malatestinić Teaching Institute of Public Health of Primorsko-goranska County, University School of Medicine Krešimirova 52/a, Rijeka, Croatia Phone: ; Fax.: ; dulija.malatestinic@zzjzpgz.hr Results Nearly half (46.3%) of the adults were smokers or had quit smoking less than 10 years ago. Prevalence of high blood pressure was high amounting to 40.6% and it was higher in middle aged males (46.7%, p<0.01) and young males (13.7%), p<0.01). Prevalence of obesity was 38.9%, highest in females aged (51.2%, p<0.001) and 65 and older (73.8%, p<0.01). Almost a quarter of respondents (23.3%) has been insufficiently physically active, especially young females 22.5%, p<0.01). Conclusion There was a significant difference in the prevalence of all observed behavioral risk factors according to the gender and age. Moreover, smoking tobacco has been found as the most frequent risk factor in the observed population. Key words: cardiovascular diseases, health survey, prevalence, risk factors Original submission: 11 February 2009; Revised submission: 17 April 2009; Accepted: 04 May Med Glas 2009; 6(2):

79 Malatestinićet al Smoking in the Croatian Western Region INTRODUCTION Cardiovascular diseases (CVDs) are the major cause of death in most European transitional countries (1). In Croatia CVDs are the leading cause of death and account for more than half of the overall mortality (2,3). Unfortunately, exact data on the spread of the most important risk factors, such as hypertension, smoking, obesity, hypercholesterolemia, hypertriglyceridemia, insufficient physical activity etc. were not available prior to Croatian Adult Health Survey (CAHS) in It provided timely, reliable, cross-sectional estimates, supporting the development of a public health information system, health promotion, with emphasis on CVD prevention, CVD risk reduction and healthier lifestyles promotion among the general population (4). Furthermore, there have been no systematic and comparable studies of the risk factors on a representative sample of the population at the national as well as the regional level. Although CVDs account for more than half of the overall mortality in Croatia, the support for the research in this area, during the last decade, has not been sufficient. In the period between 1991 and 2004, Croatia had the lowest CVD publication rates (in 2003 the estimated proportion was 1.2% as opposed to % in other analysed countries) in the MEDLINE database among the countries included into the analysis (5). The mortality caused by CVDs in the population can be significantly reduced by acquiring a healthier way of life such as non-smoking, proper diet and regular physical activity. It is well known that health promotion and primary prevention are of substantial importance in decreasing CVD mortality and morbidity (5). One of the main hypotheses in our research program on health status and health behavior in adult population of the Croatian Western region was a high prevalence of CVDs risk factors with gender and age differences and different hierarchy in the prevalence of selected behavioral risk factors for CVDs as compared with the national level. PARTICIPANTS AND METHODS Participants The data were collected in the summer of 2003, and results have been officially released in December 2003 in the cross-sectional survey entitled Croatian Adult Health Survey (CAHS) (6). The main goal of the survey was to examine the health status, risk factors and health care utilization with a focus on cardiovascular diseases (CVDs). Conceptually, the survey was based on existing studies such as the CINDI (7,8), and Short Form 36 of the World Health Organization (9). The development, design and implementation of the 2003 CAHS was conducted by the Canadian Society of International Health, Croatian Ministry of Health, Croatian Central Bureau of Statistics, Andrija Štampar School of Public Health and the National Institute of Public Health, Zagreb, Croatia, and the cooperation resulted in completion of a high quality population health survey that provided first comparable data at a regional level. A sample design for the 2003 CAHS covered approximately 98% of the Croatian population aged 18 and older, due to exclusion of people living in non-conventional dwellings, stationed in institutions, full-time members of the Croatian Armed Forces and residents of some remote regions. Observed regions The 2003 CAHS used the official definition of the five sub-national regions (groupings of counties) as proposed by the Central Bureau of Statistics. In order to ensure sufficient/representative sample for Zagreb (the capital), it was removed from the Central region and considered as the sixth region for the 2003 CAHS. Using the 2001 Census of Households, the six main regions were further stratified according to the city type (town/ municipality) and counties to account for population differences. Overall, as the country was stratified into 20 design strata a sample of 11,250 units was required to meet the survey objectives 219

80 Medicinski Glasnik, Volumen 6, Number 2, August 2009 of providing reliable estimates for six regions and taking into account anticipated non-response. For the Western region, that included three Counties (Primorsko-goranska, Ličko-senjska and Istarska) with 565,000 inhabitants, the required sample size was 1,645 units. In cooperation with the Croatian Central Bureau of Statistics, the 2003 CAHS sample of household was selected from the 2001 Census of Households according to multistage stratified cluster design. For the 2003 CAHS, one person aged 18 and over per household was randomly selected using a simple random sampling approach. A structured questionnaire was administrated face-to-face to respondents by trained public health nurses from the Counties Institutes of Public Health in Croatia. The content modules of the survey questionnaire were: 1) household, 2) socio-economic characteristics, 3) physical measurements, 4) SF-36 (general health, activity limitation, mental and physical problems), 5) access to use of health care services, 6) chronic conditions, medication, preventive examinations, 7) smoking (daily smoking, attempts to stop, second hand smoking), 8) eating habits, 9) alcohol consumption (consumption, binge drinking), and 10) physical activity (time spent at work and leisure). At the end of the interview, anthropometric measures such as height, weight, pulse and blood pressure were collected from all the respondents. The total study sample of 10,766 households was selected, and for the Western region the total sample was 1,562 households participating in the 2003 CAHS. The overall response rate was 84-3% (9,070 resp. individuals) and for the Western region it was 85-6% (1,323 resp. individuals). Selected behavioural risk factors Behavioural risk factors were observed in relation to smoking, high alcohol consumption, inadequate nutrition, physical inactivity, elevated blood pressure and obesity. For the analysis of data on health behaviour, except for smoking, high blood pressure and obesity, complex indicators were derived. The smoking status of participants was assessed on the basis of current daily smoking habit and the past habit having existed more than 10 years ago. The prevalence of smoking at the present time was recorded. Unhealthy behaviour related to alcohol consumption was defined using the WHO International Guide for Monitoring Alcohol Consumption and Related Harm Recommendations (9). The prevalence of heavy drinking was defined as drinking 6 or more glasses (regular restaurant portions) of alcohol at a single occasion at least once a week and he/she was advised by somebody (a doctor or health care personnel or family member or others) to drink less or drinking strong drinks every day whereas somebody (a doctor or health care personnel or family member or others) advised him/her to drink less. Unhealthy nutrition-related behaviour was defined as participants satisfying at least three of the following five criteria: preparing food using animal fat; drinking milk or other dairy products with more than 3.2% fat; eating fruit occasionally or not at all; eating smoked meat, sausage-meat, ham, bacon or similar products every day or almost as frequent; add salt to meals almost always before even tasting the food. Physical inactivity was defined by the respondent satisfying at least three of the following 4 criteria: a respondent goes to work by car, public transportation or similar; does not work at all or works at home; physically light work (mainly walking); doing physical exercise during leisure time for at least 30 minutes which makes him/ her at least mildly short of breath or perspires not more than once a week; doctor or other health care personnel or family have had advised him/ her to increase physical activity during the last year (12 months). High blood pressure is a category that included people with systolic blood pressure higher than 140 mmhg and diastolic blood pressure higher than 90 mmhg, and people with diagnosed hypertension that is currently under control. Obesity is category that included people with waist larger than 101 cm for men and larger than 87 cm for women (10). 220

81 Malatestinićet al Smoking in the Croatian Western Region Table 1. Estimates of prevalence (per 100 population) of selected behavioral risk factors for cardiovascular diseases in Western region of Croatia according to age No (%) of examinees according to age groups* Behavioral risk factor and older Total (n=1,1323) Smoking 225 (17.2) 336 (25.7) 45 (3.4) 606 (46.3) High alcohol 8 (0.6) 36 (2.8) 11 (0.8) 55 (4.2) Inadequate nutrition 74 (5.6) 86 (6.5) 19 (1.4) 179 (13.5) Physical inactivity 62 (4.7) 132 (10.0) 114 (8.6) 308 (23.3) High blood pressure 35 (2.7) 227 (20.9) 225 (17.0) 537 (40.6) Obesity 56 (4.2) 284 (21.5) 175 (13.2) 515 (38.9) * percentages were calculated using the number of valid answers; the percentages of missing data for the whole sample varied between 0% for inadequate nutrition, high blood pressure and obesity and 1.1% for smoking A respondent without any of the following conditions: high blood pressure, elevated blood sugar, elevated blood cholesterol, previous myocardial infraction or stroke and angina pectoris was categorized as Cardiovascular Diseases (CVD) not reported. Statistical analysis Statistical analysis was performed with SPSS statistical package for Windows, Version 11.0 (SPSS Inc., Chicago, IL, USA). Descriptive statistics was done using percentages and frequencies. The estimates of prevalence for the selected risk behaviour were defined for the Western region as a whole and then sub-grouped in respect to the gender and the age. Variable age was divided in three age strata: years, and 65 years and older. Overall differences in risk behaviour and CVDs were analysed using the chi-square test. P-value of 0.01 or less was considered significant. RESULTS Among the respondents in the Western region, there were slightly less males (47.7%) than females (52.3%) at the ratio 1:1.1. The prevalence of current daily smokers and those who quit less than 10 years ago classified as smokers in the Western region of Croatia was 46.3%. More than a quarter of them were at the age years (Table 1). Nearly every fifth smoker (17.2%) was among the youngest age group (18-34). Although the prevalence of smoking after the age of 64 significantly decreases, still 16.9% of older persons were smoking (45 out of 267 participants who were 65 years and older). As for the gender (Table 2), males older Table 2. Assessment of differences between examinees according to the age and gender in selected behavioral risk factors for cardiovascular diseases in the Western region of Croatia % Gender Statistics* Variables in the question Pearson Contingency Men Women df p Chi-square Coefficient Smoking and older < High alcohol < and older < < Inadequate nutrition < and older < Physical inactivity and older < High blood pressure < and older Obesity < and older < * df, degrees of freedom; p, value of 0.01 or less was considered significant 221

82 Medicinski Glasnik, Volumen 6, Number 2, August 2009 than 65 consumed significantly (p<0.001) more cigarettes than females of the same age. The global prevalence of high alcohol consumption was 4.2% (Table 1). It was highest in males aged (p<0.001) and also in their older age - 65 and older (p<0.001) than in females (Table 2). The prevalence of inadequate nutrition for the Western region in Croatia was 13.5% (Table 1), mostly present in the age group It was highest in males; younger males aged (28.6%) significantly differ (p<0.001) from females; and also in the middle aged males (18,5%) (p<0.001). The global prevalence of physical inactivity for the Western region in Croatia was high (Table 1), nearly quarter of respondents were insufficiently physically active (23.3%). The prevalence was lowest (10.6%) in youngest males aged against 22.5% physically inactive young females (Table 2) with significant difference (p<0.01). These findings were concordant with the previous findings (5, 6) made by other authors. Physical inactivity increased with age (Table 2). The prevalence of high blood pressure for the Western region in Croatia was very high, 40.6% among respondents (Table 1). More than every fifth person (20,9%) of the middle age stratum (35-64) had high blood pressure, the main factor in cardiovascular risks with great preventable potential. As for the gender (Table 2), it was significantly higher (p<0.01) in males (46.7%) than in females (34.8%) in the age stratum 35-64, and significantly higher (p<0.01) in the age stratum 18-34, where 13.7% of males had high blood pressure and 4.8% of females. As for obesity the category with 101 cm and more for waist circumferences in males and 87 cm in females was considered to be a better indicator instead of excessive body mass index of 30 (10). Global prevalence for the Western region in Croatia was 38.9% (Table 1). Nearly a quarter of the obese respondents were at the age group of Obesity was higher in females than in males (Table 2), with significant difference in the age groups (p<0.001) and 65 and older (p<0.01). In the middle aged respondents (35-64), the criteria variables were reported for CVDs. More than a half, 55.7% of males and 51.8% of females, smoke and every tenth male was a high consmuer of alcohol without reported CVDs (Table 3). Inadequate nutrition was present in every fifth male (19.4%) and in 5.9% of the females. Physical inactivity was present in 13.4% of males and in 18.6% of females without reported CVDs. The burden of consequent risk factors such as high blood pressure for males was 33.2% and 17.3% for females in the category of respondents without CVD (Table 3) as opposed to the category of reported CVDs where it was higher, 76.9% of males and 40.4% of females. Similar findings were present with second consequent risk factor of obesity, although with dominant occurrence in females. We have conducted the analysis considering relations between respondent s socio-demographic characteristics and high CVD risk. High risk was a complex indicator constructed by satisfying at least one of the following criteria: past myocar- Table 3. Prevalence of selected risk factors for cardiovascular diseases in Western Croatia for age according to anamnestic related diseases Risc factors Men % (95%CI) CV anamnestic healthy* CV anamnestic sick Women Men % (95%CI) % (95%CI) Behavioral risk factors Women % (95%CI) Smoking 55.7 ( ) 51.8 ( ) 45.2 ( ) 39.2 ( ) High alcohol 10.9 ( ) ( ) 0.8 ( ) Inadequate nutrition 19.4 ( ) 5.9 ( ) 16.5 ( ) 8.8 ( ) Physical inactivity 13.4 ( ) 18.6 ( ) 23.3 ( ) 28.8 ( ) Consequent risk factors High blood pressure 33.2 ( ) 17.3 ( ) 76.9 ( ) 65.6 ( ) Obesity 28.1 ( ) 38.8 ( ) 40.4 ( ) 72.8 ( ) *a person without any of the following conditions: high blood pressure, related blood sugar, elevated blood cholesterol, myocardial infarction or stroke recovery and angina pectoris; a person with at least one of the above mentioned chronic conditions 222

83 Malatestinićet al Smoking in the Croatian Western Region dial infarction, stroke, blood pressure higher than 159/99, waist circumference of more than 101 cm for males, and more than 87 cm in females. The variable gender and risk were found dependent (p<0.001), so that 50.1% of females were at high risk for CVDs and nearly 10% lower risk was found for males (Table 4) although with low contingency coefficient. The risk increases with age, so that at the age of 65 and older the risk for CVDs was higher (77.8%). The variables were found dependent with medium strong connection (C=0.394). Educational level and high risk for CVDs were found dependent (Table 4), although with little connections (C=0.279). The risk was higher for those with low level of education (uncompleted and finished elementary school). According to the marital status those at the highest risk for CVDs were widowers/ widows, 78.5% of them (C=0.334); as far as work(c=0.360) is concerned managers (84.2%) were found to be especially sensitive, followed by two thirds of housewives and retired persons, Table 4. Prevalence of high risks for cardiovascular diseases according to examinees socio-demographic status Socio-demographic status (%) Risk for cardiovascular disease* Riskless and middle High x2 Contingency Coefficient Gender male female Age and older Education incomplete elementary school elementary school high school or similar school high school graduate faculty, academy Marital status married or living in partnership single divorced widow/widower Labor activity active inactive Position in occupation housewife worker, farmer or office worker self-supporting farmer or craftsman head clerk director member of military or police forces pensioned student unemployed other Income less than 1000 Kunas between Kuna between Kuna between Kuna between Kuna between Kuna between Kuna more than Kuna no answer *values were calculated using the total number of examinees in the sample; middle risk includes examinees with presence of at least one of the following: high blood pressure, obesity and at least one of the behavioral risks (alcohol consumption, inadequate nutrition, physical inactivity, smoking); high risk include examinees with presence at least one of the following: myocardial infarction, stroke, high blood pressure, BMI>29,99 and waist > 101 for man and 87 for woman; statistically significant, p<0.001; Monthly salary in average for September 2003 was Kuna (29) 223

84 Medicinski Glasnik, Volumen 6, Number 2, August 2009 and as for the financial status (C=0.208) more than two thirds of participants had low income (monthly household income in average between 1000 and 2000 Kunas). DISCUSSION The prevalence of selected risk factors for CVDs in Western Croatia is high as expected. Our results support the findings of other authors (6, 15-18) that the prevalence of cardiovascular risk factors in Croatia is generally high, but that their ranking varies on the regions, age and sex. Equivalent to Croatian health survey in 2003, in neighbouring Slovenia there was a survey conducted in Risk Factors for Noncommunicable Diseases in Adults in Slovenia (12). Similar findings regarding to age, sex and regional differences were found, although with some speciality. In Western Croatia smoking was found to be the most frequent risk factor in both genders. The frequency of daily smokers for the Western region in Croatia was higher than that found at the national level (6). In general, more males than females were cigarette smokers, but the difference has been very narrow (18,19). Consuming tobacco, especially cigarette smoking, is the main avoidable risk factor for CVDs. The total of one third of all deaths caused by CVDs can be related to smoking cigarettes (20). The risk for myocardial infarction increases with age and number of cigarettes smoked, so that in middle age female who smoke more than 40 cigarettes every day, the risk rises for 74.6% (22). This problem is more significant due to the fact that the habit is mostly present among the youngest males and females included in this study. Therefore, we conducted the analysis with the middle aged respondents (35-64) as more prominent for presence of cardiovascular risks and related/consequent mortality rates (3). In this age group, opposite to prevalent male smokers in general, more than a half of the males and two thirds of the females are daily smokers or quitted less than 10 years ago. So far Croatia has had many attempts to promote non smoking on the National level. The largest campaign was in 2002 under the title Say yes to non smoking. Unfortunately, on a larger scale this action did not last long enough to make its impacts durable (23). On the other hand, we argue that the absence of sensibility for the problem and its true picture, lack of financial support and genuine commitment on the regional level contribute to continuously high level of cigarette smoking. High blood pressure followed by obesity was present in more than 40 % of participants. We found these risks in the middle aged population of both genders. Both of these risks are the elements of the metabolic syndrome (24). More to add, physically inactive was nearly every fourth person, especially females of younger ages. Also, every seventh person in the Region had inadequate nutrition and it can be observed as a special risk for males. In younger males, findings of inadequate nutrition are more frequent. This is an interesting finding in view of the fact that the Western region belongs to the Adriatic area where Mediterranean food is widely accessible and a part of the tradition. (6). The frequency of high blood pressure as a second risk factor for the Western region in Croatia was found little lower than that at the national level (5, 6) and the lowest among the regions (16). The prevalence of hypertension in all regions of Croatia exceeds 50% in males and 44% in females (6). The frequency of obesity as a risk factor in Western region of Croatia was higher than found at the national level (5, 6). Interestingly, the assessment of the results at the national level showed that obesity was one of the most common risk factor found for females but the least prevalent in males (5). Opposite to these findings, in the Western region obesity is a seriously widespread risk factor for males prior to physical inactivity and heavy drinking. The occurrence of heavy drinking in the Western region in Croatia was found lower than that at the national level (6). Stressing the potential but insufficient implementation of secondary 224

85 Malatestinićet al Smoking in the Croatian Western Region prevention and health promotion, high alcohol consumers were rarely counselled to reduce or stop alcohol use. The importance of heavy drinking cessation is in well known relation to cardiovascular diseases, but also notably other diseases and injuries (25,26). To somewhat summarize the occurrence of risk factors we may say that in the middle aged males, the most prominent risk factor was smoking, followed by high blood pressure, obesity, inadequate nutrition and physical inactivity. The hierarchy was somewhat different in females, except smoking which has also been the leading risk factor in young females, followed by obesity in the middle aged, high blood pressure, physical inactivity and inadequate nutrition. Can the risk factors for CVD itself, without social determinants, correctly assess the situation and be used for health care policy recommendation? The risk assessment is important but insufficient. It would be sufficient if the effects of social determinants on appearance and outcome of cardiovascular disease were unknown. However, the facts and scientific analysis speak precisely the opposite (27). Socio-economic status (SES) is consistently among the most fundamental determinants of health status (28, 29). Furthermore, SES relationship can be attributed to CVDs together with combined effects of differences in health-related behaviours, environmental conditions, social structures, and the availability and delivery of health care (29). Our findings of low education status and low income in relation to high risk for CVDs is in agreement with the findings of other authors (5, 29). They also pinpointed that education and income-related disparity influenced particularly the tobacco use and diabetes prevalence. They occur more among the people with a lower SES. The step up in lowering the frequency of CVD risk factors, and the concomitant decrease in CVD mortality among the adults is a chronic disease success story in the United States (29). It produced further good news: at least with regard to trends in blood pressure and blood cholesterol level, people with low annual incomes and low education levels were not left behind. These findings stress the need for public health efforts in finding ways to reach population with lower SES. There are some limitations to our study. First, study was based on cross-sectional samples, thus not designed to assess cause and effect between SES and cardiovascular disease risk factors. Our goal was not to assess the cause, but to determine the population burden of CVD risk factors. Finally, our assessment of disease and risk factors depended on respondents honesty. Still, thanks to the CAHS, evidence base for CVD prevention programme in Croatia and its regions was good, providing a national and regional guidance for prevention and health promotion in cardiovascular health. It emphasises the need for a holistic approach in the promotion of healthier lifestyles, especially reducing tobacco use and alcohol consumption and promoting healthy nutrition and physical activities. ACKNOWLEDGEMENT/DISCLOSURE Competing interests: none decleared. REFERENCES World Health Organization. European Health for all Database (HFA- DB). int/hfadb (5 June 2005). Croatian Central Bureau of Statistics, Statistical yearbook Zagreb: Croatian Central Bureau of Statistics, Strnad M. Čorić T. Kern J. Polašek O. Mortality due to Cardiovascular Diseases. In: Proceedings of the Symposium on Regional Distribution of Populations Cardiovascular Risk factors in Croatia, Zagreb, Croatia, 2 nd December, Academy of Medical Science Croatia, Zagreb, Croatia, 2005, p Beland Y, Bailie L, Page J. Statistics Canada, Croatian Ministry of Health, and Central Bureau of Statistics: a joint effort in implementing the 2003 Croatian Adult Health Survey. In: Proceedings of the American Statistical Association Meeting on survey research methods. Toronto, Canada, August American Statistical Association, Toronto, Lukenda J, Kolaric B, Kolčić I, Pažur V, Biloglav Z. Cardiovascular diseases in Croatia and other transitinal countries: comparative study of publications, clinical interventions, and burden of disease. Croat Med J 2005; 46:

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87 ORIGINAL ARTICLE Influence of different glass fiber reinforcements on denture base polymer strength (Fiber reinforcements of dental polymer) Denis Vojvodić 1, Dragutin Komar 1, Zdravko Schauperl 2, Asja Čelebić 1, Ketij Mehulić 1, Domagoj Žabarović 1 1 Department of Prosthetic Dentistry, School of Dental Medicine, 2 Faculty of Mechanical Engineering and Naval Architecture; University of Zagreb, Zagreb, Croatia ABSTRACT Aim Assessment of flexural strength values of dental base polymers reinforced with different glass fibers ( dental and industrial origin) after performed artificial ageing procedures. Methods Three hundred specimens (dimensions 18 x 10 x 3 mm) were produced of denture base polymers reinforced with different glass fibers. The short beam testing method was used to determine the flexural strength of the specimens after polymerization, immersion in water of temperature 37 o C for 28 days, and thermocycling. Corresponding author Denis Vojvodić Department of Prosthetic Dentistry, Department of Prosthetic Dentistry, School of Dental Medicine, University of Zagreb, Av. G. Šuška 6, Zagreb, Croatia Phone: ; Fax: ; denisvojvodic@yahoo.com Results Flexural strength of the polymer specimens was MPa, while specimens reinforced with glass fibers demonstrated rise of flexural strength values ( MPa), no matter which type ( dental or industrial ) of fibers was used. Microscopic examination revealed partial bonding between the glass fibers and polymer material. Conclusion Both of the investigated glass fibers had similar strengthening effect, but due to better investment/benefit ratio industrial glass fibers could be recommended for dental laboratory use. Prolonged polymerization of denture base materials should be proposed because it had direct impact on the improvement of flexural strength values. Key words: dental materials, polymers, glass fibers, dental prosthesis, mechanical phenomena Original submission: 06 March 2009.; Revised submission: 20 April 2009.; Accepted: 22 April Med Glas 2009; 6(2):

88 Medicinski Glasnik, Volumen 6, Number 2, August 2009 INTRODUCTION Life span of the world population has been considerably prolonged in the last century because of the huge progress of preventive and curative medicine. Therefore, the number of elderly people (60 and more years old) is increasing. There is also evidence of progressive loss of teeth connected with the age of patients, and the increased loss of teeth is dominant in the population between 50 and 60 years of age (1). Total loss of teeth is especially frequent in patients living in non-developed countries due to improper dental care and low health education of the population. Dental prostheses are at the second place in frequency of different appliances that are used by elderly population, just next to the glasses (1). Today dental prostheses are predominantly made of methyl metacrylate polymer which has proven to be the most reliable denture base material. Despite many advantages, methyl metacrylate is prone to fracturing. Therefore, fractures of the dentures are very frequent, almost equal to the number of newly made dentures (2). These dentures produced from methyl metacrylate may be strengthened by incorporation of various reinforcements into the denture base polymer, because such reinforcements enhance flexural strength and resistance to impact (3). Physical and mechanical properties of acrylic dentures can be enhanced by integration of different fibers with different fiber architectures into the denture base polymer (4-9). In order to improve flexural and impact strength of denture base polymer, graphite (7-11), glass (5,6,12-16), and organic fibers, such as, aramide (15,17,18) and polyethylene fibers with high molecular weight (9,19-27), are used. Today the most acceptable fibers for dental polymer reinforcements are glass fibers, because of their good aesthetics (6,12-14,28,29) and good bonding with polymers via silane coupling agents (30-32). Also, they can easily be adapted to the desired shape and length (33) which is then suitable for incorporation into denture base polymer material. Different procedures of pre-impregnation of glass fiber reinforcements with silane coupling agents are used to establish chemical bond with denture base polymer material. Glass fiber reinforcements produced by dental manufacturers, which are present on market, are already pre-impregnated with silane coupling agents, but they are rather expensive. Common industrial E-glass fibers are usually not pre-impregnated during manufacturing. They are rather cheap, and it is possible to pre-impregnate them with silane coupling agents in dental laboratory. The main goal of this study was the assessment of flexural strength of dental base polymers reinforced with silane pre-impregnated glass fibers of different architecture produced by dental products manufacturers. These results should be compared with the results of flexural strength of dental base polymers reinforced with industrial E-glass fibers after they are pre-impregnated in dental laboratory using silane coupling agent. Obtained results could give the preference of which glass fiber reinforcement to use, with regard to the investment/benefit ratio. MATERIALS AND METHODS Quadratic specimens with smooth surfaces and dimensions of 18 x 10 x 3 mm were made of Meliodent Heat Cure and Meliodent Rapid (Heraeus Kulzer, Hanau, Germany) polymer. Some of the specimens made from aforementioned polymers were reinforced with glass unidirectional fibers and nets produced exclusively for application in dentistry (StickTech Ltd., Turku, Finland), and some with industrial E-glass unidirectional fibers and nets (Kelteks, Duga Resa, Croatia). All the specimens were split across ten different groups with thirty specimens each (n=300). Special metal cuvette was constructed in order to produce uniform specimens. It consisted of two thick polished metal parts on the sides and two thin metal parts in the middle. The middle metal parts had ten quadratic perforations of the size of a specimen (18 x 10 mm). One thin metal part was 1 mm thick and the other thin metal part was 2 mm thick. In that way the thickness of the specimens and the proper glass fiber alignment were obtained. So, the fiber reinforcements were placed 1 mm from one side and 2 mm from the other side of a specimen. Metal parts of the cuvette were smeared twice with Ivo- 228

89 Vojvodić et al Fiber reinforcements of dental polymer clar Separating Fluid (Ivoclar Vivadent, Schaan, Liechtenstein). Pre-impregnated glass fibers for dental use (StickTech Ltd., Turku, Finland), unidirectional (Stick TM ) or net shaped (Stick TM Net) were impregnated with Meliodent Heat Cure polymer mixture of a syrup consistency (with polymer/monomer weight ratio10:8) for eight minutes. During impregnation time of the glass fibers Meliodent Heat Cure polymer was prepared according to the manufacturer s instructions and placed in both cuvette halves (consisting of one thick side part + one thin middle part). Just impregnated glass fibers, unidirectional (Stick TM ) or net shaped (Stick TM Net) were taken from polymer syrup and placed on one cuvette half. During that alignment procedure unidirectional glass fibers were laid along the specimens, in order to be orthogonal to the force to be applied. The net shaped glass fibers were placed at an angle of 45 o to the length of the specimen. The cuvette halves were then closed together and put in a hydraulic press (Zlatarne, Celje, Slovenia) under the 200 bars pressure. After pressing, the cuvette was moved to a manual bench vice and put in a polymerizing apparatus (Type 5518, KaVo EWL, Biberach, Germany) where polymerization was performed according to the manufacturer s instructions. For the specimens produced from Meliodent Rapid auto polymerizing material a similar procedure was performed. The only difference was shorter impregnation time of glass fibers (two minutes instead of eight) due to the auto polymerizing character of the material. Production of the specimens reinforced with industrial E-glass fibers was different only in the performance of pre-impregnation of the glass fibers in dental laboratory. Firstly, industrial non-impregnated fibers were weighted in weighing-machine Mettler H 311 (Mettler Instr. AG, Zurich, Switzerland) with the accuracy of 0.1 mg, to match the weight of dental glass fibers used in equivalent specimens. Then, industrial E-glass fibers were cleaned with 1.6 mol sulphuric acid for 30 sec., rinsed in distilled water, and air dried at room temperature for 24 hours. After that they were dipped into 98% γ-metacryloxypropyl-trimethoxysilane (Sigma- Aldrich Co., St. Louis, MO, USA), placed on a clean sheet of paper, put in dental sterilizer (ISO 400, Aesculap, Tuttlingen, Germany) and heated at temperature 100 o C for 2 hours. Afterwards, these so pre-impregnated glass fibers were impregnated with adequate polymer syrup (Meliodent Heat Cure or Meliodent Rapid) and the specimens were produced in the manner of the aforementioned procedures. After polymerization of the denture base materials and cooling, the cuvettes were opened and the specimens were detached. Thin polymer excess on all the specimens was removed with a carbide bur (Ivomill, Ivoclar Vivadent), and the specimen margins were finished using sandpaper (Sianor 7/0B, Frauenfeld, Switzerland). The specimens were checked with calipers (Dentarium , Dentarium, Ispringen, Germany) for the accuracy of dimensions, and the maximum allowed deviation was 0.05 mm. All the specimens were tested in the universal testing machine (Amsler, Schafhausen, Switzerland) using the short beam method (Figure 1) (34), and the moving speed of the blade was set to 1.5 mm/min in order to determine the samples flexural strength. All ten groups of specimens were subdivided into three subgroups of ten specimens each which were tested after polymerization of the specimens, immersion of the specimens in distilled water with temperature at 37 o C (thermostat Btuj, Poznan, Poland) for 28 days, and after thermocycling of the specimens according to Hansson s method (35). Specimens were placed in a testing holder, in a position where the fiber reinforcements were closer to the testing holder s posts (1mm away from the posts and 2 mm from the blade).the force causing breakdown of the Figure 1. Scheme of the specimen loading (d, thickness of the specimen, F, applied force) 229

90 Medicinski Glasnik, Volumen 6, Number 2, August 2009 specimens was recorded and the flexural strength was calculated according to the formula: F max measured force of the loader (N); l distance between posts (here 15 mm); b width of the specimen (here 10 mm); h height of the specimen (here 3 mm). Obtained numerical results were analyzed with SPSS statistical package (SPSS Inc., Chicago, USA). Statistical analysis was performed using descriptive statistics, tests of between-subjects effects, Scheffe s test, and Student s t-test. In order to reveal the quality of bonding between glass fibers and denture base polymer, glass fiber reinforced specimens were randomly chosen and sealed in Durofix material (Struers, Rodovre, Denmark). Subsequently they were ground, and polished according to the routine procedure (36) to obtain a smooth surface suitable for microscopic examination. It was performed with the use of a light microscope Olympus BH2-UMA (Olympus Optical, Tokyo, Japan) and the characteristic images were photographed with Olympus C-5050 Ultra Zoom camera (Olympus Optical, Tokyo, Japan). RESULTS During testing specimens made of Meliodent Heat Cure and auto polymerizing Meliodent Rapid polymer (control groups) demonstrated the Figure 2. Arithmetic means of flexural strength: 1, Meliodent Rapid polymer+kelteks fibers (net); 2, Meliodent Rapid polymer+kelteks fibers (unidirectional); 3, Meliodent Rapid polymer+ Stick fibers (net); 4, Meliodent Rapid polymer+stick fibers (unidirectional); 5, Meliodent Rapid polymer (control); 6, Meliodent Heat Cure polymer (control); 7, Meliodent Heat Cure polymer+kelteks fibers (net); 8, Meliodent Heat Cure polymer+kelteks fibers (unidirectional); 9, Meliodent Heat Cure polymer+stick fibers (net); 10, Meliodent Heat Cure polymer+stick fibers (unidirectional) lowest flexural strength, whereas, the specimens reinforced with both types of fibers showed higher flexural strength values (Figure 2, Table 1). Scheffe s test applied across ten investigated groups of specimens revealed a statistically significant difference (P<0.05) between some of these groups, as shown in Table 1. The test between subject s effects revealed that the type of polymer (heat cure or auto polymerizing), type of glass fibers (unidirectional or net) and ageing procedures (immersion in distilled water at temperature 37 o C for 28 days, thermocycling) had a significant influence (P<0.05) on the achieved flexural strength values, whereas, the origin of glass fibers ( dental -Stick Table 1. Descriptive statistics and Scheffe test between the groups of specimens Groups* N 95% Confidence Standard Mean Standard Interval for Mean Min. Max. Scheffe test between groups (MPa) Deviation Lower Upper - Statistically significant Error Bound Bound difference , , , , , , , , , , , , ,00 148, , , , , , ,25 122, , , , , , , , , , , , ,50 118, , , , , , ,75 160, , , , , , ,50 178, , , , , , ,25 190, , , , , , ,50 163, , , , , , ,00 236, Total , , , , , ,75 236, *1, Meliodent Rapid polymer+kelteks fibers (net); 2, Meliodent Rapid polymer+kelteks fibers (unidirectional); 3, Meliodent Rapid polymer+ Stick fibers (net); 4, Meliodent Rapid polymer+stick fibers (unidirectional); 5, Meliodent Rapid polymer (control); 6, Meliodent Heat Cure polymer (control); 7, Meliodent Heat Cure polymer+kelteks fibers (net); 8, Meliodent Heat Cure polymer+kelteks fibers (unidirectional); 9, Meliodent Heat Cure polymer+stick fibers (net); 10, Meliodent Heat Cure polymer+stick fibers (unidirectional); N, number of specimens; Mean, arithmetic mean of the recorded flexural strength values in megapascals 230

91 Vojvodić et al Fiber reinforcements of dental polymer or industrial -Kelteks) had no statistically significant influence. That was also confirmed by Student s t-test (t=1.367). Auto polymerizing polymer material was weaker than heat-cure polymerizing dental base material (P<0.05), and unidirectional glass fibers positioned perpendicularly across the applied force, on the specimen, strengthen polymer material more successfully than net shaped glass fibers positioned at an angle of 45 o (P<0.05). Thermocycled specimens had the highest flexural strength (P<0.05), whereas, there was no statistically significant difference (P>0.05) between specimens tested after polymerization and after immersion in distilled water for 28 days. After a short beam test was performed, the specimens reinforced with both types of fibers revealed mostly adhesive type of breakdown between fiber reinforcements and polymer matrix resulting in pullouts of fibers from the matrix. Microscope image analysis showed the existence of voids between glass fibers and polymer matrix and partial bonding between glass fibers and polymer material (Figure 3). DISCUSSION The prime and most frequent fracture of the upper denture occurs in the medial line and the fracture mechanism and the influence of the masticatory load which is applied onto the dentures are very similar to the three point bending test short beam test (37,38). During chewing denture base material is submitted to the flexural deformation due to applied masticatory load. Figure 3. Microscopic image of a specimen section voids between glass fibers and polymer material (magnification 1000x) (D. Vojvodić, Z. Schauperl, 2008.) Therefore, tests of the flexural strength are very appropriate for testing polymer dental materials (23, 37-41), because simulation of the masticatory load is very close to the real conditions in the mouth. Also, the influence of material fatigue on mechanical properties is decisive (42). For that reason different procedures of artificial aging, such as, underwater storage in thermally controlled conditions and cyclic temperature changes (thermocycling) are used in denture materials studies. They are used to determine the longevity of the obtained mechanical properties of dental materials in the demanding environment of the oral cavity. For that purpose different authors use different time periods of underwater storage, but the important influence of water on the flexural strength occurs during the first four weeks of immersion causing decrease of the flexural strength values. Since longer period of immersion does not perform a statistically significant decrease of flexural strength (43,44) 28 days immersion in water at 37 o C was used in this study. Immersion in water enables molecules of water to penetrate into the areas between the polymer chains, remain there, and act like wedges between these chains. Water entry in polymer material during immersion is primarily caused by diffusion, and partly by the polarity of the polymer chains that is caused by unsaturated molecules and unbalanced intermolecular forces. Penetration of water molecules may also cause softening of the denture base, as absorbed water can act as a poly(methyl methacrylate) (PMMA) plasticizer. Water absorption diminishes the mechanical properties of the material, resulting in lower flexural strength and lower modulus of elasticity (45). Also, the ageing effect of thermocycling procedure which simulate ingestion of cold and hot food/beverages can have a significant influence on mechanical properties of polymer materials (46,47). After the ageing procedures were performed no statistically significant difference (P>0.05) was found between specimens tested just after polymerization and after immersion in distilled water for 28 days. On the contrary, the flexural 231

92 Medicinski Glasnik, Volumen 6, Number 2, August 2009 strength values remained the same or were even slightly increased in some of the specimens subgroups (Figure 2). Since water absorption that occurred during the immersion of specimens in distilled water had no negative influence on the flexural strength, it could be stated that water absorption caused relaxation of the stress in polymer material that occurred during polymerization shrinkage. That could be the explanation for the increase of flexural strength values for the tested polymer materials (15,48). As mentioned, thermocycling procedure can have a significant impact on lowering the mechanical properties of polymer materials (46,47). On the contrary, in this investigation thermocycling procedure increased the flexural strength values. It appears as if the increase in temperature during thermocycing procedure resulted in the effect of prolonged polymerization, which could in turn result in the decrease of the residual monomer volume thus enhancing the mechanical properties of the polymer (increase of the flexural strength). Therefore, prolonged polymerization of the polymer material should be proposed discarding the manufacturer s instructions on the relatively short duration of polymerization, because, in the authors opinion, it had a direct impact on the improvement of mechanical properties. Glass fibers, no matter if they were of dental or industrial origin, considerably strengthened the polymer material, because testing load was shared between polymer material and the fiber reinforcements. Because of similar strengthening effect, but due to relatively high costs of the dental glass fiber reinforcements, low cost industrial glass fibers treated with self made pre-impregnation could be recommended for dental laboratories. Auto polymerizing polymer material was significantly weaker than heat-cure polymerizing dental base material (P<0.05) regardless of the details of fiber reinforcements. That fact could be attributed to the type of polymerization procedure resulting in higher residual monomer volume at auto polymerizing material that is directly influencing its mechanical properties. As expected, unidirectional glass fibers strengthen polymer material more successfully than net shaped glass fibers, because fibers were positioned perpendicularly across the applied force (opposite to the net shaped glass fibers positioned at an angle of 45 o ), thus giving better strengthening effect. Therefore they are more appropriate to be used, but in cases when the direction of the braking force is known. After short beam test was performed specimens reinforced with both, dental and industrial, fibers revealed mostly adhesive type of breakdown between fiber reinforcements and polymer material resulting in pullouts of fibers from the polymer. That arouses suspicion about inadequate bonding between glass fibers and polymer material, but there was no difference between specimens reinforced with expensive fibers produced by dental manufacturers (already pre-impregnated with silane coupling agents) and specimens reinforced with cheap industrial E-glass fibers treated with described self made pre-impregnation. Microscope image analysis of specimens reinforced with both types of glass fibers ( dental and industrial ) showed existence of voids between glass fibers and polymer revealing partial bonding between glass fibers and polymer material (Figure 3) which is, in our opinion, the result of established dental laboratory routine production, without idealization of specimens production conditions. It is the intent of the study to generate reproducible results comparable to the general laboratory practice. But even such imperfect reinforcements are good enough to significantly increase the flexural strength of the investigated polymer materials. ACKNOWLEDGMENTS/DISCLOSURE Presented results originate from the scientific project Investigation of materials and clinical procedures in prosthetic dentistry supported by the Ministry of Science, Education, and Sports of the Republic of Croatia grant No Competing interests: none declared. 232

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95 ORIGINAL ARTICLE Influence of cast surface finishing process on metal-ceramic bond strength Ketij Mehulić 1, Martina Lauš-Šošić 2, Zdravko Schauperl 3, Denis Vojvodić 1, Sanja Štefančić 2 1 Department of Prosthodontic School of Dental Medicine, University of Zagreb, 2 Dental Polyclinic, 3 Faculty of Mechanical Engineering and Naval Architecture, University of Zagreb, Zagreb, Croatia ABSTRACT Aim To investigate the influence of different cast surface finishing process on metal-ceramics bond strength. Methods Six Co-Cr alloy sample groups were cast (Wirobond C, BEGO, Bremen, Germany) and randomly selected for use in one of the six different final processing of the casting surface (oxidation, sandblasting with 110 and 250 µm Al 2 O 3, bonding agent, hydrochloric acid solution) prior to application of feldspathic ceramic (Duceram Kiss, DeguDent, Hanau-Wolfgang, Germany). The testing was carried out with a tensile testing machine (LRX with Nexygen software, Lloyd Instr., Fareham, UK) (ISO 9693). Corresponding author: Ketij Mehulić, University of Zagreb, School of Dental Medicine, Department of Prosthodontic Gundulićeva 5, Zagreb, Croatia Phone: ; Fax: ; mehulic@sfzg.hr Original submission: 19 March 2009.; Revised submission: 06 April 2009.; Accepted: 10 April Results The highest force ( N) for the separation of ceramics measured with the sample sandblasted with 250µm Al 2 O 3, oxidised and repeatedly sandblasted with 250 µm, and the lowest force ( N) with the sample treated with hydrochloric acid solution. With all sample groups except the group with the bonding agent (cohesive fracture), an adhesive fracture of the medium and an adhesive-cohesive fracture of the peripheral part of the fracture surface were observed. The oxidation, prolonged oxidation and the bonding agent do not influence the bond strength of the tested metal-ceramic system. Conclusion Different casting surface treatments have an important role on the bond strength of the ceramic-metal interface. Key words: casting surface processing, bond strength, metal-ceramic restoration, metal-ceramic interface Med Glas 2009; 6(2):

96 Medicinski Glasnik, Volumen 6, Number 2, August 2009 INTRODUCTION Ceramics are being used increasingly as a restorative material in a variety of dental restorations, including metal-ceramic crowns, all-ceramic restorations, and fixed partial dentures, mainly as a result of their excellent aesthetic properties, durability, biocompatibility and resistance to wear (1). Ceramic for dental reconstructive work are multiphase silicate ceramics, glass ceramics or monophased glasses with varying compositions (2,3). Structure composed of ceramic layers on a metal frame combined the strength of a metal substrate (dental alloy) with aesthetic of a ceramic. Currently, these ceramic fused to metal appliances are widespread in use in prosthodontics. Because of their inherently brittle nature susceptibility to their failure was identified at localized areas of high stress concentration on the ceramic surface, metalceramic interface or within the microstructure (4). In any laminate composite system the strength of the interfacial bond between the individual laminates is a major factor in determining the overall resistance of the system to deformation and failure (5,6). A strong interface should provide sufficient stress transfer between the individual laminates to allow the applied loads to be transferred and accommodated. Conversely, a weak interface will frequently result in failure by a process of delaminating under an applied load possibly arising from crack initiation and propagation within and along the layer (7). These bilayered composites have attracted considerable attention from laboratory researchers seeking to understand the failure characteristics of ceramic fused to metal systems. Alterations to the interfacial region between bilayered structures are of considerable interest and authors have reported the effects of variations in the interfacial surface roughness on the mechanical properties of metal-ceramics specimens (8). By improving a final surface treatment of metal substructure could be significantly improved functional durability of metal-ceramic appliances. Oxidation heat treatment of the metal is used to remove the entrapped gas, eliminate surface contaminants, and form the metal oxide layer. An alloy is deliberately given an oxidation treatment prior to ceramic application, or whether it oxidizes during the portion of the firing cycle before flow of the ceramics begins, the fusing ceramics comes into immediate contact with oxide rather than with metal surface (9,10). Different opinions exist as to how this oxide interacts with ceramic during the firing cycle. It is widely believed, that the fusing ceramic dissolves away the oxide originally formed and produces an interaction zone responsible for the formation of a bond (11). King rejected the oxide layer theories extend at that time (Kauzt 1936.) which postulated that a layer of oxide adherent to the metal is wetted by the ceramics and becomes the transition zone between the metal and glassy matrices (12). Pask (13) otherwise suggest a direct chemical bonding between the ceramic and metal. According to Mackert (11) the chromium-containing alloys all contain oxygen-active elements: beryl, aluminium, vanadium, titanium, and/or yttrium. Boron oxide makes these alloys self-fluxing during melting and gives them unique melting and casting behaviour. The addition of aluminium to these alloys adversely affects this behaviour because of its tendency to produce slag (14). Because of the close correspondence between oxide adherence and ceramic bonding, it can only be concluded that the adherence of the oxide plays a dominant role in ceramic bonding (11). The aim of this study was to investigate the influence of different cast surface finishing process on metal-ceramics bond strength. Table 1. Procedures of metal surface treatment for each group of samples Specimen Metals surface treatment 1 Sand blasting with 110 μm Al2O3 particles Sand blasting with 110 μm Al2O3 particles Oxidation Sand blasting with 110 μm Al2O3 particles Sand blasting with 250 μm Al2O3 particles Oxidation Sand blasting with 250 μm Al2O3 particles Sand blasting with 110 μm Al2O3 particles Extended oxidation Sand blasting with 110 μm Al2O3 particles Sand blasting with 110 μm Al2O3 particles Oxidation 5 Sand blasting with 110 μm Al2O3 particles Bonding agent 6 Etching in acid mixture 236

97 Mehulić et al Surface finishing and bond strength MATERIALS AND METHODS Six groups of same three metal cast plates, mm have been produced according to the manufacturer s instructions. The used alloy (Wirobond C, BEGO, Bremen, Germany) (weight percentage: Cr 26%, Mo 6 %, W 5 %, Si 1 %, Fe 0.5 %, Ce 0.5 %, C 0.02 %, and the rest Co) belongs to the group of cobalt-chrome alloys free of the contents of beryllium and nickel. Thus produced samples are cleaned and handled in same direction, and the surfaces to which ceram- Sample 1 Sample 2 Sample 3 Sample 4 Sample 5 Sample 6 Figure 1. Specimens surface of the sample prepared and recorded by a scanning electronic microscope (SEM) with the secondary electron detector (SE) (Faculty of Mechanical Engineering and Naval Architecture, University of Zagreb, Croatia, 2008., with permission) 237

98 Medicinski Glasnik, Volumen 6, Number 2, August 2009 ics is applied are treated by different procedures and combinations of procedures (Table 1). Sandblasting was achieved with 110 and 250 μm Al2O3 particles (Shera, Lemförde, Germany). The used bonding agent (3C-Bond, Al- Sample 1 Sample 2 Sample 3 Sample 4 Sample 5 Sample 6 Figure 2. Results of 3-point bending test performed on six groups of specimens 238

99 Mehulić et al Surface finishing and bond strength phadent N.V., Antwerpen, Belgium) is applied to the samples in group 5. The samples of group 6 are kept in the solution obtained by mixing 50 ml of distilled water and 50 ml of 32% hydrochloric acid for 30 minutes. After etching these samples are first of all washed in distilled water, and then in the compound of ethyl alcohol and acetone in ratio 1:1. Figure 1 shows the characteristic surface of the sample prepared in this way recorded by a scanning electronic microscope (Tescan Vega TS5136LS, Tescan, Brno, Czech R) with the secondary electron detector (SE). Along the middle of thus prepared metal plates the ceramics (Duceram Kiss, DeguDent, Hanau-Wolfgang, Germany) is fired (ceramic furnace Focus 2006, Shenpaz, Tel Aviv, Israel) in the length of 8 mm, width of 3 mm, and thickness of 1 mm. The ceramics corresponds to the manufacturer s instructions and belongs to the group of ceramics with the fired temperature of up to 980 C, suitable for coating of the mentioned alloy. The samples are tested by bending in three points on the tester machine (LRX Lloyd Instruments, Fareham, Great Britain) with installed Nexygen programme for the processing of results. The samples are set so that the surface with ceramics is turned opposite to the pin, and the metal part resting on the supports at a distance of 20 mm, and the diameter of pin that loads the sample is 1 mm. The shift of pin is constant during testing at a speed of 1.5 mm/min, and the testing continues until the fracture, i.e. to full separation of the ceramics from the metal. Testing procedure has been carried out according to the guidelines given in ISO 9693 (15). After testing the samples type of fracture surfaces (cohesive, adhesive or cohesive-adhesive) were examined by scanning electronic microscope (Tescan Vega TS5136LS, Tescan, Brno, Czech R). The same person has performed all the tests. The multiple range tests, Fischer s LSD test and ANOVA have been used for statistic analysis. RESULTS The results of 3-point bending test performed on 6 groups of specimens, (each group has three specimens) are presented in Figure 2. The diagrams obtained by testing on the tester and presented in Figure 2 show the same trend, i.e. the behaviour of all samples during testing is inter-compatible. Therefore, Figure 3 can generally explain the behaviour of all metal-ceramic systems in a three-point flexure bond test. According to Figure 3 it is possible to define three characteristic areas during testing. The beginning of testing where the force-deflection diagram is a horizontal line, i.e. the pin is lowered without increase of force, represents the first area. Such behaviour is caused by preparation of testing and represents the period from beginning of testing to the moment of achieving the predefined pre-load. Point A (Figure 3), where a sudden increase in force is noticed, represents the moment of contact between the pin and the sample and the actual beginning of the testing area 2. The linear part of the diagram that follows from this point represents common resistance to flexing of the metal-ceramic sample, since in this area the bond between metal and ceramics is still strong. Figure 3. Typical areas during three-point bending test Point B (Figure 3) represents the start of the third area, i.e. the moment of loosening of the bond between metal and ceramics and the moment at 239

100 Medicinski Glasnik, Volumen 6, Number 2, August 2009 which ceramics starts to get separated from metal. After point B, there is a short relaxation of the material, which is reflected in the decrease of force with simultaneous increase of deflection. After this relaxation the force-deflection diagram corresponds to the diagram for metal alone. Based on the performed analysis of the results it may be concluded that in order to make valid conclusions on the strength of the bond between the metal and the ceramics point B is the most important one, i.e. force and deflection in which the ceramics starts to get separated. Figures 4 and 5 show the diagrams of these values. The method of separation of the ceramic layer in the performed tests is almost equal for all the groups of samples. The separation always starts at the end of the sample and propagates towards the middle, which corresponds to the guidelines of standard HRN EN ISO In Figure 4, which shows the deflection that has resulted in the separation of ceramics from the metal frame, one may notice that the mean values of deflection in all the samples are approximate and range from 0.07 mm (sample 6) to 0.17 mm (sample 1). Figure 5 shows that the highest mean value of force at which ceramics separation was recorded in sample 3, whereas the minimal mean value of force is recorded in sample 6. The forcedeflection diagrams make it possible to quantify the difference in the bond strength of the tested system based on the additional parameters. The analysis of variance has been used to determine characteristics of the difference between the samples (p < 0.05) for load only, because the separation of ceramics in all the samples occurs in the approximate amount of deflection. The arithmetic means of forces significantly differ among individual groups of samples at a risk of 5%. In sample 3 the force at which the separation of ceramics comes is significantly greater compared to other tested samples. In sample 6 the bond between ceramics and metal fractures at significantly lower forces than in all the other samples. Sample 5 treated with bonding agent shows on the overall fracture area the presence of a layer, which corresponds to the fired agent. The value of force necessary to separate the ceramics from metal in this sample is not substantially different. DISCUSSION An understanding of the bonding mechanism is essential for successful metal-ceramic restorations. Although number theories and concepts have been proposed for the actual mechanism of bonding, it still remains elusive. Different tests have been used to determine metal-ceramic bond strength and beam failure loads (16). Though it is difficult to accurately quantify real bond strength, the 3-point flexural test is frequently used. Flexural tests were subjected to criticism because maximal tensile stresses were created the surface of the ceramics and resulted in predictable tensile failures. The validity of these tests to evaluate different alloys has been questioned because ceramic breakage depended on the modulus of elasticity of the metal tested. An alloy with an elevated modulus of elasticity would resist flexural to a greater extent, Figure 4. Deflection values (during a separation of ceramics from the metal frame) Figure 5. Load in which ceramics were separated 240

101 Mehulić et al Surface finishing and bond strength creating a higher bond (17). It is difficult to quantify the real bond strength because in vitro testing is not usually in correlation with ceramic breakdown in function. The shear strength of the metalceramic bond was evaluated by the Shell-Nielsen test described by Dent (18) method similar to that used by Anthony (19), Moffa (20), Diaz (21), Anusavice (22), Warpeha (23), Miller (24), Riley (25). The authors suggested that the differences in oxide composition and amount, influenced by different surface finishing procedures. Sandblasting the finished surface is though to remove furrows, overlaps, and flakes of metal created by the grinding process. A sandblasted surface may have higher surface energy that alloys increased wetting of the metal during ceramic application. Evidence suggested that this roughened surface could also provide mechanical interlocking and increase the surface area for metal-ceramic bonding (26). According to Brantley (27), oxide layer is different before and after sandblasting. Graham (28) suggested final finishing process in the order: sandblasting, grinding, sandblasting and oxidation. Smoother surface achieved the lowest values of bond strength and bonding agent did not improve bond strength because of hermetical sealing of cast surface (29). It created alumina layer on cast surface and thus change oxide ratio on it (30). The gold rich bonding agent reduced the interfacial stress by improving the compatibility between ceramic and metal (31). Basic elements oxidised selective; and created Fe 2 O 3, In 2 O 3 i SnO 2 on cast surface (32). The amount of oxides is not always in proportions with elements, which were added. Rake (33) suggested opaque in two layers on unutilised surfaces. In Ni-Cr alloy (34) and alloy with Pd (35) ceramic fired in vacuum produced excessive amount of oxides, and argon reduced their appearance. The most reliable evaluation of metal-ceramic bond strengths should be based on minimal experimental variables and least residual stresses at metal-ceramic interfaces. Evaluation for types of metal-ceramic failures is critical even though cohesive failures within ceramic have been an indication of clinically acceptable metal-ceramics bond. Although laboratory studies offer predictable guidance to comprehensive selection of materials, clinical longitudinal studies should also be encouraged to complement laboratory results and enhance clinical standards (17). It can be concluded that the analysis of all the parameters used in assessing the strength of the bond between metal and ceramics has confirmed that the bond is the strongest in the surface treatment procedure sandblasting with 250 µm Al 2 O 3, oxidation, and sandblasting again with 250 µm, and significantly weaker in the etched sample. It should be noted that, in spite of the recommendations of the producer of materials and the usual practice, the application of the bonding medium has not shown any influence on the bonding strength of the tested metal-ceramic system. The metal samples revealed an adhesive mode of failures on the most part of surface, and adhesivecohesive on the edges. ACKNOWLEDGEMENT Grant No , and No from the Ministry of Science, Education and Sports of the Republic of Croatia supported this work. Competing interests: none declared. REFERENCES Mehulić K, Čvrljak Tomić I, Schauperl Z, Komar D. Wear Characteristics of Esthetical Prosthetic Materials. Acta Stom Croat 2006; 40:56-64 Mehulić K. Glassceramics. Acta Stom Croat 2005; 39: Musić S, Živko-Babić J, Mehulić K, Ristić M, Popović S, Furić K. Microstructure of leucite glass-ceramics for dental use. Materials Letters 1996; 27: Fleming GJ, Nolan L, Harris JJ. The in-vitro clinical failure of all-ceramic crowns and the connector area of fixed partial dentures: the influence of interfacial surface roughness. J Dent 2005; 30: Thomson GA. Influence of relative layer height and testing method on the failure mode and origin in a bilayered dental ceramic composite. Dent Mater 2000;16:

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103 ORIGINAL ARTICLE Use of digital photography in the reconstruction of the occlusal plane orientation Nikola Petričević 1, Marko Guberina 2, Robert Ćelić 1, Ketij Mehulić 1, Marko Krajnović 2, Robert Antonić 3, Josipa Borčić 4, Asja Čelebić 1 1 Department of Prosthodontics, School of Dental Medicine, University of Zagreb, Zagreb, 2 Private Dental Practice, Zagreb, 3 Department of Prosthodontics, School of Dental Medicine, University of Rijeka, Rijeka, 4 Department of Oral and Maxilofacial Surgery, School of Dental Medicine, University of Rijeka, Rijeka; Croatia ABSTRACT Aim This study evaluated whether the occlusal plane measurements on digital photographs were reliable for the reconstruction of occlusal plane. Corresponding author: Nikola Petričević, Department of Removable Prosthodontics, School of Dental Medicine, University of Zagreb Gundulićeva 5, Zagreb, Croatia Phone ; Fax.: ; petricevic@sfzg.hr Original submission: 13 September 2008; Revised submission: 11 December 2008; Accepted: 12 January Methods Forty-two subjects (25 female and 17 male subjects, aged 19 to 30 years) with all teeth and Angle Class I participated. Irreversible hydrocolloid impressions were made and the casts were poured in dental stone (ISO Type I) and finally mounted in the S.A.M. 2 P, articulator (S.A.M. Praezisiontechnik, GmbH, Munich, Germany) by a quick mount face-bow transfer. Lateral digital photographs were taken from a distance of 1.5 m in a natural head position with a subject in erect posture. A Fox plane was placed over the maxillary dental arch. A quick-mounting face-bow was positioned. The angles between the articulator horizontal plane and the occlusal plane (AHP-OP), as well as those between the face bow and the Fox plane (FB-FP) were measured, and the significance of the difference between the means was tested by the t-test (p<0.05). Results The mean value of AHP-OP angle was 8.56 ± 3.1 degrees and the mean value of FB-FP angle was 8.80 ± 4.2 degrees. There was no significant difference between the male and the female subjects (p<0.05). There was no significant difference between AHP- OP and FB-FP angles (p<0.05). Conclusion Measurements of occlusal plane inclination from digital photographs could be helpful in future prosthodontic reconstruction treatment. Key words: occlusal plane, inclination, prosthodontic, digital photograph, Angle Class I Med Glas 2009; 6(2):

104 Medicinski Glasnik, Volumen 6, Number 2, August 2009 INTRODUCTION Correct occlusal plane orientation in prosthodontic reconstructive treatments is one of the most important factors for the stability of the removable dentures and for the achievement of good esthetics, phonetic and masticatory function, as well as for the patient s satisfaction (1-8). A faulty orientation of occlusal plane will jeopardize the interaction between the tongue and the buccinator muscle. Where the occlusal plane is too high, the tongue cannot rest on the lingual cusps of the mandibular denture teeth and prevent its displacement. There is also a tendency for accumulation of the food in the buccal and lingual sulci (4). An occlusal plane that is too low could lead to the tongue and cheek biting (9). When the occlusal plane orientation is lost by complete or partial edentulism, it should be relocated correctly by means of a prosthodontic restoration. Over the last century, investigators have used various methods and advocated many anatomical landmarks to set a correct occlusal plane orientation and position to be able to set artificial denture teeth appropriately (1-5, 9-26). A record of the occlusal plane orientation of an individual with natural teeth should be helpful in any reconstructive treatment. Therefore, the position of the occlusal plane in both, fixed and removable denture wearers could be as close as possible to the position which was previously occupied by the occlusal plane of the natural teeth. This also enables better denture stability and decreases patients adaptation to dentures (27-29). Recent developments of digital photography and wide use of personal computers have made these techniques and equipment widely available. Photographic analysis of craniofacial characteristics has already been used in dentistry, mainly in orthodontics, and it is considered to be acceptably reproducible (30-35). The aim of this study was to check the reliability of measurements on the digital photographs for possible reconstruction of occlusal plane in the future. PATIENTS AND METHODS Forty two subjects (25 female and 17 male subjects, aged between 19 and 30 years, average 24 years) were selected from dental students at the University of Zagreb, Croatia, according to the following eligibility criteria: complete natural dentition (except for occasionally missing third molars) and normal occlusion (bilateral Angle Class I molar and canine relation). All subjects were well-informed and gave a written consent to participate in the study. The study was approved by the Institutional Ethics Committee. Irreversible hydrocolloid impressions of the maxillary and mandibular jaw were made (Alginoplast fast set, Heraeus Kulzer, Hanau, Germany) and the casts were poured in dental stone (ISO Type I, Vel-Mix Stone, Kerr Italia S. p. A., Salerno, Italy). The casts were mounted in the S.A.M. 2 P articulator (S.A.M. Praezisiontechnik, GmbH, Munich, Germany) through a transfer with a quick-mounting face-bow. Prior to measurement a transparent triangular plate was placed over the maxillary teeth, so that the contacts of cusps of the maxillary posterior teeth with a transparent triangular plate could be observed. Most often, the first posterior contact was between the triangular plate and the mesiopalatal cusp of the first molar. In 3 subjects the first posterior contact was between the triangular plate and the premolar palatal cusp. The occlusal plane was defined on the cast of the maxillary jaw as the plane connecting the incisal edge of the maxillary central incisors and the mesiopalatal cusp of the left maxillary first molar (or the first cusp of the posterior teeth in contact with a transparent triangular plate placed over the maxillary teeth). The following was measured: the vertical distance between the incisal edge of the maxillary central left incisor and the articulator horizontal plane, the vertical distance between the mesiopalatal cusp of the first left maxillary molar (or the first cusp of the posterior teeth in contact 244

105 Petričević et al Occlusal plane and digital photography with a transparent triangular plate) and the articulator horizontal plane, as well as the horizontal distance between the incisal edge of the left maxillary central incisor and the mesiopalatal cusp of the first left molar (or the first cusp of the posterior teeth in contact with a transparent triangular plate). Measurements were made with a calliper of 0.1 mm precision (MEBA, Zagreb, Croatia). Values obtained were transferred to a sheet of paper, calibrated in millimeters, and the lines were drawn. The occlusal plane was drawn and the angle (AHP-OP angle) between the horizontal line (representing articulator horizontal plane) (AHP) and the occlusal plane (OP) (representing the distance between the left maxillary central incisor and the first lateral cusp in contact with a transparent triangular plate, mostly mesiopalatal cusp of the first maxillary molar) was measured to the nearest 0.5 degree mark. Lateral digital photographs of each subject were obtained in accordance with the following procedure: A quick-mounting face bow of the S.A.M. articulator was placed on the subject s face; olives (plastic auriculars) were gently introduced into the meatus accousticus externus and the arch was fixed on the soft nasion. The Fox plane (Candulor AG, Wangen, Switzerland) was also placed in the mouth (Fig. 1). The subjects were standing barefoot on the ground in front of a mirror, with his/her feet slightly apart and divergent externally, and both arms hanging loosely. Following that, the subject was asked to look straight into the mirror (1.5 m x 0.5 m) at the reflection of his/her pupils and to assume a relaxed and normal erect posture of the head and shoulders. This is considered to be a natural head position (NHP) (22, 23). The mirror was positioned 1.5 m away, in front of the subject. All digital photographs were taken from a distance of 1.5 m with the subject standing, clenching the Fox plane and with the facial arch positioned. Digital photographs were obtained by using a digital camera (Fuji Finepix A310, 3.1 Megapixel 3x Optical/2.9x Digital Zoom) on an adjustable tripod (Manfrotto Tripod Digi MN714- SHB) conveniently adjusted so that the camera was at the height of the subject s ala-tragus line. The images were transferred by an USB cable to a personal computer in JPEG format. The ISSA computer program was used for direct measurements on the screen (VAMS, Zagreb, Croatia): the grid-lines were drawn by superimposing the Fox plane (FP) and the face-bow plane (FB); and the angle between FP and FB planes was measured (FB-FP angle). Statistical analysis included testing the normality of distribution by the one sample Kolmogorov-Smirnov test and descriptive statistics. The significance of the differences between males and females was assessed by the independent Student s t test. The significance of the differences between the occlusal plane inclination measured in the articulator and on the digital photographs was tested by the Student s t test for dependent samples. The significance was set at 95% probability level. RESULTS The data was distributed normally, as revealed by the one-way Kolmogorov-Smirnov test (p>0.05). Therefore, parametric tests were used for further statistic analysis. There was no significant difference between men and women (for AHP-OP angle: t = 0.81, d.f. = 40, p = 0.412; for FB-FP angle: t = 1.16, d.f. = 40, p = 0.23), as revealed by the independent Student s t test. Descriptive data (x ± SD) is shown in the Table 1. There was no significant difference between the articulator AHP-OP angle (angle between the articulator horizontal plane and the maxillary Table 1. Angle between the horizontal plane and the occlusal plane* Angle x SD No AHP-OP FB-FP *AHP-OP, angle measured after mounting in the articulator; FB-FP, angle measured on digital photographs; x, mean; SD, standard deviation; No, number of measurements 245

106 Medicinski Glasnik, Volumen 6, Number 2, August 2009 occlusal plane), and the digital photograph FB- FL angle (between the Fox plane and the quickmounting face bow plane) (p>0.05) (Table 2). DISCUSSION One of the major problems in prosthodontics therapy is the lack of reproducible referencestructures for determining orientation and position of the occlusal plane. Not only did different authors use different landmarks and methods to establish the occlusal plane, but also the definition of the occlusal plane varied throughout the literature (1-5, 9-26). Although almost all textbooks on prosthodontics advocate the orientation of the occlusal plane parallel to the Camper s line, many authors found significant differences between the orientation of the natural occlusal plane and the ala-tragus line (4,11,13,18,22,26,31). Another widely used landmark for the establishment of the artificial occlusal plane is the retromolar pad, although it was found out that orientation upon retromolar pad could place the occlusal plane a little too low posteriorly from the natural occlusal plane (3,20). Nissan (4) concluded that the cephalometric analysis alone cannot determine the location of the occlusal plane in edentulous patients and, consequently, he advocated that intraoral structures, which had been described by other authors should be considered (2,3,14). Bassi concluded that the cephalometric parameters do not correspond to the clinical positioning of the posterior teeth in a successful rehabilitation with complete denture (21). Although many articles in the literature describe the establishment of the occlusal plane in completely or partially edentulous patients, none of the described methods seem to be completely satisfactory. Much of the controversy results from the small number of subjects examined, Table 2. Significance of the differences between AHP-OP and FB-FP angle* In order to test the accuracy of the inclinax diff t df p AHP-OP:FB-FP (>0.05, N.S.) *AHP-OP, measured on casts mounted in the articulator; FB-FP, measured on digital photographs; x diff, mean difference between angles; t, t-value; df, degree of freedom; p, p-value great variability of the location of anatomical structures and the lack of an agreement on the definition of the exact anatomical structures. In order not to change proprioceptive regulatory mechanisms which ensure normal function of the cheek, tongue and other masticatory muscles, establishment of the occlusal plane should be reconstructed as close as possible to the position prior to the loss of teeth,. Therefore, a record of the patient s occlusal plane orientation would be helpful in a reconstructive therapy. Recent development of digital photography and wide use of personal computers and their low cost have made these techniques and equipment widely available. Therefore, this study was made with the idea that general practitioners can easily obtain the profile digital photographs of their patients with a Fox plane in the mouth (representing the occlusal plane). Such photographs could be used for measurement of the occlusal plane inclination in a possible reconstructive treatment. Photographic evaluation of craniofacial characteristics has been already purposefully used in orthodontics and other fields of dentistry, and has showed to be acceptably reproducible in earlier studies (30-35). The method of digital photography used in this study has already been standardized (8, 30-35) and has proved to be reproducible and reliable (26, 20-35), which is in agreement with the results of this study. We placed a Fox plane and a facial arch on the face of each subject in order to obtain objective data and information valuable for a clinical practice. Great advantage of digital photography in comparison with the cephalometric analysis is that it avoids exposition of subjects to potentially harmful radiation and it was therefore used in the present study. The results of this study revealed no significant differences between genders (P>0.05) for the angle between the articulator horizontal plane and the occlusal plane (for both measurements), which is in agreement with other similar studies (3,13,17,19,30). 246

107 Petričević et al Occlusal plane and digital photography also proves that digital photographs with a subject biting on the Fox plane could be helpful in any possible future reconstructive prosthodontics procedures where the occlusal plane must be reestablished. ACKNOWLEDGEMENTS/DISCLOSURE Figure 1. Subject with a quick-mounting face bow and Fox plane positioned (left profile) (N. Petričević, 2008., with patient s permission) tion of the occlusal plane on digital photographs, the AHP-OP angle (between the articulator horizontal plane and occlusal plane) and FB-FP angle (face-bow-fox plane on digital photographs) were measured, and the significance of the differences between them was tested by using the Student s t test for dependent samples. The mean difference between the angles was only degrees, which is of no clinical relevance. However, there was no significant difference between the angles measured on the articulator and on the digital photographs (Table 1 and Table 2). This study confirms the hypothesis that digital photographs are reliable for craniometric analysis. It REFERENCES This study was partly presented as a part of: Petričević N., Čelebić A., Poljak-Guberina R., Knezović Zlatarić D., Komar D. Accuracy of digital photograph measurements in reconstruction of occlusal plane orientation. Proceeding of the 12 th Meeting of International College of Prosthodontics, Fukuoka, Japan, September 5, The authors wish to acknowledge the support of the Ministry of Science, Education and Sports of the Republic of Croatia, Project No : Influence of Prosthetic Therapy and Other Factors to Orofacial System and Health (original title: Utjecaj protetskog rada i drugih faktora na stomatognati sustav i zdravlje) and Project No : Stess, occlusal trauma and oral-surgical patology (original title: Stres, okluzijska trauma i oralno-kirurška patologija). Subject on the Figure 1 was well-informed and gave a written consent to publish photograph of his face in this journal. Competing interests: none declared Carey PD. Occlusal plane orientation and masticatory performance of complete dentures. J Prosthet Dent 1978; 39: Alfano SG, Leupold RJ. Using the neutral zone to obtain maxillomandibular relationship records for complete denture patients. J Prosthet Dent 2001; 85: Celebic A, Valentic-Peruzovic M, Kraljevic K, Brkic H. A study of the occlusal plane orientation by intra-oral method (retromolar pad). J Oral Rehabil 1995; 22: Nissan J, Barnea E, Zeltzer C, Cardash HS. Relationship between occlusal plane determinants and craniofacial structures. J Oral Rehabil 2003; 30: Williams DR. Occlusal plane orientation in complete denture construction. J Dent 1982; 10: Celebic A, Knezovic-Zlataric D. A comparison of patient s satisfaction between complete and partial removable denture wearers. J Dent 2003; 31: Celebic A, Knezovic Zlataric D, Papic M, Carek V, Baucic I, Stipetic J. Factors related to patient satisfaction with complete denture therapy. J Gerontol A Biol Sci Med Sci 2003; 58:M Wright, C.R. Evaluation of the factors necessary to develop stability in mandibular dentures. J Prosthet Dent 2004; 92:

108 Medicinski Glasnik, Volumen 6, Number 2, August Monteith, B.D. A cephalometric method to determine the angulation of the occlusal plane in edentulous patients. J Prosthet Dent 1985; 54:81-7. Foley PF, Latta GH. A study of the parotid papilla relative to the occlusal plane. J Prosthet Dent 1985; 53: Guillen GE, Staffanou RS. Occlusal plane modification of an existing maxillary complete denture prior to removable partial denture construction: a case report. Quintessence Int 1991; 22: Van Niekerk, FW, Miller VJ, Bibby RE. The alatragus line in complete denture prosthodontics. J Prosthet Dent 1985; 53:67-9. Karkazis HC, Polyzois GL. Cephalometrically predicted occlusal plane: Implications in removable prosthodontics. J Prosthet Dent 1991; 65: Rich H. Evaluation and registration of the H.I.P. plane of occlusion. Aust Dent J 1982; 27: Sloane RH, Cook J. A guide to orientation of the occlusal plane. J Prosthet Dent. 1953; 3:53 Celebic A, Brkic H, Kaic Z, Vojvodic D, Poje Z, Singer Z. Occlusal plane Orientation in Klinefelter Syndrome (47, XXY males). J Oral Rehabil 1997; 24: Vojvodic D, Celebic A, Valentic Peruzovic M, Cifrek M, Kraljevic K, Stipetic J. A study of the occlusal plane orientation by extraoral method (Camper s line). Coll Antropol 1996; 20 (Suppl.): Koller MM, Merlini L, Spandre G, Palla S. A comparative study of two methods for the orientation of the occlusal plane and the determination of the vertical dimension of occlusion in edentulous patients. J Oral Rehabil 1992; 19: Karkazis HC, Polyzois GL. A study of the occlusal plane orientation in complete denture construction. J Oral Rehabil 1987; 14: Ismail YH, Bowman JF. Position of the occlusal plane in natural and artificial teeth. J Prosthet Dent 1968; 20: Bassi F, Deregibus A, Previgliano V, Bracco P, Preti G. Evaluation of the utility of cephalometric parameters in constructing complete denture. Part I: placement of posterior teeth. J Oral Rehabil 2001; 28: Petričević N, Stipetić J, Antonić R, Borčić J, Strujić M, Kovačić I, Čelebić A. Relations between Anterior permanent teeth, dental arches and hard palate. Coll Antropol 2008; 32: Rener-Sitar K, Petričević N, Čelebić A, Marion Lj. Psychometric properties of Croatian and Slovenian short form of oral health impact profile questionnaires. Croat Med J 2008; 49: Petričević N, Čelebić A, Ibrahimagić-Šeper L, Kovačić I. Appropriate proportions as guidelines in selection of anterior denture teeth. Med Glas 2008; 5: Ibrahimagić-Šeper L, Čelebić A, Petričević N, Selimović E. Anthropometric differences between males and females in face dimensions and dimensions of central maxillary incisors. Med Glas 2006; 3: Petricevic N, Celebic A, Celic R, Baucic-Bozic M. Natural head position and inclination of craniofacial planes. Int J Prosthodont 2006; 19: Celebic A, Valentic-Peruzovic M, Alajbeg ZI, Mehulic K, Knezovic Zlataric D. Jaw elevator silent periods in complete denture wearers and dentate individuals. J Electromyogr Kinesiol 2008; 18: Celebic A, Alajbeg ZI, Kraljevic-Simunkovic S, Valentic-Peruzovic M. Influence of different condylar and incisal guidance ratios to the activity of anterior and posterior temporal muscle. Arch Oral Biol 2007; 52: Alajbeg IZ, Valentic-Peruzovic M, Alajbeg I, Illes D, Celebic A. The influence of dental status on masticatory muscle activity in elderly patients. Int J Prosthodont 2005; 18: Ciancaglini R, Colombo-Bolla G, Gherlone EF, Radelli G. Orientation of craniofacial planes and temporomandibular disorder in young adults with normal occlusion. J Oral Rehabil 2003; 30: Ferrario VF, Sforza C, Serrao G, Ciusa V. A direct in vivo measurement of the three-dimensional orientation of the occlusal plane and of the sagittal discrepancy of the jaws. Clin Orthod Res 2000; 3: Ferrario VF, Sforza C, Miani A, Tartaglia G. Craniofacial morphometry by photographic evaluations. Am J Orthod Dentofacial Orthop 1993; 103: Ferrario VF, Sforza C, Germano D, Dalloca LL, Miani A. Head posture and cephalometric analyses: an integrated photographic / radiographic technique. Am J Orthod Dentofacial Orthop 1994; 106: Chiu CS, Clark RK. Reproducibility of natural head position. J Dent 1991; 19: Celebic A, Stipetic J, Nola P, Petricevic N, Papic M. Use of digital photographs for artificial tooth selection. Coll Antropol 2004; 28:

109 ORIGINAL ARTICLE A three-dimensional evaluation of microleakage of the class V cavities restored with flowables Paris Simeon 1, Silvana Jukić-Krmek 1, Goranka Prpić-Mehičić 1, Ivica Smojver 2, Ivica Anić 1, Ivica Pelivan 3 1 School of Dental Medicine, University of Zagreb, Department of Endodontics and Restorative Dentistry, 2 Faculty of Mechanical Engeneering and Naval Architecture, 3 School of Dental Medicine, University of Zagreb, Department of Prosthodontics; Zagreb, Croatia ABSTRACT Aim To evaluate the dye leakage of three flowables with a new three dimensional method. Corresponding author: Paris Simeon School of Dental Medicine, University of Zagreb, Department of endodontics and restorative dentistry, Gundulićeva 5, Zagreb, Croatia Phone: ; Fax: paris.simeon@zg.t-com.hr Original submission: 16 March 2009; Revised submission: 05 May 2009; Accepted: 06 May Methods Three groups of twelve premolars have received Class V cavities of uniform standardized dimensions. The cavities were restored with: Clearfil liner Bond 2 with adhesive SE Bond (group I), Definite flow with Etch & Prime 3.0 (group II) and, the Tetric flow with Syntac Single Component (group III). The teeth were immersed in a contrast dye for 24 hours, rinsed and immersed in the 5% nitric acid for 72 hours. The teeth were acid-softened and the fillings were pulled out of their cavities. The inner surfaces of extracted fillings were photographed in three different rotated positions (3 x 120 o ). The photographs were transferred to a computer image and the deepest point and the area of the leaked surface were determined. Results All restorative systems showed leakage at the adhesive level. The statistical analysis showed significant differences between group I and group III in the number of leaked samples, in the depth of leakage and in the leaked surface. Best results in all three ways of leakage evaluation were obtained in group I (Clearfil liner Bond 2 with adhesive SE Bond). Conclusion In this study, flowable Clearfil liner Bond 2 with adhesive SE Bond had leaked in all three ways of evaluation significantlly less than Tetric flow in combination with Syntac Single Component. Key words: microleakage, dye leakage, class V restorations, flowable resins Med Glas 2009; 6(2):

110 Medicinski Glasnik, Volumen 6, Number 2, August 2009 INTRODUCTION Restorative materials and procedures often fail to produce an intact marginal integrity (1). The cervical region cavities are burdened with a highly stressful position and with a non-homogenous tooth structure (2). The cervical cavities have also a stressful cavity configuration and are usually restored with resin restoratives which exhibit shrinkage during polymerization causing failures (3). Marginal gaps between restoratives and the cavity walls are a consequence of such a failure, thus causing leakage (4). Nonetheless the properties of flowable composites make them suitable as a restorative material for this highly stressful region (5,6). The researches of marginal leakage usually describe a two-dimensional, linear and very unclear pattern of microleakage (7). Dye leakage is the oldest and most common method of detecting a leakage in vitro. The main disadvantages of this method are its qualitative nature and the fact that damage to the sample occurs due to sectioning, eventually leading to false results (8,9). The ideal way of understanding would be if we could somehow extract the filling undamaged from its cavity and see the leakage in its full physical appearance. The aim of this study was to investigate and evaluate the leakage of three flowables with a new three dimensional method which enables the extraction of the filling out of the cavity and the direct measurement of the died surface and maximum depth of the leakage. Thus the dye leakage becomes a fully quantitative method. MATERIALS AND METHODS Thirty-six intact premolars, extracted for orthodontic reasons were randomly divided in three groups of twelve each. All teeth have received buccal cervical Class V cavities of uniform standardized dimensions (3 x 2 x 1.5 mm) with margins in enamel and cementum. These cavities were prepared with a diamond-coated bur (# ,2ML, Diatech SDI, Switzerland) mounted on a water-cooled air-driven handpiece. The specific shape of the bur enabled easy, almost automatic, preparation of the cavity simply by pressing the rotating bur at the previously determined cervical buccal position of each tooth. The same bur was used for 12 cavities and replaced with a new one. Following the preparation prior to the restoration groups 1 and 2 required no additional etching because of the self-etching primer included in their adhesive systems. Cavities of the group 3 were additionally etched for 20 seconds with 37% ortophosphoric acid. All cervical cavities were restored according to the manufacturer s instructions. Restorative material used in this research consisted of three groups of adhesive restorative systems produced by three different manufacturers. Each group was restored with the - respectively - sameproducer flowable restorative and same-producer adhesive system, as follows: Group I Group II SE Bond (Lot.41116) and Clearfil liner Bond 2 (Lot 0045A) (Kuraray, Osaka, Japan); Etch & Prime 3.0 (Lot C) and Definite flow (Lot.12003B) (Degussa, Hanau, Germany); Group III Syntac Single Component (Lot D33562) and Tetric flow (Lot. E38161) (Vivadent, Schaan, Liechtenstein). The cavities were restored in one single injected layer of flowable composite and subsequently polymerized for 40 seconds all with the same-type Elipar II (ESPE, Germany) halogen light device. All restorations were finished and polished. The specimens were then thermally cycled for 1000 cycles in cold (5 o C) and warm (55 o C) baths with dwell times of 60 s and the transfer time of 10 s. After thermocycling, the root surfaces and the adjacent enamel were coated with two layers of nail varnish, except the filling surface and the area 1 mm around the filling. The specimens were then immersed in a contrast liquid (acid resistant - Rotring Ink, Stanford GmbH, Hamburg, Germany) for 24 hours, rinsed with tap water, and immersed in 5% nitric acid. After

111 Simeon et al Three-dimensional evaluation of microleakage hours the teeth were softened enough to pull the fillings out from their cavities with ease, just with a sharp excavator. The extracted fillings have thus become the specimen ready for observation. Leakage pattern was observed and measured through a dissecting microscope equipped with a digital camera (Olympus SZX-12 and Olympus DP-12, Olympus Optical Co.GmbH, Hamburg, Germany). Photographic images of the inner surface (tooth-faced) of the specimens were taken in three different positions, the different position meaning a one-third rotation (for 120 o ) in order to encompass the entire filling mantle (360 o ). The photographs were then transformed into computer images. A CAD (computer-aided design) computer program was used to observe the leakage pattern and to measure died surfaces. The first method of assessment was to measure the maximum depth point of leakage, and ordinal rating scores or levels - ranging from 0 to 3 - were attributed to the marginal dye leakage. The ordinal rating scores are defined as follows (Figure 1): The second method of leakage evaluation was to measure the inner surface of the filling colored with the contrast dye on three different surfaces of each specimen. The colored surface was measured in mm² and the total inner surface of (specimen s) filling s mantle was totaling 15.1 mm². For the statistical analysis of the depth of microleakage the highest (maximal) result measured in one specimen was used. For the analysis of surface of microleakage the sum of the leaked area from all three differently angled images was calculated. As a post-hoc test Kruskal-Wallis with Mann-Whitney U tests were used. RESULTS The observation showed that all restorative systems had leaked at the adhesive level, e.g. between the restorative material and the walls of the prepared cavity (Figure 2). The values of the deepest point and the area of leakage of all tested samples and materials are shown in Table 1. In Group I only one (1) restoration (8%) had leaked, in Group II four (4 viz. 33%) restorations, and in Group III seven (7 viz. 58%) restorations Figure 1. The scheme of a specimen with marked levels (in order to measure the point of leakage) (P. Simeon, 2005.) score 0 no leakage; score I leakage deep up to 1/3 of internal surface (up to 0.71 mm); score II leakage deep up to 2/3 of internal surface (up to 1.42 mm); score III leakage deep through entire lateral surface and filling s bottom (up to 2.14 mm). Figure 2. Filling from the Group II specimen No 3, with dye leakage in surface 1 and level of depth 2. Arrows are pointing the area of marginal leakage and the triangle is pointing the maximal depth of leakage. The blue and red marks are the orientation marks. (P. Simeon, 2005.) 251

112 Medicinski Glasnik, Volumen 6, Number 2, August 2009 out of total of 12 respectively. Comparing the frequency of leaked samples, a statistically significant difference was found between Group I and Group III (χ 2 = 6.75; df = 1; p = 0.009). Considering the area of the leaked surface, a statistically significant difference was found between the groups (χ 2 = 6.34; df = 2; p = 0.042). The Mann-Whitney test showed a significant difference between Group I and Group III (U = 3; Z = 2.58; p = 0.010). The ranking of the groups according the mean range of the Kruskal Wallis test showed the best range of the Group I (s.r. = 13.88), then Group II (s.r. = 18.67) and, as the last, the range Group III (s.r = 22.96). This means that Group III had a larger area of the leaked surface than Group I, but it could not be said of the leaked area of Group II to be larger than Group I or smaller than Group III. Comparing the three groups of material according to the deepest point of leakage, the Kruskal Wallis test showed a difference between the groups (χ 2 = 5.99; df = 2; p = 0.050). The Mann-Whitney test showed the statistically significant difference between group I and III (U = 36.5; Z = 2.44; p = 0.015). The differences between Group I and Group II, and Group II and Group III were not statistically significant. The ranking of groups according to the mean range of the Kruskal Wallis test showed the best range of the Group I (s.r. = 14.04), then Group II (s.r. = 18.58) and, as the last, Group III (s.r = 22.88). DISCUSSION Adhesive restorative dentistry today has not - in the clinical environment - accomplished its goal i.e. the optimal adhesion that would totally endure the stress forces generated in the cervical area of the tooth (8,9). The capability of the restorative materials to seal the restoration borders is influenced by the resin composition and the filler, the material s plastic deformability and ability to flow, the thermal expansion coefficient, the Young modulus, the choice of the enameldentin adhesive system and restorative technique used, the mechanical stress due to the cavity shape (8), and eventually the quality of the hard dental tissue (8,9). Table 1. Microleakage of flowable restorative materials according the leaked surface and deepest point of leakage for each specimen that expressed leakage Flowable restorative material Group I Group II Group III Specimen that leaked Spec. No. 1 Spec. No. 2 Spec. No. 3 Spec. No. 6 Spec. No. 7 Spec. No. 1 Spec. No. 2 Spec. No. 3 Spec. No. 4 Spec. No. 5 Spec. No. 6 Spec. No. 7 *the maximal depth of microleakage used for statistical analysis Leaked surface (mm2) Total leaked surface Deepest point of leakage (mm) Surface 1 Surface 2 Surface 3 (mm2) Surface 1 Surface 2 Surface * Level * Level Level 2 *1.78 Level 3 *0.62 Level 1 *0.20 Level 1 *0.81 Level 1 *0.92 Level 2 *1.10 Level Level 2 *1.76 Level 3 *2.14 Level 3 *2.14 Level Level Level Level Level 2 252

113 Simeon et al Three-dimensional evaluation of microleakage The flowable composite, because of its injectability in the cavity, surpasses one of composite s clinically less practical characteristics (stickiness for example) (10,11). It is a kind of material capable of flowing like honey with a lower Young modulus (11) which combines the good characteristics of the hybrid composites and their applicability (10). The material is therefore recommended for Class V restorations (12,13). The reason for this recommendation in the toothneck area is the non-directional loading of the occlusal and articulation forces. On the other hand, the cervical area is highly burdened depending on the stressed cavity configuration and the flexional forces (because of the tooth deflection). It is indicated because of its favorable mechanical properties of the flowables during and after the polymerization process (lower modulus, lower rigidity of the hardened material). The material s property to flow during the pre-gel phase reduces the overall tension on the hybrid layer, on the polymerized material itself, and finally on the cavity walls. The leakage of oral fluid, together with bacteria and their by-products, happens in the majority of the present-day restorations (7,14) regardless of their adhesive mediators. The microleakage comprehension and its evaluation is important because of the ability of the process itself to weaken and - finally - get the restoration lost during its clinical lifetime. The microleakage and nanoleakage research more or less objectively confirm the presence of a leakage but do not show how severe the leakage is influencing the final evaluation of a certain restorative system or material (8,15). A great part of laboratory researches objectively observe and prove the leakage by a contrast-fluid penetration (4,13). The reading of the leakage is a two-dimensional process based on the fact that a specimen is mostly sectioned or broken, or prepared to obtain several cuts thru the cavity and restoration surface, in order to see and measure the leakage. However, it is not objective, no matter how precisely it is done. The specimen used in a three-dimensional method of research (8,9) shows higher a quality of evaluation of the microleakage thru the tissue-restoration margins bond. This method enables the «reading» of the leakage of the contrast dye through marginal microcraks. Of course, there is a certain danger of non-objectivity in the use of the mentioned twodimensional procedure and the fact that a partial picture of the leakage is taken as the measure of the evaluation of a restorative material and therefore conclusions of this kind could be misleading for a reader. Further, the observation and measurement of microleakage routinely requires a dissecting microscope with a mounted digital camera (13). The leakage is measured in two ways: a maximum depth point of leakage with the associated rating scores or levels, ranging from 0 to 3, and the other way is to measure the surfaces colored with the contrast dye leak. An alternative could be to measure the concentration of the contrast dye with a spectrophotometer (4) or a similar device. This data is to be statistically analyzed and qualified conclusions to be drawn. All of the aforementioned researches are similar for the most part in their respective procedures. In order to evaluate leakage a most objective method is needed. One part of this mosaic is surely the need to understand the problem of the marginal cracks. The essential defining characteristics of the threedimensional method is to bring the elevated and more realistic value of the results because of the possibility to observe the leakage rather clearly, and because of the combination of two methods of evaluation and subsequent quantification of the leakage. The pattern of microleakage according to the research could be superficial with only slight marginal coloring. But this slight superficial marginal coloring could penetrate from this margin into the deep dentinal surfaces near the pulp with all consequences (8). This understanding of different patterns of leakage justifies the use of the two different methods of leakagemeasurement, in order to find the most objective way to evaluate the quality of a certain material. If we were to apply only one of the aforementioned methods, for example, a small edge-superficial leakage could mean only a small irrelevant dam- 253

114 Medicinski Glasnik, Volumen 6, Number 2, August 2009 age to a restoration. On the other hand, it could mean a deep thin highway straight down into the pulp. So clarified a picture of leakage enables in vitro research making better use of the contrast dye which could leave better traces on the inner surface of the restoration investigated by making it easier to observe and read. Improvement could be achieved by a better observation of the aforementioned leaked surfaces (a digital threedimensional scanner instead of a three-angled digital photography of each filling) and subsequent transfer of the data to a computer, in order to get a best-quality computer-image of the innerrestoration surfaces. The results of this research are hardly comparable to other related researches because of the difference in the methods applied. The analysis of the results of this study, according to the dual criteria, showed that statistically-significant better flowable-restoratives were Clearfil liner and ormocer Definite flow. Between these two flowables there were no statistically significant differences, although the former had showed a lesser leakage, the least of all. The question is how a resin flowable material indicated to be a lining material would eventually behave as a definitive restorative material in terms of its physical and mechanical properties. That is the reason why we give the advantage to the Definite flow ormocer definitive flowable resin. Significantly less successful was Tetric flow restorative in comparison to the Clearfil liner, but not when compared with the ormocer Definite flow. Regardless of the method of research, our results are similar to those of other related research investigating the resin composites (16,17) attributing good properties to the composites, but different from the results of those research where other materials had been more successful (18). According to Eliades (19) the clinical relevance of the formulation and testing of the dentine bonding systems is not clear and not always comparable to the clinical situation, but certainly significant and necessary as a part of the mosaic of evaluation and recommendation of restorative systems, and therefore a good indicator for the convenience of both the manufacturers and clinicians (7,14,19, 20). The quality-marks of different restoratives are vital in the clinical restorative dentistry and are the results of both in vitro and in vivo researches and the data so obtained. According to this, and by the rules prescribed by various dental associations (ADA, IADR, CRA), a clear picture of a given restorative is made. More research is needed to better illustrate the marginal leakage and to give an evaluation of the investigated flowable restoratives. ACKNOWLEDGEMENTS/DISCLOSURE Competing interests: none declared. REFERENCES Irie M, Suzuki K, Watts DC. Marginal gap formation of light activated restorative materials: effects of immediate shrinkage and bond strength. Dent Mater 2002; 18: Retief DH. Do adhesives prevent microleakage? Int Dent J 1994; 44: Van Meerbeek B, Braem M, Lambrechts P, Vanherle G. Evaluation of two dentin adhesives in cervical lesions. J Prosthet Dent 1993; 70: Aguiar FHB, Ajudarte KF, Lovandino JR. Effect of light curing modes and filling techniques on microleakage of posterior resin composite restorations. Oper Dent 2002; 27: Wattanawongpitak N, Yoshikawa T, Burrow MF, Tagami J. The effect of bonding system and composite type on adaptation of different C-factor restorations. Dent Mater J 2006; 25: De Boever JA, Mc Gall WD, Holden S, Ash MM. Functional occlusal forces: An investigation by telemetry. J Prosthet Dent 1987; 40: Rueggeberg FA. Substrate for adhesion testing to tooth structure - Review of the literature. Dent Mater 1991; 7:2-10. De Munck J, Van Landuyt, Peumans M, Poitevin A, Lambrechts P, Braem M, Van Meerbeek B.A critical review of the durability of adhesion to tooth tissue: Methods and results. J Dent Res 2005; 84: Hilton TJ. Can modern restorative procedures andmaterial reliably seal cavities? In vitro investigations. Part 2. Am J Dent 2002; 15: Bayne SC, Thompson JY, Swift EJ, Stamatiades P, Wilkerson M. A characterization of first generation flowable composites. J Am Dent Assoc 1998; 129:

115 Simeon et al Three-dimensional evaluation of microleakage Chuang SF, Liu JK, Jin YT. Microleakage and internal voids in class II composite restorations with flowable composite linings. Oper Dent 2001; 26: Frankenberger R, Lopes M, Perdigao J, Ambrose WW, Rosa BT. The use of flowable composites as filled adhesives. Dent Mater 2002; 18: Yazici AR, Baseren M, Dayangac B. The effect of flowable resin composite on microleakage in class V cavities. Oper Dent 2003; 28:42-6. Pashley DH, Sano H, Ciucchi B, Yoshiyama M, Carvalho RM. Adhesion testing of dentin bonding agents: A review. Dent Mater 1995; 11: Silveira de Araujo C, Incerti da Silva T, Ogliari FA, Meireles SS, Piva E, Demarco FF. Microleakage of seven adhesive systems in enamel and dentin. J Contem Dent Pract 2006; 7: Ernst CP, Cortain G, Spohn M, Rippin G, Willerhausen B. Marginal integrity of different resin based composites for posterior teeth: an in vitro dye penetration study on eight resin composite and compomer adhesive combinations with a particular look at the additional use of flow composites. Dent Mater 2002; 18: Jang KT, Chung DH, Shin D, Garcia-Godoy F. Effect of eccentric load cycling on microleakage of class V flowable and packable composite resin restorations. Oper Dent 2001; 26: Schneider BT, Baumann MA, Watanabe LG, Marshall GW. Dentin shear bond strength of compomers and composites. Dent Mater 2000; 16:15-9. Eliades G. Clinical relevance of the formulation and testing of dentine bonding systems. J Dent 1994; 22: Folwaczny M, Mehl A, Kunzelmann KH, Hickel R. Clinical performance of a resin modified glass ionomer and a compomer in restoring non carious cervical lesions five year results. Am J Dent 2001; 13:

116 ORIGINAL ARTICLE Oral health of the Croatian army recruits in 2001 Tomislav Badel 1, Jadranka Keros 2, Vjekoslav Jerolimov 1, Nikša Dulčić 1, Snježana Restek Despotušić 3 ¹Department of Prosthodontics, 2 Department of Dental Anthropology; School of Dental Medicine, University of Zagreb, Zagreb, 3 Dental Office, Barracks Ban Krsto Frankopan, Koprivnica; Croatia ABSTRACT Aim Oral health of Croatian Army recruits has been researched. In 2001, year-old recruits in the barracks in Koprivnica were clinically examined and asked about their health care habits. Methods The oral status of all teeth (except wisdom teeth) was described by the DMFT index (decayed, missing, and filled teeth) and compared with the FST index (filled and sound teeth). The level of the recruits restored teeth was calculated by the formula FTx100/DFT. Corresponding author: Tomislav Badel, Department of Prosthodontics, School of Dental Medicine, University of Zagreb Gundulićeva 5, Zagreb, Croatia Phone: , Fax: badel@sfzg.hr Original submission: 16 July 2008; Revised submission: 12 December 2008; Accepted: 03 March Results The research showed the average DMFT index value of The average value of the FST index was 23.56, and 47.8% of the teeth were restored. A statistically significant difference according to domicile was determined in the DT, MT, FT and FST index. The subjects from rural environments had more teeth affected by caries, and those from urban environments had more restored teeth (66.59%). The health condition of the subjects from urban environments is better (higher values of the FT index and slower cumulative distribution and statistical significance of the FST index). Conclusion The FST index is more adequate than the DMFT index for application in populations with a higher level of teeth affected by caries. The research conducted contributes to the determination of dental health of the Croatian Army recruits as well as to the organisation of optimal preventive programs. Key words: dental caries, recruits, DMFT, epidemiology Med Glas 2009; 6(2):

117 Badel et al Oral health of the army recruits INTRODUCTION Oral health assessment is based on the examination of incidence and frequency of dental caries. Its spread is determined by regional factors and dynamic migration of people and it is directly determined by nutrition, oral hygiene and type of fluoridation. Epidemiological studies of caries use methodological standards, especially the decayed, missing, and filled teeth (DMFT) index as an indicator of the cumulative effect of caries on permanent teeth during life. The data about smaller and specific groups are especially important, due to the interaction of various socioeconomic states and habits (1, 2). The system of health protection oriented towards planning and implementation of preventive measures against caries brought about the tendency of decline in caries prevalence in children and adolescents in all European countries (2-5). Oral health of recruits was a subject of many epidemiological studies carried out in Australia (6, 7), the Czech Republic (8), Denmark (9), Germany (10, 11), Italy (12), Norway (13), Switzerland (14, 15), UK (16), Turkey (17), and Croatia (18, 19). The purpose of our study was to assess oral health of Croatian army recruits by establishing the DMFT value depending on age and social communities the subjects came from. Caries was diagnosed by standard instruments, diagnostic light and Kuhhorn probe. Caries was described by the DMFT index as follows: D=decayed, M=missing, F=filled and T=teeth. Teeth with diagnosed decay (D) were classified in D 2 4 according to Marthaler (20). This clinical classification includes caries lesions with cavitations that can be identified by probing. Initial lesions were not considered. The level of restored teeth was calculated by the formula FTx100/DFT. Wisdom teeth were not examined, and the study also comprised subjects without caries (DMFT=0). The FST index (F=filled and S=sound teeth). Clinical examination included the evaluation of dental status and was performed always in the same way, starting from the lower right quadrant. The data on potential risk factors for caries were entered in a form made for this purpose, containing the living areas, and oral and hygienic habits of the subjects (number of toothbrushing per day and reasons to visit the dentist per year). No radiographs were taken. Multi-examiner training, calibration and validation courses were arranged by two independent examining teams, each consisting of a dentist and a dental nurse. Dental examinations were carried out by the authors of this study, who were calibrated between themselves by asking each other to examine the same group of recruits and to compare the findings. The statistical analysis (average, standard deviation, t-test, chi-square tests, one phase variance analysis) was performed by means of the programme STATISTICA for Windows, Release 5.5 A ( 99 Edition). Statistical significance of p<0.05 was used. PATIENTS AND METHODS Caries prevalence was examined in the Dental Clinic of the Recruits Centre in Koprivnica. The study was carried out in 2001 and comprised 505 randomly chosen recruits at the age of 19. The subjects were classified according to their area of living (urban and rural). RESULTS Inter-examiner agreement between researchers S.R.D. and T.B. was measured with the t-test statistics on 50 subjects. It showed a statistical high correlation for the DMFT index (correlation was with p<0.001) (Table 1). The mean value for the DMFT was 7.32, ranging from 0 (9.3% of the subjects) to 28 (5.0% of the subjects). There were 3.15 decayed teeth Table 1. Inter-examiner agreement between researchers for the DMFT index of 50 subjects by means of t-test* Investigators Mean Standard deviation Standard error mean T.B S.R.D *DMTF, Decayed, Missing, Filled, Teeth 257

118 Medicinski Glasnik, Volumen 6, Number 2, August 2009 Table 2. Test differences of the DMFT index and FST index in subgroups of the subjects (average and standard deviations for caries frequency) by means of variance analysis* Subgroups No of subjects DMFT DT MT FT FST Total ± ± ± ± ±4.13 Urban ± ± ± ± ±3.51 Rural ± ± ± ± ±4.28 Probability statistical test <0.001 =0.004 <0.001 <0.001 *DMTF, Decayed, Missing, Filled, Teeth; DT, Decayed, Teeth; MT, Missing, Teeth; FST, Filled and Sound Teeth (DT), 1.29 extracted and/or missing teeth (MT), and 2.88± teeth with fillings (FT). The mean value for the FST index was The average of the DMFT value for each subjects area of living is shown in Table 1. The subjects of the rural area had more decayed teeth, and the urban subjects had more teeth with fillings (DT p<0.001, FT p<0.001). The subjects of the rural area had more missing teeth (MT p=0.004) and a lower FST index (p<0.001). But there was no statistically significant difference for the DMFT index. The subjects (only 5.35%) who brush their teeth three or more times per day had significantly higher values of the FT (p=0.008) and FST index (p=0.026), and a lower DT index (p=0.007) than the subjects who brushed their teeth less than three times per day (Table 3). The subjects (only 18.2%) who visit the dentist regularly have had a lower DT index (p<0.001) than the subjects who visited the dentist irregularly during a year. The FT and FST index were also significantly higher in the subjects who visit the dentist regularly (p<0.001). There was no difference for the MT index between these two subgroups (p=0.155). The average amount of restored permanent teeth was 48.92%. The subjects from the rural environment have more teeth affected by caries and less restored teeth (33.25%), and those from the urban environment have more restored teeth (decayed, missing and filled teeth (66.59%), which was statistically significant (p<0.001). Two way analysis of variance (ANOVA) confirmed the independent statistical significance for the restored teeth and the subjects area of living (p<0.001), and according to the number of toothbrushing (p=0.048), domicile (p<0.001) and the reason to visit the dentist (p<0.001). The health condition of the subjects from the urban environment was better (higher values of the FT index and slower cumulative distribution and statistical significance of the FST index) than the subjects of the rural area. There was no statistically significant difference in the cumulative distribution of the DMFT index in relation to the subjects from different environments (Figure 1A, Figure 1B, Table 2). Figure 1. A) Cumulative distribution of the DMFT (Decayed, Missing, Filled, Teeth) index in relation to subjects from different environments; B) Cumulative distribution of the FT (Filled, Teeth) index in relation to subjects from different environments 258

119 Badel et al Oral health of the army recruits DISCUSSION Croatia is a country with characteristics of a socio-economic transition. The DMFT index is a good indicator of the degree of development and an important factor in designing and planning the national programmes of oral health starting from the earliest age (4). The data about the DMFT values in populations of recruits are relatively numerous. Morgan et al. (6) determined the DMFT values of 6.8 in Australian Navy recruits. In subjects of Australian army recruits Hopcraft and Morgan (7) determined a decline in the level of caries experience, with the mean DMFT scores for recruits aged between was For the subjects of the same age (17-25 years of age) in this study DMFT scores were only Krejsa et al. (8) established the average DMFT value of 6.22 (DT: 0.87, MT: 0.02 and FT: 5.33) in 18-year-old Czech recruits. Antoft et al. (9) found a decrease of caries of 63% in Danish recruits in the period between (DMFT 6.2. in 1993), Willerhausen et al. (10) found the average DMFT index of 13.0 in German recruits, and Klimek et al. (11) the value of 7.5. In Italian recruits the determined DMFT was 7.14 (12). Asmyhr et al. (13) revealed caries decrease in Norwegian recruits to the DMFT value of In the study of Swiss recruits Menghini et al. (14) established the DMFT values of 10.1 (DT: 4.2, MT: 0.5 and FT: 5.4), and in 1996 Menghini et al. (15) found a decline in caries of 48%, which amounted to In Royal Air Force recruits Richardson and McIntyre (16) revealed a caries decrease to DMFT 6.5. A study of Turkish recruits (17) established the DMFT values of 6, with a significant relationship between the DMFT value and sugar consumption. A common characteristic of all results in these studies is significant caries decrease in recruits, and the determined DMFT values which are mostly lower than those determined in our study of the Croatian army recruits. Specific characteristics of various age and social populations in Croatia with respect to age and social status are discussed in many studies. In the former Yugoslavia there were great differences between caries prevalence between the developed and undeveloped Federal Republics. The average DMFT value for 12-year-olds amounted to 6.1 and for 18-year-olds as high as 10.9 (21). The spread of caries was exceptionally wide, and therefore, the DMFT index for the age range between 19 and 29 amounted between and (22). Lobnik- Gomilšek (23) found the average DMFT index of 8.06 for military recruits in the Federal Republics of the former Yugoslavia (for the subjects from rural areas 7.64 and urban areas 8.52). In Croatia the average DMFT value was 8.41 (DT: 3.87, MT: 1.15 and FT: 3.39), with a higher value in urban areas. According to our results for Croatian recruits, a decline in caries of 7.7% was found. In the analysis of single values share of DMFT it can be concluded that there is a decline of decayed teeth for 48%, whereas there is an increase of filled teeth for 25%. An increase in the number of missing teeth for 30% is unfavourable. Previous studies of Croatian recruits showed that the most common oral disease was dental caries (value 5.84), and in 2000 healthy teeth were found in only 4% subjects, but with better values of the investigated indices. The DMFT=6 value was lower, and the FST=25 value was higher (18, 19). Epidemiological studies provide valuable data on the health status of groups of inhabitants, assessing the prophylactic measures that have been implemented. They also provide guidelines for improvement of oral health, which is especially important in European countries in transition (24-26). Table 3. Test differences of the DMFT index and FST index in subgroups of the subjects according to the number of toothbrushing per day (average and standard deviations for frequency) by means of variance analysis* Subgroups No of subjects DMFT DT MT FT FST 1 time per day ± ± ± ± ± times per day ± ± ± ± ± times per day ± ± ± ± ±3.16 Probability statistical test =0.007 =0.571 =0.008 =0.026 *DMTF, Decayed, Missing, Filled, Teeth; DT, Decayed, Teeth; MT, Missing, Teeth; FST, Filled and Sound Teeth 259

120 Medicinski Glasnik, Volumen 6, Number 2, August 2009 ACKNOWLEDGMENT / DISCLOSURE This study is a part of the scientific project No supported by the Ministry of Science, Education and Sports, the Republic of Croatia. The manuscript was presented as a part of: Badel T, Restek-Despotušić S, Keros J, Azinović Z, Dulčić N. Oralno zdravlje novaka Hrvatske vojske. Proceeding of the Third International Congress of Croatian Dentists, Zagreb/Croatia, October 6-8, Acta Stomatol Croat 2003; 37: [Abstract]. Competing interests: none declared. REFERENCES 1. Thylstrup A, Fejerskov O. Textbook of clinical cariology. Copenhagen: Munksgaard, Freire MC, Sheiham A, Netuveli G. Relationship between height and dental caries in adolescents. Caries Res 2008; 42: Petersson HG, Bratthall D. The caries decline: A review of reviews. Eur J Oral Sci 1996; 104: Marthaler TM. Changes in dental caries Caries Res 2004; 38: Reich E. Trends in caries and periodontal health epidemiology in Europe. Int Dent J 2001; 51: Morgan MV, Stonnill A, Laslett AM. Dental caries amongst Royal Australian Navy recruits, Aust Dent J 1992; 37: Hopcraft M, Morgan M. Dental caries experience in a young adult military population. Aust Dent J 2003; 48: Krejsa O, Mrklas L, Broukal Z. Caries of 18-yearold recruits in the Czech Republic in 1995 [Abstract]. Caries Res 1996; 30: Antoft P, Rambusch E, Antoft B, Christensen HW. Caries experience, dental health behaviour and social status-three comparative surveys among Danish military recruits in 1972, 1982 and Comm Dent Health 1999; 16: Willerhausen B, Ernst C-P, Seifert Y, Nauth C. Zur Mundgesundheit von Rekruten der Bundeswehr. Acta Med Dent Helvet 1997; 2: Klimek J, Ganß C, Alffen T. Kariesbefall, Restaurationsarten und Fissurenversiegelungen bei deutschen Rekruten in den Jahren 1992 und Deutsch Zahnärzt Z 1999; 54: Polastri F, Cerato E, Gallesio C, Pancotti G. Stato di salute orale in un campione di reclute delle varie regioni d Italia. Minerva Stomatol 1991; 40: Asmyhr O, Grytten L, Grytten J. Changing trends in caries experience among male military recruits in Norway. Community Dent Oral Epidemiol 1994; 22: Menghini GD, Marthaler TM, Steiner M, Bandi A, Schürch E Jr. Kariesprävalenz und gingivale Entzündung bei Rekruten im Jahre 1985: Einfluss der Vorbeugung. Schweiz Monatssch Zahnmed 1991; 101: Menghini GD, Steiner M, Marthaler TM, Weber M.W. Rückgang der Kariesprävalenz bei Schweizer Rekruten von 1970 bis Schweiz Monatssch Zahnmed 2001; 111: Richardson PS, McIntyre IG. Dental treatment needs of a cohort of Royal Air Force recruits over 5 year. Community Dent Health 1996; 13:11-6. Ceylan S, Acikel CH, Okcu KM, Kilic S, Tekbas OF, Ortakoglu K. Evaluation of the dental health of the young adult male population in Turkey. Mil Med 2004; 169: Škec V, Macan JS, Susac M, Jokić D, Brajdić D, Macan D. Influence of oral hygiene on oral health of recruits and professionals in the Croatian Army. Mil Med 2006; 171: Badel T, Restek-Despotušić S, Kern J, Keros J, Šegović S. Karijes novaka Hrvatske vojske u godini. Acta Med Croat 2006; 60: Marthaler TM. A standard system of recording dental conditions. Helv Odontol Acta 1966; 10:1-18. Vulović M, Rajić Z, Popić B, Aurer-Koželj J, Nečeva L, Redžepagić S et al. Stanje oralnog zdravlja u SFRJ. Zobozdravstveni Vestnik 1988; 1:3-10. Artuković D. Prevalence of periodontal diseases of adult population of Zagreb, according to the criteria by WHO. University of Zagreb, Zagreb 2001; Master s thesis Lobnik-Gomilšek B. Epidemiological examination and caries prevalence in conscripts in 1989, 1990, and University of Zagreb, Zagreb 1993; Master s thesis Künzel W. Rise and fall of caries prevalence in Eastern Europe reasons and consequences. Acta Stomatol Croat 1998; 32: Szöke J, Petersen PE. Evidence for dental caries decline among children in an East European country (Hungary). Community Dent Oral Epidemiol 2000; 28: Vrbič V. Reasons for the caries decline in Slovenia. Community Dent Oral Epidemiol 2000; 28:

121 ORIGINAL ARTICLE Security Perception of a Portable PC User (The Difference Between Medical Doctors and Engineers): A Pilot Study Krešimir Šolić, Vesna Ilakovac Department of Biophysics, Medical Statistics and Medical Informatics, J.J. Strossmayer University of Osijek, School of Medicine, Osijek, Croatia ABSTRACT Aim The aim of this pilot study was to compare knowledge on security threats and habits in dealing with computer security issues. Corresponding author Krešimir Šolić J. J. Strossmayer University of Osijek, School of Medicine Josipa Huttlera 4, Osijek, Croatia; Phone: ; Fax: ; kresimir@mefos.hr Original submission: 15 June 2008; Revised submission: 18 September 2008; Accepted: 02 February Methods Two groups of researchers and teaching staff, portable personal computer (PC) users, coming from different environments were included in the study: School of Medicine (n=19) and School of Electrical Engineering (n=20). Participants were asked to complete an anonymous questionnaire consisting of 21 questions about basic demographic data, years of using PC, years of owning/using portable PC, position at the School, habits in dealing with security issues and knowledge about potential security threats. Results Both groups demonstrated similar pattern of behaviour in dealing with security issues. Participants from the School of Electrical Engineering showed a higher level of knowledge in three questions about security experts terminology (Fisher s exact P<0.05 for all questions). Results also showed a very low frequency of making security backups. Conclusion The results of this pilot study indicate that working environment and background do not have a great impact on behaviour of highly educated portable PC users in connection with security issues. However, it seems that information about the importance of security backups should be presented more often to each PC user. Keywords: security, security risk, portable PC, backup, ICT security. Med Glas 2009; 6(2):

122 Medicinski Glasnik, Volumen 6, Number 2, August 2009 INTRODUCTION Security vulnerabilities concerning personal data in medical information and communication technology (ICT) systems can negatively impact patient healthcare, but may also represent a potential law violation (1). Data stored in the institutional ICT systems are more or less protected by different mechanisms and supervised by system administrators. Main computers with databases are usually stored in secured, air-conditioned and dedicated locations. Data safety and security mostly depend on institutional policy by means of time and resources invested in data protection. This is not the case with data stored in portable personal computers (PC). While wide adoption of mobile computing technology can improve information access and enhance workflow (2), the data stored in portable computing devices are more accessible to potential attackers and thieves, physically but also using malicious software (3). In addition, such data are at higher risk of physical damage due to frequent transportation. The user and his/her portable device constitute a closed system, which is often connected to other systems using different network environments with various security levels. The vulnerability of such systems depends immensely on user awareness about potential threats. Security risk behaviour of portable PC users might be a threat for their working environment. The aim of this pilot study was to compare knowledge about security threats, analyse the frequency of making backups and investigate user habits in dealing with security issues in two research groups and teaching staff, namely the portable PC users coming from the following different environments: School of Medicine and School of Electrical Engineering. Users working in the field of medicine are compared with users that have a much better technical knowledge background. PARTICIPANTS AND METHODS There was a total of 39 researchers and teaching staff of the J.J. Strossmayer University in Osijek - School of Medicine (n=19) and School of Electrical Engineering (n=20) who own or use official (school owned) portable PC on a regular basis and who participated in this pilot study. Participants were asked to complete an anonymous questionnaire comprising 21 questions about basic demographic data, years of using a PC, years of owning/using portable PCs, position at the School, habits in dealing with security issues and knowledge about potential security threats. The main demographic characteristics, years of using a PC, years of owning/using a portable PC and a distribution by the position at the School did not differ between groups (Table 1). Data were presented as absolute frequencies, means with standard deviations and medians with interquartile range where appropriate. Differences in numerical attributes were tested with Student s t-test and Mann-Whitney U test. Differences in categorical attributes were tested with Fisher s exact test. All P values were two tailed. Analyses were conducted using the SAS software (version 8.02, Cary, NC, USA), with significance level set at P<0.05. RESULTS More than a half of the participants in both surveyed groups used an Internet banking service, and about the same number were users of inter- Table 1. Characteristics of participants included in the study Participants School of Medicine (n=19) School of Electrical Engineering (n=20) Age, mean (SD*) 34.1 (7.2) 32.7 (9.8) Gender, M/F 10/9 16/ Years of using PC, mean (SD) 14.3 (6.4) 13.6 (5.3) Years of owning/ using portable PC, median (interquartile range) 5.0 ( ) 5.5 ( ) No of participants by the position junior researcher 6 8 assistant lecturer assistant professor 3 3 full professor 3 1 *SD, standard deviation; Student s t-test; Fisher s exact test; Mann- Whitney U test p 262

123 Šolić et al Security Perception of a Portable PC User Table2. Internet related security issues Security issue School of Medicine (n=19) School of Electrical Engineering (n=20) Accessing Internet services via public computers with questionable protection on an exceptional basis 8 10 very rarely often 5 4 don t care about that 1 0 Internet banking user User of international Web-based >0.950 services Shopping on the Internet * Fisher s exact test national Web-based services (like Gmail, Yahoo or other). However, public computers with questionable protection were used for accessing Internet services on an exceptional basis or very rarely in both groups of participants (Table 2). Eleven out of 19 participants from the School of Medicine and 17 out of 20 from the School of Electrical Engineering set a password on their portable PCs (Fisher exact test, p=0.082). Four participants from each institution respectively made security backups more than once a week. The distribution of frequency of making security backups did not differ between the institutions Table 3. Security issues related to backing up data and protection against malicious software Security issue School of Medicine (n=19) School of Electrical Engineering (n=20) Frequency of making security backups of important documents, No never 1 1 very rarely 2 1 from time to time 12 7 once in a month once in a week 0 2 more frequently 4 4 Type of security software installed, No antivirus anti-spyware spam filter firewall Number of various types of security software installed, No none * Fisher s exact test p* p* (Fisher s exact test, p=0.083). The same could be said for the distribution of number of various types of security software installed (Fisher s exact test, p=0.263). The majority of participants from both institutions were using antivirus software (Table 3). Most participants in both surveyed groups (14 at School of Medicine group and 18 at School of Electrical Engineering group) were familiar with the fact that messages were easily intercepted in Internet communication (Fisher s exact test, p=0.235). Participants from the School of Electrical Engineering showed a higher level of knowledge in three questions dealing with terms hoax, phishing and encryption (Table 4) DISCUSSION The results of this pilot study indicate that working environment and background do not have a great impact on behaviour of highly educated portable PC users in connection with security issues. Internet security presents a challenge in many fields of human activity, from commerce, education to e-health (4,5). Accessing Internet services such as Internet banking or Web based client via public computers with questionable protection is a serious threat to users important and Table 4. Answers to knowledge questions School of Question Medicine (n=19) Do you know what is HOAX? School of Electrical Engineering (n=20) never heard of it 12 8 heard, but don t know 4 0 heard, but not sure 1 3 yes, I know 2 9 Do you know what is PHISHING? never heard of it 10 2 heard, but don t know 4 3 heard, but not sure 1 2 yes, I know 4 13 Do you know what the data encryption is? never heard of it 3 0 heard, but don t know 3 0 heard, but not sure 0 2 yes, I know * Fisher s exact test p*

124 Medicinski Glasnik, Volumen 6, Number 2, August 2009 private data. The fact that majority of participants from both groups do it on an exceptional basis or very rarely implies their awareness of this major security issue. Furthermore, the Internet is a well known source of malicious software. Medical information systems are becoming more and more vulnerable to attacks by malicious software (6). Unaware and unprotected portable PC users who have access to a medical information system also pose a security threat. The only participant from the School of Medicine who has no security software on his portable PC is secured by using the LINUX operating system, whereas the majority of the persons tested have at least 2 types of security software installed on their portable PCs. Even though the reason for such consciousness might be just self-protection, it results in a reduced risk not only for the user and his portable PC, but also for the environment in which they operate. The frequency distribution of making security backups in both groups is very low. Only 4 participants from the School of Medicine and 6 from the School of Electrical Engineering make security backups of their important documents once a week or more frequently. The difference in answers on questions about security experts terminology between groups investigated was the only difference found between them. It seems that information about the importance of backups and instructions on how to do backup should be presented to each PC user. It is considered to be even more important for users having a mobile device because their portable PCs are not (all the time) part of the secured corporate ICT system. Those instructions should come in the same box with portable PCs (7). The study shows that users awareness on security risks plays an important role in securing the data (8,9). Participants did present great knowledge in this matter and their systems are secured. The number of portable PC users is growing every day, as well as their impact on the security of surrounding ICT systems. Although based on a rather small sample, the results of the pilot study may serve as a starting point for further research in security matters in systems consisting of portable PCs and their users, as well as in their security behavior in different environments. ACKNOWLEDGMENT / DISCLOSURE Competing interests: none declared. REFERENCES Dantu R, Oosterwijk H, Kolan P, Husna H. Securing medical networks. Network Security 2007; 2007: Yen-Chiao L, Yan X, Andrew S, Julie A.J. A review and a framework of handheld computer adoption in healthcare. Int J Med Inform 2005; 74: Potter B. Mobile security risks: ever evolving. Network Security 200; 2007: Hawkins S, Yen D.C, Chou D.C. Awareness and challenges of Internet security. Information Management & Computer Security 2000; 8: Kluge E-H.W. Secure e-health: Managing risks to patient health data. Int J Med Inform 2007; 76: Gobuty D. Defending medical information systems against malicious software. International Congress Series 2004; 1268: Phippen A, Furnell S. Taking Responsibility for online protection - why citizens have their part to play, Computer Fraud & Security 2007; 2007: TechNet Security Centre, Microsoft. microsoft.com/technet/security/ (10 th of August 2007) Sophos Security Information web page. (10 th of August 2007) 264

125 CASE REPORT Akutna bruceloza udružena sa Coombs-pozitivnom autoimunosnom hemolitičkom anemijom i diseminiranom intravaskularnom koagulacijom Nerma Mušić, Eldira Hadžić Služba za zarazne bolesti, Kantonalna bolnica Zenica, Bosna i Hercegovina Corresponding author: Nerma Mušić, Služba za zarazne bolesti, Kantonalna bolnica Zenica, Crkvice 67, Zenica, Bosna i Hercegovina Phone: ; Fax: nermamusic@hotmail.com Originalna prijava: 06. decembar 2008.; Korigirana verzija: 12. januar 2009.; Prihvaćeno: 06. mart Med Glas 2009; 6(2): SAŽETAK Hematološke manifestacije bruceloze su različite. Akutna hemoliza i diseminirana intravaskularna koagulopatija (DIK) jako se rijetko viđaju kod bruceloze. Terapijski pristup ovakvim formama bolesti je jako kompleksan. U ovom radu prikazan je slučaj bolesnika s akutnom brucelozom udruženom s Coombs-pozitivnom autoimunom hemolitičkom anemijom i diseminiranom intravaskularnom koagulacijom. Pacijent je dobro reagirao na terapiju antibioticima i kortikosteroidima. Ključne riječi: bruceloza, autoimuna hemolitička anemija, diseminirana intravaskularna koagulopatija UVOD Bruceloza je širom svijeta raširena zoonoza uzrokovana mikroorganizmom iz roda Brucella (1). Bolest zahvata mnoge organe i organske sisteme, gastrointestinalni, kardiovaskularni, hematopoetski, nervni, koštani, respiratorni, kožni i očni (2). Hematološke manifestacije bruceloze su različite i obično se manifestiraju kao blaga anemija i leukopenija (1-3). Anemija u pacijenata sa brucelozom rezultat je poremećaja metabolizma željeza tokom infekcije, hipersplenizma, krvarenja, supresije koštane srži ili autoimune hemolize (2). Teška trombocitopenija, pancitopenija, bicitopenija, akutna hemoliza i diseminirana intravaskularna koagulacija (DIK) su rijetke (2, 3). Infekcija uzrokovana vrstama Brucella može rezultirati pojavom sistemskog autoimunosnog odgovora kod ljudi (3). Pojava Coombs-pozitivne autoimunosne hemolitičke anemije kod akutne bruceloze je rijetka (3). Mehanizam nastanka trombocitopenije nije potpuno jasan, ali neki predloženi mehanizmi su hipersplenizam, diseminirana intravaskularna koagulacija, supresija koštane srži tokom septikemije, hemofagocitoza i imuna destrukcija trombocita (4). Autoimunosna hemolitička anemija (AIHA), kao jedna od posljedica imunosnog razaranja eritrocita, odlikuje se skraćenim vijekom eritrocita preko mehanizama domaćinovih antitijela koja reagiraju sa vlastitim antigenima (5). Može biti dio kliničke slike infekcija uzrokovanih različitim uzročnicima. Najčešće su to vrste Plasmodium, Haemophilus influenzae, Escherichia coli, Salmonella spp., Shigella spp., Mycoplasma pneumoniae, citomegalovirus, varicella zoster i herpes simplex virus, virus influenzae A (5). Klinički autoimuna hemolitička anemija može se očitovati različito, kao teška hemolitička anemija sve do akutnih hemolitičkih kriza sa hemoglobinurijom i akutnom bubrežnom insuficijencijom (5). Diseminirana intravaskularna koagulacija (DIK) često nastaje u sistemskoj upali uzrokovanoj endotoksinom koji istovremeno aktivira imunosni (monocitno-makrofagni) sistem i endotelne stanice koji luče tkivni faktor (6). Između ostalih, i intravaskularna hemoliza jeste stanje kod kojeg se javlja DIK (6). U ovom radu prikazan je slučaj bolesnika s teškim oblikom akutne bruceloze udružene sa kliničkim i hematološkim nalazom Coombspozitivne autoimunosne hemolitičke anemije i diseminirane intravaskularne koagulacije. 265

126 Medicinski Glasnik, Volumen 6, Number 2, August 2009 PRIKAZ SLUČAJA Šezdesetdvogodišnji muškarac hospitaliziran je u Službi za zarazne bolesti Kantonalne bolnice Zenica krajem treće sedmice od pojave simptoma gubitka apetita, opće slabosti i malaksalosti. Dan prije dolaska u bolnicu imao je povišenu temperaturu i otežano kretanje. Na dan prijema bolesnik je bio dezorijentiran, te je teško uspostavljen kontakt. Bolesnik je stanovao u seoskom domaćinstvu, bavio se uzgojem ovaca i preradom mesa. U objektivnom statusu dominirala je febrilnost, dezorijentacija i dehidratacija. Na odjelu je šest dana bila prisutna visoka febrilnost, a četvrtog dana po prijemu razvili su se znakovi afekcije jetre, hemolitičke anemije i diseminirane intravaskularne koagulacije. Bolesnik je povremeno bio konfuzan (Tabela 1). Dijagnoza bruceloze postavljena je na osnovu pozitivnih seroloških testova, Rose Bengal testa, RVK na brucelozu čiji je titar iznosio 1:128, te ELISA-testa na brucelozu koji je bio pozitivan na sve tri frakcije (IgM, IgG i IgA). Serološki testovi na viruse hepatitisa A, B i C bili su negativni. Dijagnostička procedura nadopunjena je ultrazvučnim nalazom hepatosplenomegalije, kompjuteriziranom tomografijom (CT) mozga i citobiohemijskim nalazom likvora koji su bili uredni. Direktni Coombsov test bio je pozitivan četvrtog dana hospitalizacije, kada je došlo i do pojave ikterusa. Laboratorijski nalazi ukazivali su na značajnu leziju jetre, hemolizu i znakove DIK-a. U perifernom razmazu krvi ustanovljeno je parcijalno megaloblastno sazrijevanje, trombocitopenija i toksične granulacije (Tabela 2). Petog dana nakon što je započeto liječenje kombinacijom gentamicina i doksiciklina uz kortikosteroide, bolesnik je postao afebrilan i postepeno je došlo do oporavka. Tabela 1. Objektivni patološki nalaz kod bolesnika oboljelog od bruceloze kod prijema u bolnicu Organski sistem Glava i vrat Trbuh Koža Nalaz ikterus očnih sklera znaci dehidratacije sluznica meteorizam hepatosplenomegalija ikterus, petehije Šest mjeseci nakon otpusta iz bolnice, bolesnik je imao uredne kontrolne nalaze (krvna slika, transaminaze, bilirubin, laktatdehidrogenaza, gama glutamil-transferaza, alkalna fosfataza, proteinogram, željezo u krvi, protrombinsko vrijeme), ultrazvuk abdomena i negativan Coombsov test. Brucele su fakultativno intracelularne bakterije (7). Neki faktori virulencije brucela esencijalni su u invaziji domaćinovih ćelija, dok su drugi odlučujući u izbjegavanju eliminacije od strane domaćina (8). Ove bakterije posjeduju nekonvencionalni non -endotoksični lipopolisaharid koji im omogućava otpornost na antimikrobijalni napad i modulira domaćinov imunosni odgovor (7, 8). Ključni aspekt virulencije brucela jeste njena sposobnost proliferacije unutar profesionalnih i neprofesionalnih fagocita (9). U uslovima gdje je tijelo izloženo lipopolisaharidu, pretjerano ili sistematski, kao kad lipopolisaharid uđe u krvnu struju, može doći do sistemske upalne reakcije dovodeći do multiplog organskog oštećenja, šoka, a potencijalno i smrti (9). Tabela 2. Rezultati značajnih laboratorijskih nalaza pri prijemu i otpustu bolesnika oboljelog od bruceloze Vrsta analize Rezultati analiza Referentne kod prijema otpusta kod vrijednosti Sedimentacija (mm /sat) Eritrociti (x 106/L) 3,14 4,42 4,30 5,70 Hemoglobin (g/l) ,1 14,0 18,8 Leukociti (x 103/L) ,0 Trombociti (x 103/L) Protrombinsko vrijeme (%) 17 13, Aktivirano parcijalno tromboplastinsko , vrijeme (sec.) Fibrinogen (g/l) 1,00 3, D-dimeri (ng/l) <500 Bilirubin ukupni (mikromol/l) ,8 Bilirubin konjugovani (mikromol/l) Aspartataminotransferaza (U/L) Alaninaminotransferaza (U/L) Laktatdehidrogenaza (U/L) Gamaglutamiltransferaza (U/L) Alkalna fosfataza (U/L) Željezo u serumu mikromol/l 38,4 14, ,6 Ukupni proteini (g/l) Albumini (g/l) Globulini (g/l)

127 Case report Akutna hemoliza i DIK jako se rijetko viđaju kod oboljelih od bruceloze, u 0,5%, odnosno 0,1% slučajeva (2). Poremećaji koagulolitičkog sistema kod bruceloze također su veoma rijetki i javljaju se u oko 1% slučajeva. U tom slučaju nastale promjene posljedica su oštećenja zidova kapilara brucelama, kao i stvorenim imunim kompleksima u toku infekcije (10). U Službi za zarazne bolesti Kantonalne bolnice u Zenici, u periodu od do godine od bruceloze je liječen 631 bolesnik, a samo jedan bolesnik (0,16%) je imao kliničke i laboratorijske parametre i AIHA i DIK-a (neobjavljeni podaci, N. Mušić, Služba za zarazne bolesti, Kantonalna bolnica Zenica, 2008.). Dijagnoza bruceloze prikazanog bolesnika postavljena je na osnovu pozitivnih seroloških testova, a dijagnoza AIHA i DIK-a na osnovu laboratorijskih analiza i pozitivnog Coombsovog testa. U kliničkoj slici bolesnika dominirali su simptomi lezije jetre udružene sa febrilnošću i hemolitičkom anemijom. Serološkim testovima isključena je mogućnost hepatalnog oštećenja hepatotropnim virusima. Bolesnik je liječen kombinacijom antibiotika. Afebrilnost koja je nastala petog dana liječenja, uz postepeno poboljšanje općeg stanja i laboratorijskih nalaza, te porasta trombocita nakon terapije kortikosteroidima, uz laboratorijski verificiran prestanak hemolize i negativizaciju Coombsovog testa, sugerirali su imunološku osnovu ovih zbivanja Alici O, Kasapoglu B, Alkan R, Sarifakioglu E, Akgedik R, Bozalan R, Kosar A,. Sahin H. Case report: An unusual presentation of acute brucellosis with thrombocytopenia and maculopapular rash. J Infect Developing Countries 2007; 1: Dilek I, Durmus A, Krahocagil M K, Akdeniz H, Karsen H, Baran AI, Evirgen O. Hematological complications in 787 cases of acute brucellosis in Eastern Turkey. Turk J Med Sci 2008; 38: Sari I, Kocygit I, Altuntas F, Kaynar L, Eser B. An unusual case of acute brucellosis presenting with Coombs-positive autoimmune hemolytic anemia. Intern med 2008; 47: Yalaz M, Mehmet T, Arslan, Kurugol Z. Thrombocytopenic purpura as only manifestation of brucellosis in a child. Turk J Pediatr 2004; 46: Nemet D. Anemije nepoznatog i višetrukog mehanizma nastanka U: Božidar Vrhovec i suradnici. Interna medicina. Treće promjenjeno i dopunsko izdanje. Zagreb: Naklada Ljevak, 2003: Stančić V. Sindrom diseminirane intravaskularne koagulacije (DIK) ili sindrom potrošne koagulopatije. Stančić%20Vladimir.doc. (Datum pristupa ). Lapaque N, Moriyon I, Moreno E, Gorvel JP. Brucella lipopoliysaccharide acts as a virulence factor. Curr Opin Microbiol 2005; 8:60-6. Fugier E, Pappas G, Gorvel JP. Virulence factors in brucellosis:implications for aetiopathogenesis and treatment. Expert Rev Mol Med 2007; 9:1-10. Cardoso PG, Macedo GC, Azevedo V, Oliveira SC. Brucella spp. noncanonical LPS: structure, biosynthesis and interaction with host immune system. Microb Cell Fact 2006; 5:13. Čengić Dž. Poremećaji hemostaze i fibrinolize u infektivnim bolestima. Sarajevo: Nacionalna i Univerzitetska biblioteka Bosne i Hercegovine, 1997: 61, 85. Kod bolesnika sa temperaturom, znacima oštećenja jetre i hematološkim poremećajima, pa čak i onim rijetkim kao što je AIHA i DIK, u diferencijalnoj dijagnozi treba uvijek imati na umu brucelozu, posebno u endemskim geografskim područjima kao što je Zeničko-dobojski kanton. ZAHVALE / IZJAVE Komercijalni ili potencijalni dvostruki interes ne postoji. LITERATURA 1. Acute brucellosis associated with Coombs-positive autoimmune hemolytic anemia and disseminated intravascular coagulation (DIC) Nerma Mušić Department for Infectious Diseases, Cantonal Hospital Zenica, Bosnia and Herzegovina ABSTRACT Hematological manifestations of brucellosis are different. Acute hemolysis and disseminated intravascular coagulopathy are rarely seen in the course of brucellosis. Therapeutic approach to 267

128 Medicinski Glasnik, Volumen 6, Number 2, August 2009 these forms of diseases is extremely complex. This paper presents a case of acute brucellosis manifested by coombs-positive autoimmune hemolytic anemia and disseminated intravascular coagulation. The patient responded well to antibiotic and corticosteroid therapy. Key words: brucellosis, autoimmune hemolytic anemia, disseminated intravascular coagulopathy Original submission: 06 December 2008; Revised submission: 12 January 2009; Accepted: 06 March CASE REPORT Pneumolabirint \enad Hodžić, Služba za bolesti uha, grla, nosa i maksilofacijalnu hirurgiju, Kantonalna bolnica Zenica, Zenica, Bosna i Hercegovina Corresponding author: \enad Hodžić, Služba za bolesti uha, grla, nosa i maksilofacijalnu hirurgiju, Kantonalna bolnica Zenica, Crkvice 67, Zenica Tel.: ; Fax.: eminh@bih.net.ba Originalna prijava: 25. februar 2009.; Korigirana verzija: 01. april 2009.; Prihvaćeno: 19. april Med Glas 2009; 6(2): SAŽETAK Nalaz zraka u labirintu uha, nakon frakture temporalne kosti glave, pomoću kompjuterizovane tomografije (CT), rijetka je klinička manifestacija. Prateći simptomi su vertigo, oštećenje sluha i često perilimfatična fistula. U radu je opisan slučaj petnaestogodišnjeg mladića kojem su kliničke tegobe nastale nakon pada sa visine. Pneumolabirint je ostao neprepoznat gotovo dvije sedmice, a otkriven je na CT snimcima temporalne kosti. Pacijent je liječen konzervativno. Budući da je liječenje započeto kasno, krajnji rezultat ove ozljede bio je potpuni i trajni gubitak funkcije ozlijeđenog uha. Ključne riječi: pneumolabirint, fraktura temporalne kosti, gubitak sluha, perilimfatična fistula UVOD Termin pneumolabirint prvi put se spominje godine, kada su Mafee i sur., na snimcima dobijenim kompjuterizovanom tomografijom (CT) temporalne kosti, opisali pacijenta kod kojeg je nastala nagla nagluhost i vertiginozne smetnje, prisustvo zraka u labirintu i fraktura pločice stapesa (1). Naime, malo je objavljenih radova koji prikazuju slične slučajeve. Opisani su slučajevi pneumolabirinta kod pacijenata sa barotraumom, frakturom temporalne kosti, perilimfatičnom fistulom, komplikacijama stapedektomije (2-6). U ovom radu opisat ćemo slučaj pacijenta sa frakturom lijeve temporalne kosti, udružene sa nastankom pneumolabirinta, nagle gluhoće i šuma lijevog uha, te vertiginoznih smetnji. PRIKAZ SLUČAJA Petnaestogodišnji mladić, prilikom pada sa visine od oko dva metra, u etiliziranom stanju, zadobio je udarac u glavu, rame i potkoljenicu, na lijevoj strani tijela. Neposredno nakon pada, uz simptome vrtoglavice, mučnine, povraćanja (nekoliko puta), pacijent je bio nestabilan u hodu i nije se sjećao pada. Sljedeća dva dana ležao je kod kuće, uz izraženu vrtoglavicu prilikom pokretanja glave, dezorijentiranost, somnolenciju i nestabilnost u hodu. Šum u lijevom uhu i naglo slabljenje sluha u lijevom uhu, pojavili su se sljedećeg dana poslijepodne. U sljedeća 2-3 sata pojavilo se pojačanje šuma, koji je od tada postao stalan, te jako izražen oslabljen sluh na lijevom uhu. Sljedeća 3-4 dana pacijent je imao osjećaj da ponešto i čuje na lijevo uho, ali nakon toga, i taj osjećaj je nestao. Istovremeno je subjektivno došlo do smanjenja vrtoglavice, mučnine i nestabilnosti u hodu. Otoskopijom, kod kliničkog pregleda, ustanovljen je obostrano intaktan bubnjić normalnog izgleda. Nije bilo znakova izljeva u srednje uho, test na prisustvo fistule bio je negativan, a Romberg test pozitivan sa zanošenjem udesno i natrag. Nije bilo znakova pareze nervusa facialisa. Osnovni laboratorijski nalazi urađeni su u nekoliko navrata i bili su u fiziološkim granicama. Tonalni audiogram pokazao je desno uredan prag sluha, a lijevo gluhoću. Timpanometrija je pokazala desno tip A timpanometra, a lijevo tip B timpanometra. CT piramida temporalne kosti pokazao je transverzalnu frakturu piramide i mastoida lijeve 268

129 Case report temporalne kosti, uz prisutnost tečnog sadržaja u većini mastoidnih ćelija. Fraktura je bila usmjerena kroz labirint vestibuluma, kohleu, dno meatus acusticus internus, a u labirintu je ustanovljeno prisustvo zraka, te dijagnosticiran pneumolabirint. Na osikulama i unutar kavuma nije bilo vidljivih promjena (Slika 1). Pacijent je liječen ambulantno, uz simptomatsku terapiju i kućnu njegu. Nakon mjesec dana, subjektivno vertiginozne smetnje kod pacijenta su nestale, ali je ostao stalno prisutan šum u lijevom uhu, te potpuni gubitak sluha na lijevom uhu. Dijagnoza frakture lijeve temporalne kosti sa pneumolabirintom, postavljena je nakon 12 dana od dana ozljeđivanja, na osnovu CT nalaza piramida temporalnih kostiju. Dijagnoza je bila otežana uslijed nepostojanja vidljive lezije na koži glave, etiliziranosti pacijenta i urednog otoskopskog nalaza. Intenzivne tegobe od strane labirinta, nastale su poslije više od 24 sata, u vidu naglo nastalog šuma u lijevom uhu i intenzivne nagluhosti na istom uhu, kada je nastao gubitak sluha na lijevom uhu i ostao samo subjektivni osjećaj percepcije zvuka. Trajanje ovog pogoršanja stanja sluha od svega nekoliko sati i njegov nagli nastanak upućivali su na mogućnost da pneumolabirint nije nastao odmah nakon povrede, nego da je možebitno isprovociran nekim postupcima bolesnika (povraćanje, saginjanje, naprezanje). Implozivne i eksplozivne sile su potencijalni uzrok nastanka perilimfatične fistule i pneumolabirinta (10). Implozivne sile mijenjaju tlak u srednjem uhu koji pritišće ovalni i okrugli prozor (fossulu ante fenestram), te Hyrtleovu fissuru. Porast tlaka može uzrokovati kompresivna trauma uha, Valsalvina proba, kihanje sa zatvorenim nosom (10). Slika 1. Kompjuterizovana tomografija lijeve temporalne kosti. Strelica pokazuje prisustvo zraka u labirintu unutrašnjeg uha. Lanac slušnih koščica je intaktan i u bubnjištu nema tečnog sadržaja. Desno od strelice je frakturna pukotina. (Služba za bolesti uha, grla, nosa i maksilofacijalnu hirurgiju, Kantonalna bolnica Zenica, 2008.) Eksplozivne sile u vidu kašljanja, kihanja ili defekacije, koje se prenose u unutrašnje uho kroz kohlearni akvedukt i laminu kribrozu, povećavaju pritisak cerebrospinalnog likvora (CSL) (10). Potencijalna ulazna vrata za ulazak zraka u labirint mogu biti ovalni i okrugli prozor, mikrofisure između stražnjeg polukružnog kanala i okruglog otvora (fossula ante fenestram) (9). Prisustvo zraka u skali timpani ili skali mediji znak je traume kohleje (16). Mjehurić zraka onemogućava širenje putujućem valu duž bazilarne membrane, što se očitovalo na timpanogramu i kod našeg pacijenta (timpanogram tip B) (16). Curenje perilimfe uzrokuje relativno povećanje endolimfatičnog tlaka, što, uz nizak tlak perilimfe, uzrokuje endolimfatični hidrops, a koji je uzrok vrtoglavice i nestabilnosti pacijenta (8, 17). Jednom nastala komunikacija između unutrašnjeg i srednjeg uha dovodi do gubitka perilimfe, relativnog hidropsa endolimfe i pratećih simptoma. Prisustvo pneumolabirinta može olakšati dijagnozu perilimfatične fistule (11, 15), koja sama po sebi ne znači i postojanje pneumolabirinta, dok prisustvo pneumolabirinta obavezno ukazuje i na postojanje perilimfatične fistule. Nalaz zraka u bazalnom zavoju kohleje jeste dobar pokazatelj postojanja perilimfatične fistule, te upućuje na potrebu eksploracije i kirurškog zbrinjavanja fistule (12, 17). Rano kirurško zbrinjavanje perilimfatične fistule onemogućava teška oštećenja uha, kao što su gluhoća, meningitis, labirintitis (12, 17). Iako, u našem slučaju, nismo našli pozitivan znak postojanja perilimfatične fistule, nalaz tečnosti u mastoidnim ćelijama upućivao je na njezino postojanje, a lokalizacija frakturne pukotine ukazivala je na postojanje komunikacije između mastoidnih ćelija i labirinta. Frakturna pukotina nije zahvatala dio labirinta koji ujedno predstavlja i medijalni zid bubnjišta. Intaktan lanac slušnih koščica i bubna opna dodatno su potvrdili odsustvo znaka postojanja perilimfatične fistule. Poredeći konzervativni i kirurški tretman, neki autori, u odsustvu znakova postojanja perilimfatične fistule i težih simptoma, preporučuju inicijalno konzervativno liječenje 269

130 Medicinski Glasnik, Volumen 6, Number 2, August 2009 (14). Konzervativno liječenje, pored antibiotika, kortikosteroida i simptomatske terapije, podrazumijeva i postupke kojima se prevenira nastanak eksplozivnih i implozivnih sila za koje se pretpostavlja da su bitan faktor u nastanku pneumolabirinta - mirovanje, antiemetici, sedativi, položaj glave u blagoj elevaciji, regulisanje stolice (6). Eksperimentalno, kod zamoraca, utvrđeno je kako se, nakon resorpcije zraka, funkcija labirinta u potpunosti povratila (4, 5). U unutrašnjem uhu, zrak se može kretati ovisno od položaja glave, a što se može vidjeti na CT snimcima visoke rezolucije (13). Stoga je za dijagnostiku pneumolabirinta ključna pretraga CT-om sa tankim slojevima (od 1 do 1,5 mm debljine), radi boljeg kontrasta u prikazivanju odnosa zrak-kost, nego nuklearna magnetna rezonanca (MRI) (7). Rano otkrivanje pneumolabirinta i perilimfatične fistule, rani početak terapije, bilo konzervativno ili hirurški, kao i niz drugih postupaka i preporuka u njihovom tretmanu, jako su važni u liječenju teških oštećenja sluha i prevenciji mogućih teških komplikacija. U ovom slučaju od velike koristi bila bi rano započeta konzervativna terapija (odmah nakon ozljeđivanja) antibioticima, kortikosteroidima, uz regulaciju probave, mirovanja, blagu elevaciju glave, antitusika, antiemetika, te praćenje audioloških parametara kao što su tonalna audiometrija, BERA (engl. brain steam auditory evoked potentials), timpanometrija. ZAHVALE/IZJAVE Komercijalni ili potencijalni dvostruki interes ne postoji. LITERATURA 1. Mafee MF,Vaslvassori GE, Kumar A, Yannisas DA, Marcus RE. Pneumolabyrinth. A new radiologic sign for fracture of the stapes footplate. Am J Otol 1984; 5: Scheid SC, Feehery JM, Willcox TO, Lowry LD. Pneumolabyrinth: A late complication of stapes surgery. Ear Nose Throat J 2001; 80: Isaacson JE, Laine F, Williams GH. Pneumolabyrinth as computed finding in poststapedectomy vertigo. Ann Otol Rhinol Laryngol 1995; Yanagihara N, Nishioka I. Pneumolabyrinth in periliymphatic fistula: report of tree cases. AmJ Otolaryngol 1987; 8: Nakashima T, Kaida M, Yanagita N. Round window membrane rupture and inner ear damage due to barotrauma. Acta Otolaryngol Suppl. 1992; 493: McGee MA, Dornhoffer JL. A case of barotrauma-induced pneumolabyrinth secondary to perilyphatic fistula. Ear Nose Throath J 2000; 76: Nishizaki K, Yamamoto T, Akagi H, Ogawa T, Masuda Y. Pneumolabyrinth: imaging case of the month. Am J Otol 1998; 19: Lyos AT, Marsh MA, Jenkins HA, Coker NJ. Progressive hearing loss after transverse temporal bone fracture. Arch Otolaryngol Head Neck Surg 1995; 121: Meyerhoff WL, Marple BF. Perilymphatic fistula. Otolarygol Clin North Am 1994; 27: Goodhill V. Sudden deafens and round window rupture. Laryngoscope 1971; 81: Sheridan MF, Hetherington HH, Hull JJ. Inner barothrauma from scuba diving. Ear Nose Throath J 1999; 78: Pullen FW, Rosenberg GJ, Cabeza CH. Sudden hearing loss in dives and flyers. Laryngoscope 1979; 9: Kobayashi T, Sakurada T, Ohyama K. Inner ear injury caused by air intrusion to the scala vestibuli of the cochlea. Acta Otolaryngol 1993; 113: Lao WW, Niparko JK. Assesment of changes in cochlear function with pneumolabyrinth after middle ear trauma. Otol Neurotol 2007; 28: Lo S-H, Huang Y-C, Wang P-C. Pneumolabyrinth associated with perilymph fistula. Chang Gung Med J 2003; 26: Nomura Y. Perlymph fistula: concept, diagnosis and management. Acta Otolaryngol (Suppl) 1994; 514: Nomura Y, Okuno T, Hara M, Young YH. Floating labirynth. Pathophysiology and treatment of perilymph fistula. Acta Otolaryngol 1992;112: Pneumolabyrinth \enad Hodžić Department for Ear, Nose, Throat and Maxillofacial Surgery, Cantonal Hospital Zenica, Bosnia and Herzegovina ABSTRACT Computed tomography (CT) revealing pneumolabyrinth after temporal bone fracture is a rare clinical finding. Accompanying symptoms are: vertigo, hearing loss and very often perilymphatic fistula. This paper presents a case of a 270

131 Case report fifteen-year old boy with clinical discomfort after falling down from a height. Pneumolabyrinth was diagnosed by CT scan of the temporal bone and it had remained unrecognized for almost two weeks. The patient was treated conservatively. As the hospital treatment started too late the final result of this injury was complete and permanent hearing-loss of the impaired ear. Key words: pneumolabyrinth, temporal bone fracture, hearing loss, perilymphatic fistula Original submission: 25 February 2009; Revised submission: 01 April 2009; Accepted: 19 April 2009; CASE REPORT Sphenochoanal polyposis Ivana Pajić-Penavić 1, Davorin \anić 1, Ljubica Fuštar-Preradović 2 1 Department of Otorhinolaryngology Head and Neck Surgery, 2 Department of Pathology and Cythology; General Hospital Dr. Josip Benčević Slavonski Brod, Croatia Corresponding author: Ivana Pajić-Penavić, Department of Otorhinolaryngology Head and Neck Surgery General Hospital Dr. Josip Benčević Andrije Štampara 42, Slavonski Brod, Croatia Phone: ivana.pajic-penavic@sb.t-com.hr Original submission: 04 December 2008; Revised submission: 09 February 2009; Accepted: 04 May Med Glas 2009; 6(2): ABSTRACT The reports of sphenochoanal polyps in the literature are relatively rare. Computed tomography and nasal endoscopy contribute in diagnosis of sphenochoanal polyps. Simple polypectomy which partialy leaves a polyp inside the sphenoid sinus increases the risk of a relapse. Using powered instrument-assisted endoscopic sinus surgery we surgically removed sphenochoanal polyp in a ten year old boy. We wide opened the orifice of sphenoid sinus and removed the cystic polyp part from sphenoid sinus. At the annual follow-up examination, this patient remains free of signs of polyp recurrence. Key words: spenochoanal polyposis, endoscopic surgery, computed tomography (CT scan) INTRODUCTION Choanal polyps are rare benign mucous tumors of the nose and the paranasal sinus which grow from the sinus orifice and spread through nasal meatus into choanae and nasopharynx. They make 3-6 % of all the polyps of the nose (1). Based on the origin of the polyp s petiole, polyps are divided into the three types: antrochoanal (originating in the maxillary sinus), ethmoidochoanal (originating from the etmoid sinus) and sphenochoanal (originating from the sphenoid sinus) (2). Polyps whose source is in the orifice or in sphenoid sinus are distinctly rare (2,3). They were first described by Zuckerkandl in 1892 (4,5). Clinically, the glistening and pale masses are identical to typical nasal polyps; careful inspection using endoscopy can disclose a stalk leading to the sinus of origin (6). It must be emphasized that the stalk of antrochoanal polyp can be easily visualized but it is very difficult to visualize sphenoid orifice in children even without polyps. In case of sphenochoanal polyp only stalk from sphenoethmoid recess can be seen. In general, the diagnosis of choanal polyps is established by nasal endoscopic examination and CT (3,6). In this paper we presented a case of endoscopic treatment of a ten year old male with the sphenochoanal polyp. CASE REPORT The patient was a 10-year old male, with symptoms of heavy respiration through his nose, with incessant frontal rhinorrhea, purulent mucous discharge with occasional snoring. He had been adenoidectomised three years earlier in another hospital due to heavy respiration through the nose. After the operation he showed no postoperative improvement. Using a front rhinoscopia, an abundance of mucous and purulent secretion was found in both nasal cavities. Physical examination by endoscope revealed an intranasal pearly 271

132 Medicinski Glasnik, Volumen 6, Number 2, August 2009 polyp creation in the right nasal cavity, situated in a lower nasal meatus and in left nasal cavity, obstructing the entire nasal cavity (Figure 1). Computed tomography (CT scan) of nasal cavity and paranasal sinuses in axial and coronary projection confirmed a shading of both sphenoid sinuses, partly of frontal ethmoids left, and both nasal cavities alongside unobstructed maxillary sinuses. With the powered instrument-assisted endoscopic approach under general endotracheal anesthesia, we identified a pearly polyp-creation centered between the middle nasal concha and a septum of the left nasal cavity with a petiole spanning from the left sphenoid sinus. We endoscopically removed the polyp petiole intersection which originates from the anterior wall of the left sphenoid sinus. Enlarging the orifice of the sphenoid sinus we removed a cystic part of intrasinusal polyp along with mucous membrane (Figure 2). Free orifices of the maxillary sinuses are mutually displayed. Pathologic analysis of the creation confirms a diagnosis of chronic polypus inflammation. The patient experienced successful operative and post operative process without any complications. One year after this procedure was completed, the left sphenoid sinus orifice is wide opened and both nasal cavities and the sinus remain free of recurrence. A polyp which grows from the singular sinus and spreads out through choanes into nasopharynx is called the choanal polyp. Sphenochoanal polyps are the polyps whose roots arrive from sphenoid sinuses. Compared with common nasal polyposis and antrochoanal polyps, sphenochoanal polyps are relatively rare, with only 35 cases reported in the English literature to date (7). Histologically, majority of all the polyps look similar and include a cystic center that is usually caused by gland hyperplasia which is surrounded by edematous parenchyma with infiltration of inflammatory cells whereas the polyp surface is covered with respiratory epithelium. This histological appearance is not always present in case of sphenochoanal polyps (8). There are numerous theories explaining polyp development. Two of them are mostly described by Berg and Mills (8,9). Sphenochoanal polyp occurs evenly in male and female population from childhood till the fourth decade of life (10). The polypoide mass in our case contains few mucous glands and has a myxoid stroma, with variable densities of inflammatory cells concentrated near the surface which can confirm the polyp development as a result of mucocela expansion caused by blockade and burst of acino-mucus glands in bacterial rhinitis phase during the period of recovery from a chronic infection according to Mills (9). Clinically, choanal polyps produce symptoms of nasal obstruction, rhinorrhea, pain in the facial area, partial deafness caused by dysfunction of Eustachian tube, otalgia, snoring and a presence of a creation in nasal and oral cavity (11). Sphenochoanal polyps as the one from our case are clinically present as unilateral, solitary, bluish or yellowish mass involving the nasal fossa between the middle nasal concha and septum and the choana can differ from antrochoanal polyp which takes up osteomeatus complex between the middle nasal concha and the lateral wall of the nasal cavity. Both polyp types can obstruct nasal cavities Figure 1. Endoscopic picture of a sphenochoanal polyp centered between a middle nasal concha and septum (I. Pajić Penavić, 2007.) Figure 2. Endoscopic view of wide opened orifice of a sphenoid sinus (I. Pajić-Penavić, 2007.) 272

133 Case report and may spread through the choanae into the nasopharynx, then spread down into the oral cavity. Frontal rhinoscopy, flexible and rigid nasal endoscopies are obligatory for the diagnosis of sphenochoanal polyposis. They are recommended for the description of a location of the polyp petiole. To distinguish an antrochoanal polyp from sphenochoanal polyps computed tomography or magnetic resonance of the paranasal sinuses can be used (12). Sphenochoanal polyp can be seen on CT scan as an opacification of the sphenoid sinus and mass in common meatus extending to the nasopharynx without evidence of pathology in maxillary sinuses. When choanes are filled with necrotic sphenochoanal polyposis CT with contrast or angiographia towards suspect angiofibroma is recommended (13). Surgical treatment of sphenochoanal polyp involves endoscopic removal of choanal portion of the polyp and widening ostium of sphenoid sinus with no need of middle meatal antrostomy as it is suggested in treatment of antrochoanal polyp (6,13). A comprehensive discussion of the differential diagnosis should include the possibility of an antrochoanal polyp, hypertrophic adenoid, Tornwald s cyst, pituitary tumor, lymphoma, meningoencephalocela, angiofibroma, inverted papilloma and fungal rhinosinusitis (13,14). Treatment of sphenochoanal polyposis involves complete surgical removal. Choanal polyposis recidivate in 25% cases if removed by simple intranasal removal of polyp by forceps. Endoscopic approach with a review of petiole and wide microdebrider or forceps removal of the polyp in nasal cavity and in the sinus is a surgical technique of choice (15). In conclusion, sphenochoanal polyp is an extremly rare type of choanal polyp and it can be easily confused with antrochoanal polyp. An adequate preoperative preparation with CT scan and endoscopy is crucial to establish an exact diagnosis and for planning of an adequate surgical technique to reduce the percentage of possible polyp recidivism. ACKNOWLEDGMENT / DISCLOSURE Competing interests: none declared. REFERENCES 1. Eloy PH, Evrard I, Bertrand B, Delos M. Choanal polyp of sphenoid origin. Acta Otolaryngol Belg 1996; 50: Chen JM, Schloss MD, Azouz ME. Antrochoanal polyp: a 10 year retrospective study in the pediatric population with a review of the literature. J Otolaryngol 1989; 18: Lessa Marcus M, Voegels Richard L, Padua F, Wiikmann C, Romano Fabrozio R, Butugan O. Sphenochoanal polyp: diagnose and treatment. Rhinology 2002; 40: Stammberger H. Functional Endoscopic Sinus Surgery. Pennsylvania, Philadelphia: BC Decker, Prasad U, Sagar PC, Shahul Hameed O.A.N. Choanal polyp. J Laryngol Otol1970; 84: Tosun F, Yetiser S, Akcman T, Özkaptan Y. Sphenochoanal polyp: endoscopic surgery. Int J Pediatr Otorhinolaryngol 2001; 58: Tsai CH, Hsu M-C, Liu C-M.Sphenochoanal polyp.tzu Chi Med J 2008; 20: Berg O, Carenfelt C, Silfversward C. Origin of the choanal polyp. Arch Otolaryngol Head Neck Surg 1988; 114: Mils CP. Secretory cysts of the maxillary antrum and their relation to the development of antrochoanal polyp. J Laryngol Otol 1959; 73: Cook PR, Davis WE, Mc Donald R, Mc Kinsey JP. Antrochoanal polyposis: a review of 33 cases. ENT J 1993; 72: Crampette L, Mondain M, Rombaux P. Sphenochoanal polyp in children. Diagnosis and treatment. Rhinology 1995;33: Weissman JL, Tabor BK, Curtin HD. Sphenochoanal polyps:evaluation with CT and MR imaging. Radiology 1991;178: Soh KBK, Tan KK. Sphenochoanal polyps in Singapore: diagnosis and current management. Singapore Med J 2000; 41: Yanagisawa E, Yanagisawa K. Endoscopic view of thornwald cyst of the nasopharynx. Ear Nose Throat J 1994; 73: Bozzo C, Garrel R, Meloni F, Stomeo F, Crampete L. Endoscopic treatment of antrochoanal polyps. Eur Arch Otorhinolaryngol 2007; 264:

134 Medicinski Glasnik, Volumen 6, Number 2, August 2009 CASE REPORT Primary extranodal Natural Killer/ T-cell lymphoma of the ethmoid sinus masquerading as orbital cellulitis Davorin \anić 1, Ana \anić Hadžibegović 1, Ivana Mahovne 2 1 Department of Otorhinolaryngology, Head and Neck Surgery, 2 Department of Pathology; General Hospital Slavonski Brod, Slavonski Brod, Croatia Corresponding author: Davorin \anić, Department of Otorhinolaryngology, Head and Neck Surgery General Hospital Slavonski Brod, A. Štampara 42, Slavonski Brod, Croatia Phone/ Fax: davorin.djanic@sb.t-com.hr Original submission: 29 January 2009; Revised submission: 14 April 2009; Accepted: 22 April Med Glas 2009; 6(2): ABSTRACT This report presents a case of an exceptionally rare primary Natural Killer/T cell (NK/T) lymphoma of the right paranasal frontal and ethmoid sinuses in a patient treated previously for right side chronic sinusitis. It highlighted the importance of adequate tissue biopsy and patohistological examination in patients with chronic sinusitis or orbital cellulitis that fail to respond to traditional management. Key words: NK/T cell lymphoma, paranasal sinuses INTRODUCTION In 1897 Mc Bride first described a patient with surface crusting, widespread necrosis and inflammation, aggressive and rapid destruction of nose and face midline, and lethal course (1). In the past, the term lethal midline granuloma was usually used but it included three histologically different lesions: Wegener s granulomatosis, polymorphic reticulosis and malignant lymphoma (2). Neoplasms of paranasal sinuses are very rare, comprising less than 3% of all autodigestive tract tumors (3). Lymphoma of the primary paranasal sinuses are even rarer and represent only 0-17% of all lymphomas in Kiel Lymph Node registry and account for only 5-8% of the extranodal lymphomas of the head and neck area (3). Based on morphology and cell lineage there are currently 3 types of lymphoma: B cell, T cell and Hodgkin lymphoma. In addition, many proliferating T cells have shown to express an additional marker (CD56), which suggests an NK cell origin. These tumors are classified as NK/T lymphomas (4). Most common presenting signs and symptoms of primary paranasal sinuses lymphoma are nonspecific and fall into several categories: nasal: epistaxis, nasal obstructions, congestion, extension into the nasal cavity; facial: unilateral facial or cheek swelling, facial asymmetry, pain, infraorbital nerve hypoesthesia; and ocular: unilateral tearing, diplopia, fullness of lids, pain, and exophthalmia (5). In this paper we presented an uncommon case of primary NK/T lymphoma of the ethmoid sinus masquerading as chronic rhinosinusitis and orbital cellulitis. CASE REPORT A 60 year-old man presented with progressive well marked periorbital edema and erythema of the right eye, and thickness of the nasal dorsum and right cantal region. During the last 5 years he had been treated for right side chronic sinusitis. He had six millimeters proptosis and relative upper eyelid ptosis of the right eye. Nasal endoscopy revealed anterior deviation of nasal septum, obstructed ostiomeatal complex with a black mass and purulent discharge in the middle and common meatus. Computed tomography (CT) scan of the paranasal sinuses and orbit showed soft tissue mass filling the right ethmoid, frontal and maxillary sinus, eroding the anterior part of the right lamina papiracea, and infiltrating right medial rectus muscle (Figure 1). Retention cyst in max- 274

135 Case report illary sinus, polypoid mucosa of ethmoid sinuses and orbital soft tissue swelling without focal abscess were found during a functional endoscopic surgery. All of necrotic tissue was removed and first histological examination showed chronic inflammation of paranasal sinusal mucosa. Bacterial and fungal cultures were negative. Four months later the patient developed fever, swelling, surface crusting, and widespread necrosis of the right periorbital and nasal area (Figure 2). Multiple biopsies of the paranasal sinuses were performed and diagnosed as nonspecific granulomatous inflammation. Finally, diagnosis of NK/T cell (CD 56+) lymphoma was made by histological and imunohistochemical reexamination of the paraffineembeded tissue obtained from the first biopsy of the ethmoid sinus and orbit. There were necrotic changes of varying degrees and a polymorphous pattern of proliferation involving large atypical cells with an occasional multilobated nucleus and various numbers of lymphocytes, plasma cells and macrophages. Features of vascular invasion by neoplastic lymphocytes were apparent. Occasionally, angiocentric pattern of proliferation was observed. Large atypical cells were positive for the NK-cell marker CD 56 (Figure 3). Patient had IV-A stage lymphoma and was EBV positive. Neck lymph nodes were negative. Thoracic and abdominal CT scans as well as bone marrow biopsy were all negative. He was scheduled for continuous chemotherapy with 8 CHOP 14-day cycles. Unfortunately, the patient s condition deteriorated rapidly after the development of liver failure and respiratory failure and after 18 months he died. Primary paranasal sinus NK/T-cell (CD 56 positive) lymphoma is a polymorphic extranodal lymphoma, expressing NK or rarely cytotoxic T- cell phenotype (3). It is an uncommon disease, and generally highly aggressive in its clinical course. Primary paranasal sinus T-cell lymphomas are much more frequent in Asian and Latin American countries, present at younger age, and usually arise from nasal cavity than the paranasal sinuses. In contrast, lymphomas of B-cell phenotype predominate in Western population and usually arise from paranasal sinuses. T-cell lymphomas are characterized by progressive ulceration and necrosis that are not typical for B-cell lymphomas (3, 5). Numerous studies showed that patients with NK/T lymphomas of the sinusonasal area had a high incidence of Epstein-Barr virus infections (6). The majority of lymphomas involving the ocular adnexa are of B-cell lineage. However, NK/T- cell lymphoma is associated only infrequently with orbital or adnexal involvement (7). Life style and environmental factors significantly increased risk for developing NK/T-cell lymphoma among individuals exposed to pesticides (8). Figure 1. Axial CT scan showing soft tissue mass in the right ethmoid, frontal, and maxillary sinus eroding bony structures and infiltrating right medial rectus muscle (D. \anić, 2007.) Figure 2. Widespread necrosis of the right periorbital and nasal area in a patient with primary paranasal NK/T-cell lymphoma (D. \anić, 2007., with patient s permission) 275

136 Medicinski Glasnik, Volumen 6, Number 2, August 2009 Figure 3. Immunohistological section of the lymphoma showing strong positive staining for cytoplasmic CD56 (CD56; x40) (I. Mahovne, 2007.) This patient lived in a rural area, worked in agricultural environment and was exposed to pesticides for many years. The diagnosis of lymphoma cannot be made from clinical findings solely and thus biopsy and imaging of the lesions is mandatory prior to any treatment (9). During the diagnostic procedure adequate tissue biopsy must be taken to differentiate lymphoma from destructive inflammatory diseases or malignant tumors (9). Biopsy must be adequate, not too small or too superficial because sinusonasal lymphomas are subepithelial lesions, often with perfectly normal overlying mucosa, unlike carcinoma, which are usually ulcerative (9). Repeated biopsy may sometimes be needed. Cross-sectional imaging findings like pathologic contrast enhancement or bone changes may reveal the malignant nature of the disease but there is significant overlapping between those possible pathologies that can arise in this region (9). Physiologic imaging, like perfusion CT and proton MR spectroscopy, in the extracranial head and neck can be implemented in any CT or MRI survey, provide functional information of the lesion, and may be helpful to differentiate benign from malignant disease as well as guide therapeutic decisions (10). The optimal treatment for primary nasal lymphoma remains unknown (11). Surgical resection of paranasal sinusal lymphoma is not recommended unless the tumor spreads to critical locations resulting in impending death (11). Complete response rate after radiotherapy is much higher as compared to chemotherapy although radiotherapy planning for primary nasal lymphomas may be difficult because these lymphomas often encroach on such radiosensitive critical structures as the optic chiasm, optic nerve and eyeballs and exact dose-tumor response relationship is unknown (11). Addition of chemotherapy to radiotherapy did not improve survival rate with early stage NK/T cell lymphoma (12). Ocular manifestation prior to systemic ones may be useful to monitor the response to therapy (12). Prognosis associated with sinonasal NK/T cell lymphomas varies. Dissemination is infrequent, but when it occurs it typically involves other extranodal sites (12). In conclusion, this case highlights the importance of adequate tissue biopsy and patohistological examination in patients with chronic sinusitis or orbital cellulitis that fail to respond to traditional management. ACKNOWLEDGMENT / DISCLOSURE Competing interests: none declared. REFERENCES McBride P. Photographs of a case of rapid destruction of the nose & face. Laryngol 1987;12:64 6. Kassel S, Echevaria RA, Guzzo FP. Midline malignant reticulosis (so-called lethal midline granuloma). Cancer 1969;23: Aozasa K, Takakuwa T, Hongyo T, Yang WI. Nasal NK/T-cell lymphoma: epidemiology and pathogenesis. Int J Hematol 2008; 87: Swerdlow SH, Campo E, Harris NL, Jaffe ES, Pileri SA, et al. WHO Classification of Tumours, Volume 2 [IARC WHO Classification of Tumours, No 2], Vidal RW, Devaney K, Farkiti A, Rinaldo A, Carbone A. Sinusonasal malignant lymphomas: a distinct clinicopathological category. Ann Otol Rhinol Laryngol 1999; 108: Van de Rijin M, Bhargava V, Molina-Kirsc H, Carlos-Bregni R, Warnke RA, Cleary ML. Extranodal head and neck lymphomas in Guatemala: high frequency of Epstein-Barr virus associated sinusonasal lymphomas. Hum Pathol 1997; 28: Charton J, Witherspoon SR, Itani K, Jones FR, Marple B, Morse B. Natural Killer/T cell lymphoma masquerading as orbital cellulitis. Ophtal Plast Reconst Surg 2008; 24: Hardell L, Erickson MA. A case-control study of non-hodgkins lymphoma and exposure to pesticide. Cancer 1999; 85:

137 Case report 9. Chen SH, Wu CS, Chan KH, Hongh YT, Shun CT, Liu CM. Primary sinusonasal non-hodgkins lymphoma masquerading as chronic rhinosinusitis: an issue of rutine histopathological examination. J Laryngol Otol 2003; 117: Bisdas S, Fetscher S, Feller AC, Baghi M, Knecht R, Gstoettner W, Vogl TJ, Balzer JO. Primary B cell lymphoma of the sphenoid sinus: CT and MRI characteristics with correlation to perfusion and spectroscopic imaging features. Eur Arch Otorhinolaryngol 2007; 264: Yu K. Primary nasal lymphoma. J HK Coll Radiol 2001; 4: Yao B, Song YW, Jin J, Wang WH,Wang SL Sun YT et al. Treatment option and outcome for patients with primary non-hodgkins lymphoma of the nasal cavity. Zhonguhua Zhong Liu Za Zhi 2006; 28: CASE REPORT Knee disarticulation Ognjen Živković¹, Antun Muljačić², Renata Poljak- Guberina³ ¹Institute for Rehabilitation and Orthopaedic Aids of the University; Hospital Center, Zagreb, ²University Hospital of Traumatology, Zagreb, ³Private Practice, Zagreb; Croatia Corresponding author: Renata Poljak Guberina Private practice Rockefellerova 23a, Zagreb, Croatia Phone: renata.poljak@zg.t-com.hr Original submission: 16 September 2008; Revised submission: 29 December 2008; Accepted: 04 February Med Glas 2009; 6(2): ABSTRACT In this paper we presented three patients with knee disarticulation performed according to Baumgartner. The Baumgartner tehnique and the application of knee disarticulation prosthesis appeared to be superior in comparisson with other methods. Key words: knee, disarticulation, Baumgartner tehnique INTRODUCTION Knee disarticulation is a rarely used method in amputation surgery, primarily due to the operative technique itself and secondly, due to poor understanding of prosthetic replacement possibilities (1). Technological progress and new developments in the prosthetics have opened new possibilities of amputation methods and consequently in the choice of the amputation level (1). A prosthesis for patients with knee disarticulation has been designed, with construction being based on the operative method of knee disarticulation according to Baumagartner (2). The energy expenditure during walking with knee disarticulation prosthesis is a little more than 40%, the same as for below-knee prosthesis (3). Disarticulation of a knee is recommended for high traumatic amputation of the below-knee, crush injury, complex injuries and tumors of the belowknee (1). Surgeons have been in dilemma between the method of transcondylar amputation and knee disarticulation (2). Knee disarticulation proves to be superior due to the possibilities of prosthetic replacement. The advantages are: a long and strong stump with a tip that can endure full-weight bearing and is suitable for a knee disarticulation prosthesis, the energy expenditure during walking equal to walking with below-knee prosthesis, normal function of the abbove-knee muscles (2). Unpopularity of knee amputation over many years was caused by bad experience with primary wound healing and the resulting stump of poor quality with regard to its function (2). In order to prevent these complications some surgeons introduced modifications in operative method (4-6). These methods are surgically more demanding and associated with a higher risk of complications (7). Baumgartner 1971 describes the method of knee disarticulation as a surgically simple procedure that creates a functionally satisfactory stump with regard to further prosthetic fitting (2). The simplicity of the technique is reflected in every aspect - skin, cartilage, bone, muscles (2). During the last 10 years the Clinic of Traumatology Zagreb has been using the technique of knee disarticulation described by Baumgartner. However, we have introduced some minor modifications. Instaed of sutturing the patellar ligament as Baumagartner was practising, we cut ligament at the top of the patella. So we additionally increas the contact and weight-bearing surface 277

138 Medicinski Glasnik, Volumen 6, Number 2, August 2009 of the stump. These modifications have yielded good results in the application of disarticulation prosthesis for the knee. Three patients (of different age but with the same successful therapy results) with knee disarticulation performed are presented in this study ( Figure 1). A skin incision is performed in two directions from the outer side of the medial and lateral condyle using an anterior long semicircular incision at 3-5 cm distally below the tibial tuberosity and posteriorly at the level of the sagittal line along the midline of the popliteal fossa Procedure I. A skin flap is raised and the knee joint exposed. The exposed collateral ligaments and hamstring tendons are resected. The patellar ligament is cut off at the patellar tip. The patella is placed in the position of patella alta, which additionally increases the contact and weight-bearing surface of the stump. A transverse wide capsulotomy is done to expose the knee joint with menisci and ACLs that are resected -Procedure II. The knee joint is flexed, the notch is exposed, the PCL is removed and the femoral condyle surface is left intact. Nerves and blood vessels in the popliteal fossa are exposed, ligated and transsected. Intact cartilage and femoral condyles are covered with the anterior skin flap which is sutured tension - free to the posterior skin flap in the popliteal fossa Procedure III. A free drain is placed below the fascia along the entire scar length. The drain is removed after two days and sutures after 14 days. Case one: A 61-year-old male patient fell under a motor excavator and a distal third of the right below-knee was crushed. Primary amputation at the level of the middle third of the belowknee was performed. The wound was left open and local therapy instituted. Due to complications in terms of bone protrusion on the fibular and tibial stumps, musculo-cutaneous defect and impossibility of wound closure, reamputation at the proximal third level was indicated. Knee disarticulation was done and after the wound healing the patient was admitted to the rehabilitation and fitted with a disarticulation prosthesis. After the prosthetic rehabilitation the patient was able to use the prosthesis during the whole day, walk independently, use a walking cane for longer walks and work on the land. Case two: A 36-year-old male patient substained a traumatic amputation of distal third of the right below-knee, and a femoral fracture due to a mine explosion. Transtibial amputation was performed at the level of the proximal third and the right femur was treated by internal fixation according to the AO method. After several months of treatment, the stump was in flexion contracture greater than 30 degrees. Prosthetic fitting was not possible so surgeon recommended the knee disarticulation. After the completed healing of the stump, prosthetic rehabilitation began. Following rehabilitation the patient was able to use the prosthesis for all daily activities and to walk unassisted. Case three: A 45-year old male patient was injured in a traffic accident as a car driver and substained an open fracture of the right belowknee. An operative treatment of this complex fracture was attempted but due to infection appearing in the postoperative course the amputation was indicated. The knee disarticulation was performed and prosthetic rehabilitation began (Figure 2). After a rehabilitation the patient wore the prosthesis during the entire day, used a walking cane for longer walks and worked actively. According to American authors, knee dis- Figure 1. Modified technique of knee disarticulation by Baumgartner (R. Poljak Guberina, 2004., with patient s permission) 278

139 Case report articulation is described as a simple, safe operative procedure, which has advantages in the prosthesis application but which is not widely applied (8). Some authors recommend the Mazet and Hennessy as well as the Burgess or Bowker methods (5,9). For those methods it is significant that due to cartilage removal a large bone surface is created, which increases the risk for bleeding and consequently associated complications (5). Thus, the end-bearing of the distal stump portion is significantly reduced. In the prosthetic sense, a disarticulation stump of the knee is obtained and it can accept only an above-knee prosthesis with weight-bearing tuberosity of the ischial bone. This is the very reason for application of those methods only for palliative indications where no prosthesis will be applied because patients will use wheel chairs (8). However, according to the International Society for Prosthetics and Orthotics (ISPO) Consensus Conference on Amputation Surgery 1990, knee disarticulation has an absolutely important place in the practice as an amputation technique (10). It is recommended in younger and elderly patients with indications like trauma, tumors, below-knee infections or circulatory problems in diabetics (11). Our ten-year experience (150 transcondylar amputations and 15 knee disarticulations) of the application of knee disarticulation as well as the application of the disarticulation prosthesis for the knee shows the advantage of this technique in relation to transcondylar amputation of the femur. Our patients with knee-disarticulation prosthesis showed in average a 50% increase in walking speed in comparison with patients with above-knee prosthesis. Our conclusions are Figure 2. Knee-disarticulation stump and prostheses (R. Poljak Guberina, 2004., with patient s permission) equivalent with results of authors that showed that energy consumption during walk with disarticulation prosthesis was increased by 40% and with the above-knee prosthesis by 70 80% (3). The advantages of knee-disarticulation are the simplicity of operative procedures, good quality of stump and successfully application of prosthesis which result in inproved quality of life. ACKNOWLEDGEMENT/DISCLOSURE Written consent was obtained from the patient for publication of the Figure 1 and Figure 2. REFERENCES 1. Murdoch G, Bennett WA, Jr. Amputation- Surgical practice and patient management. Oxford: Butterworth-Heinemann Co, Baumgartner RF. Knee disarticulationem versus above-knee amputation. Prosthet Orthot Int 1979; 3: Nader M, Nader HG. Otto-Bock prosthetic compendium: lower extremity prostheses. Berlin: Schiele and Schon GmbH., Faber David C, Fielding P. Gritti-Stokes (Through-Knee) amputation: should it be reintroduced? South Med J 2001; 94: Mazet R, Hennessy CA. Knee disarticulation: a new technique and a new knee joint mechanism. J Bone Joint Surg 1966; 48: Vaucher J, Blanc Y. Les desarticulation du genou. Technique operatoire-appareillage (Disarticulation of the knee. Surgical and prosthetic techniques.) Rev Chir Orthop1982; 68: Duerksen F, Rogalsky RJ, Cochrane IW. Knee disorticulation with intercondylar patellofemoral arthrodesis. Clin Orthop1990; 256: Smith DG, Micheal JW, Bowker JH. Atlas of Amputations and Limb Deficiencies: Surgical, Prosthetics, and Rehabilitation Principles. American Academy of Orthopeadics Surgeons, Rosemont, Illinois, Murdoch G, Bennett WA, Jr. A primer on amputations and arteficial limbs. New York: Charles Thomas Co., Jensen SJ, Lyquist E: Through-knee amputations. International Society for Prosthetics and Orthotics ( ISPO) Consensus Conference on Amputation Surgery. University of Strathclyde, Dundee, Scotland, 1990: DL, Taylor SM, Hamontree SE, Langan EM, Snyder BA, Sullivan TM, Youkey JR. A reappraisal of a modified through-knee amputation in patients with peripheral vascular disease. Am J Surg 2001;182:

140 Medicinski Glasnik, Volumen 6, Number 2, August 2009 CASE REPORT Izvanmaternična trudnoća izliječena metrotreksatom Ljiljana Bilobrk Josipović, Branka Lovrinović, Anton Galić Ginekološko-porođajni odjel, Hrvatska bolnica Dr. Fra Mato Nikolić, Nova Bila, Bosna i Hercegovina Corresponding author: Ljiljana Bilobrk Josipović, Ginekološko-porođajni odjel, Hrvatska bolnica Dr. Fra Mato Nikolić, Dubrave bb, Nova Bila, Bosna i Hercegovina Phone: ; Fax.: bjljiljana@hotmail.com Originalna prijava: 19. mart 2009.; Korigirana verzija: 20. april 2009.; Prihvaćeno: 06. maj Med Glas 2009; 6(2): SAŽETAK Prikazan je slučaj pacijentkinje čija je tubarna trudnoća liječena medikamentozno, metrotreksatom. Pacijentkinja se, 10+1 tjedan po izostanku menstruacije, javila ginekologu zbog blažih bolova pri dnu trbuha i oskudnog vaginalnog krvarenja. Pregledom, ultrazvučno i biokemijskim biljezima, dijagnosticirana je izvanmaternična (tubarna) trudnoća u desnom jajovodu. Početna razina β hcg-a bila je 5005 miu/ml, a veličina gestacijskog miješka 51 x 47 mm, sa embrijem dužine 5 mm. Pacijentkinja je uspješno izliječena jednom ampulom metrotreksata od 50 mg, koja je aplicirana intramuskularno. Ključne riječi: izvanmaternična trudnoća, metrotreksat, vrijednosti β hcg-a Uvod Izvanmaternična ili ektopična trudnoća jeste ona trudnoća koja se implantira i razvija izvan šupljine maternice. Najčešće je smještena u jajovodu (97%), ali može biti i u ovariju, trbušnoj šupljini, atretičnom rogu maternice, grliću i u plica lata (1). Kod tubarne trudnoće zametak je najčešće smješten u ampuli, poslije toga u istmičnom dijelu jajovoda, abdominalnom ušću, fimbrijama i intersticijskom dijelu jajovoda (1). Poseban slučaj predstavlja heterotopična trudnoća koja podrazumijeva istovremeno i unutarmaterničnu, i izvanmaterničnu trudnoću (2). Glavni klinički simptomi ektopične trudnoće su izostanak menstruacije, bol u trbuhu i vaginalno krvarenje (3). Dijagnosticira se ginekološkim i ultrazvučnim pregledom, biokemijskim biljezima (β hcg, estriol, progesteron), kiretažom maternice i kuldocentezom (3). Određivanje kreatinin kinaze i onkofetalnog fibronektina, kao biokemijskih biljega izvanmaternične trudnoće, je napušteno (4, 5). Danas je uobičajena vaginalna sonografija i Doppler u boji, u kombinaciji sa određivanjem razine β hcg-a (6, 7, 8). Izvanmaterničnu trudnoću uobičajeno je liječiti kirurški, laparoskopijom ili laparotomijom, poslije čega uslijedi salpingektomija ili rjeđe salpingotomija. Laparoskopija je, u zadnje vrijeme, češći pristup zbog brojnih prednosti pred laparotomijom (9). Relativne kontraindikacije jesu prethodni operativni zahvati, odnosno priraslice u trbuhu, tubarna trudnoća, veća od 6 cm i β hcg veći od miu/ml. Ponekad se primjenjuje i kombinirana laparoskopskofarmakološka metoda (9). Manji broj pacijentkinja vodi se ekspektativno ili medikamentozno. Uvjeti za medikamentozni postupak su nerupturirana izvanmaternična trudnoća manje od 4 cm, isključena heterotopična trudnoća, vrijednost β hcg miu/ml (po nekim autorima miu/ml), vrijednost progesterona manja od 40 nmol/l i hemodinamski stabilna pacijentkinja (10). U medikamentoznom liječenju mogu se primijeniti metrotreksat, aktinomicin D, NaCl, hipertonička otopina glukoze (50%), prostaglandini E 2 i F 2α i mifepriston (10). Osim tubarne trudnoće, medikamentozno se najčešće liječe cervikalna i intersticijska trudnoća (10). U kliničkoj primjeni uglavnom se koristi metrotreksat (11). U ovom radu prikazali smo slučaj uspješnog medikamentoznog liječenja tubarne trudnoće metotreksatom. PRIKAZ SLUČAJA Tridesetogodišnja pacijentkinja primljena je na Ginekološko-porođajni odjel Hrvatske bolnice Dr. Fra Mato Nikolić zbog krvarenja i bolova u trbuhu koji su počeli dva dana ranije, s amenorejom tjedan, hemodinamski stabilna pri prijemu. Iz reproduktivne anamneze pacijentkinja je navela dva poroda (prvi završen vaginalno; 280

141 Case report drugi operativno, carskim rezom, zbog parcijalne abrupcije posteljice tijekom poroda), jedan spontani pobačaj u 8. tjednu trudnoće, menstrualni ciklusi 30/5. Od ranijih oboljenja, pacijentkinja je imala česte vaginalne infekcije, a povremeno infekcije mokraćnog sustava. Obiteljska anamneza bila je bez osobitosti. Ginekološkim nalazom ustanovljeno je slijedeće: cilindričan grlić, dužine dva članka prsta, uz zatvoreno vanjsko ušće; krvarenje ex utero u tragu oskudnim tamnocrvenim iscjetkom; uterus, veličine guščijeg jajeta, mekše konzistencije, desno adneksalno kobasičasta tumefakcija, palpatorno bolno osjetljiva; lijeva adneksa i parametrija urednog palpatornog nalaza, Douglasov prostor slobodan. Transvaginalnim kolor-doplerom (TVCD) ustanovljeno je slijedeće: uterus 62 x 62 x 36 mm; endometrij 12,6 mm, inhomogen, bez vidljive intrauterine trudnoće; desni ovarij 35 x 23 mm, a ispod desnog ovarija inhomogena izdužena struktura sa gestacijskim miješkom 51 x 47 mm, embrionalni odjek 5 mm, bez vidljivih srčanih otkucaja; uz rub gestacijskog miješka, obilan protok sa RI: 0,667; lijevi ovarij 21,7 x 21,8 mm, bez vidljive slobodne tekućine u abdomenu. Uz informirani pristanak, pacijentkinji se, isti dan po prijemu, ordinira 50 mg metrotroksata intramuskularno, te intravenski (i.v.) 80 mg garamycina, dva puta dnevno (cave penicillin), 500 mg metronidazola, tri puta dnevno, po 500 ml ringer solutio i 5% glukoze jedanput dnevno. Drugi dan Slika 1. Ultrazvučni nalaz pacijentkinje: maternica i desni jajnik (izvanmaternična trudnoća u desnom jajovodu) kod prijema (lijevo); maternica i desni jajnik, devet mjeseci nakon liječenja (desno) (Ginekološko-porođajni odjel, HB Dr. fra Mato Nikolić Nova Bila, BiH, 2008.) po prijemu, pacijentkinji se peroralno ordinira folacin od 5 mg 3 x 2. Leucovorin (kalcij folinat) ampule nismo uspjeli pronaći u ljekarnama. Trećeg dana hospitalizacije pacijentkinja se žali na nešto intenzivnije bolove u trbuhu. Ultrazvučno je uočena oskudna količina tekućine u Douglasovom prostoru. Pošto je pacijentkinja i dalje bila hemodinamski stabilna, nastavljeno je praćenje, te su bolovi prestali slijedeći dan. Pacijentkinja je otpuštena sedmog dana po prijemu, sa zadovoljavajućim vrijednostim β hcg i krvne slike (Tablica 1), veličine gestacijskog miješka od 10,7 x 16,7 mm, bez vidljive slobodne tekućine u trbuhu. Ginekološki nalaz kod otpusta bio je slijedeći: grlić maternice cilindričan, bez vaginalnog krvarenja, maternica veličine guščijeg jajeta, tvrde konzistencije; desna adneksa i parametrija zadebljana za veličinu od jednog poprečnog prsta; lijeva adneksa i parametrija uredna. Iz bakteriološke kulture cervikalnog brisa, izoliran je Streptococcus foecalis i Neisseria gonorrhoeae, poslije čega je uključena i antibiotska terapija po antibiogramu. Tablica 1. Vrijednosti laboratorijskih parametara kod prijema i tokom liječenja Dani nakon početka liječenja Vrijednosti parametara* Kod prijema 2. dan 3. dan 5. dan 7. dan 10. dan 30. dan β hcg (miu/ml) ,2 Er (10 12 /L) 4,19 4,13 3,58 3,45 3,69 Hb (g/l) Hct ( L/L) 0,37 0,37 0,32 0,31 0,33 Le (10 9 /L) 8,7 4,3 Tr (10 9 /L) MCV (fl) 89 MCH (pg) 32 MCHC (g/l) 352 Fibrinogen (g/l) 5,1 Se (mm/h) 14 CRP (mg/dl) 24 Urea (mmol/l) 2,3 Kreatin (µmol/l) 53 Ukupni bilirubin(µmol/l) 12 AST (U/L) ALT (U/L) GGT (U/L) 10 Alkalna fosfataza (U/L) 36 *β hcg, β humani korionski gonadotropin; Er, eritrociti; Hb, hemoglobin; Hct, hematokrit; Le, leukociti; Tr, trombociti; MCV, mean corpuscular volume; MCH, mean corpuscula hemoglobin; MCHC, mean corpuscular hemoglobin concentration; Se, sedimentacija, CRP, C reaktivni protein; AST, aspartat aminotransferaza; ALT, alanin aminotransferaza; GGT, gamma glutamiltransferaza 281

142 Medicinski Glasnik, Volumen 6, Number 2, August 2009 Desetog dana, nakon započetog liječenja (ambulantno), vrijednost β hcg iznosila je 89 miu/ml, a nakon mjesec dana 1,2 miu/ml (Tablica 1), što je ukazivalo na uspješnost primjenjenog liječenja koje nije imalo nikakvih posljedica po zdravlje pacijentkinje. Šest mjeseci poslije sprovedene terapije, nije ustanovljena prisutnost β hcg (0 miu/m). Palpatorno je ustanovljena diskretna osjetljivost desnih adneksa i parametrija, a ultrazvučna slika maternice i jajnika bila je uredna, bez vidljivog proširenja desnog jajovoda. Pacijentkinji je predložena histerosalpingografija, koju ona odbije jer nije planirala dalje trudnoće. Ponovljeni palpatorni i ultrazvučni pregled, devet mjeseci nakon liječenja, ostao je isti. Transabdominalnim ultrazvukom moguće je dijagnosticirati izvanmaterničnu trudnoću kod vrijednosti β hcg miu/ml, a transvaginalnim ultrazvukom kod vrijednosti β hcg-a miu/ml (12). Medikamentozno liječenje ektopične trudnoće u svijetu je čest postupak. Po jednoj shemi metrotreksat se daje u četiri doze, prvi, treći, peti i sedmi dan, a leucovorin drugi, četvrti, peti i osmi dan. Po drugoj shemi, daje se 50 mg metrotreksata intramuskularno jednokratno (12). Ista doza lijeka može se ponoviti, ako se vrijednost β hcg ne snizi za l5% od početne vrijednosti za sedam dana, do ukupno četiri doze. β hcg se određuje svaki tjedan do negativizacije nalaza. Potpuna negativizacija β hcg-a očekuje se za 4-6 tjedana. Najmanje tri mjeseca iza primjene metrotreksata, treba odgoditi slijedeću trudnoću (12). Mi smo primijenili drugu shemu, bez potrebe ponovnog davanja lijeka za sedam dana. U nekim studijama daje se prednost medikamentoznom načinu liječenja pred kirurškim postupcima i to kod pacijentkinja koje zadovoljavaju kriterije za medikamentozno liječenje (13, 14). Medikamentozno liječenje je svakako manje traumatičan, jeftiniji i jednako uspješan način liječenja u slučajevima kada je izvanmaternična trudnoća dijagnosticirana na vrijeme. U jednoj studiji, koja je obuhvatila 350 pacijentkinja, ovaj način liječenja bio je uspješan kod 92% pacijentkinja, sa vrijednostima β hcg manjim od miu/ml, a uspješnost je bila 98% sa vrijednostima β hcg manjim od miu/ ml (15). Smatra se i da je u većini slučajeva dovoljna jednokratna primjena metrotreksata (16). Kombinirana primjena metroksata i mifepristona povećava uspjeh terapije (17). U našem radu prikazan je slučaj pacijentkinje sa uspješno izliječenom tubarnom trudnoćom metrotreksatom. Međutim, metrotreksat je uključen pri nešto većoj vrijednosti β hcg-a i pri znatno većoj dimenziji gestacijskog miješka od preporučenih (10). Razlozi zbog kojih smo se odlučili za medikamentozno liječenje kod naše pacijentkinje bili su prethodni operativni zahvat (carski rez), neobučenost ginekologa za endoskopske kirurške zahvate, hemodinamska stabilnost bez vidljivog krvarenja u trbuh. Trećega dana hospitalizacije, registrirana je kratkotrajna bol u trbuhu zbog manjeg istjecanja hemoragičnog sadržaja iz jajovoda u Douglasov prostor, što je dijagnosticirano ultrazvučno. Vrijednosti β hcg-a pokazivale su kontinuirani pad što ne mora biti uobičajeno kod ove terapije (12). Ponekad se može pojaviti i kratkotrajno povećanje vrijednosti β hcg-a, između 3. i 5. dana liječenja, što ne mora biti razlog za hitnu kiruršku intervenciju, ako je pacijentkinja hemodinamski stabilna i bez većeg pada vrijednosti hemograma (12). Naša pacijentkinja bila je pod intenzivnim bolničkim nadzorom sedam dana od postavljanja dijagnoze, nakon čega je otpuštena na kućno liječenje uz redovite kontrole. Od očekivanih nuspojava razvila se kratkotrajna neutropenija i trombocitopenija, kao i kratkotrajno povećanje vrijednosti transaminaza, zbog primjene citostatika (11). Nakon mjesec dana, uključena je i dodatna antibiotska terapija zbog dijagnosticirane kontaminacije cervikalne sluznice bakterijama. Infekcije adneksa najčešće nastaju ascendentnim putem, mada se ne može isključiti ni mogućnost hematogenog, limfogenog širenja infekcije ili perkontinuitatem (12). Palpatorni i ultrazvučni nalaz desnih adneksa, nakon šest mjeseci, bio je zadovoljavajući, osim što je palpatorno ustanovljena manja rezistencija desnog jajovoda, vjerojatno zbog kroničnog upalnog procesa, koji je i uzrokovao ektopičnu trudnoću. Nažalost, nije urađena his- 282

143 Case report terosalpingografija koja bi evaluirala konačni učinak terapije. U Bosni i Hercegovini cervikalne trudnoće, koje su inače rijetke, tretiraju se medikamentozno (12). Međutim, medikamentozno liječenje tubarne trudnoće, kao češće patološke pojave, nije uobičajeno. S obzirom da se radi o neagresivnoj, uspješnoj i jeftinoj terapiji, trebala bi se češće primjenjivati kod pravilno odabranih slučajeva. Literatura 1. Grizelj V. Izvanmaternična trudnoća.u: Dražančić A i sur. Porodništvo. Zagreb: Školska knjiga, 1994: Čanić T, Ciglar S, Kašnar V. Combined intrauterine and tubal ectopic pregnancy. Ginecol Clin Oncol 1997;18: Stowall TG, McCord ML. Early pregnancy loss and ectopic pregnancy. U: Berek JS. Gynecology. Baltimor: Williams and Wilkins, 1999: Lavie O, Beller U, Neuman M, Ben-Chentrit A, Gotteshalk S,Diamant Y. Maternal serum creatinine kinase: a possible predictor of tubal pregnancy. Am J Obstet Gynecol 1993; 169: Ness R, Mclaughlin M, Heine R, Bass D, Mortimer L. Fetal fibronectin as a marker to discriminate between ectopic and intrauterine pregnancies. Am J Obstet Gynecol 1998; 179: Kupešić S, Kurjak A. Uloga ultrazvuka u otkrivanju i liječenju ektopične trudnoće. U: Kurjak A. i sur. Ultrazvuk u ginekologiji i perinatologiji. Zagreb: Medicinska naklada, 2007: Jurković D, Jauniaux E, Kurjak A, Hustin J, Campbell S, Nicolaides KH, Transvaginal color Doppler assessment of uteroplacental circulation in early preganacy. Obstet Gynecol 1991;77: Shepard RW, Paton PE, Novy MJ, Burry KA. Serial beta hcg measurments in the early detection of ectopic pregnancy. Obstet Gynecol 1990, 75: Čanić T, Fistonić I. Laporoskopsko liječenje ektopične trudnoće. Gynecol Perinatol 2008; 17: Šimunić V. Izvanmaternična trudnoća.u: Šimunić V i sur. Ginekologija. Zagreb: Naklada Ljevak, 2001: Bradamante V. Kemoterapijski lijekovi protiv zloćudnih tumora. U: Medicinska faramakologija. Zagreb: Medicinska naklada, 1999: Farquhar MC. Ectopic pregnacy. Lancet; 2005; 366: Mol BW, Hajenius PJ, Engelsbel S, Ankum WM, Hemrika DJ, Van der Veen F, Bossuyt PM. Treatment of tubal pregnancy in The Netherlands: an economic comparison of systemic methotrexate administration and Laparoscopic salpingostomy. Am J Obstet Gynecol 1999; 181: Sowter M, Farquhar C, Petrie K, Gudex G. A randomised trial comparing single dose systemic methotrexate and laparoscopic surgery for the treatment of unruptured tubal pregnancy. Br J Obstet Gynecol 2001; 108: Lipscomb GH, McCord ML, Stovall TG, Huff G, Portera SG, Ling FW. Predictors of success of methotrexate treatment in women with tubal ectopic pregnancies. N Engl J Med 1999; 341: Barnhart KT, Gosman G, Ashby R, Sammel M. The medical management of ectopic pregnancy: a meta-analysis comparing single dose and multidose regimens. Obstet Gynecol 2003; 101: Perdu M, Camus E, Rozenberg P, Goffinet F, Chastang C, Philippe HJ, Nisand I. Treating ectopic pregnancy with the combination of mifepristone and methotrexate: a phase II nonrandomized study. Am J Obstet Gynaecol 1998; 179: Extrauterine pregnancy treated with Metrotrexat Ljiljana Bilobrk Josipović, Branka Lovrinović, Anton Galić Department of Obstetric and Gynecology, Croatian Hospital Dr Fra Mato Nikolić Nova Bila, Bosnia i Herzegovina ABSTRACT In this report we are describing a case of a patient whose tubal pregnancy was medicated with metrotrexat. The patient visited gynecologist 10+1 week after the last period, complaining about mild pain in the lower abdomen area and scarce vaginal bleeding. After ultrasound and biochemical markers examinations the extrauterine (tubal) pregnancy in the right oviduct was diagnosed. The initial level of β hcg was 5005 miu/ml and the size of gestational sac was 51 mm x 47 mm with an embryo 5 mm long. The patient was successfully cured with one 50 mg ampule of metrotrexat, intramuscularly applied. Key words: extrauterine pregnency, metrotrexat, values of β hcg-a Original submission: 19 March 2009; Revised submission: 20 April 2009; Accepted: 06 May 2009.; 283

144 ERRATUM Quality of the cardiovascular drugs prescribing in Zagreb during the period Danijela Štimac 1, Josip Čulig 1 Ivan Vukušić 1, Albert Cattunar 2,Dražen Stojanović 2 1 Zagreb Institute of Public Health, Zagreb, Croatia, 2 Department of Epidemiology, School of Medicine, University of Rijeka Volume 6 Number 1, 2009., page 118: 2 Department of Health Ecology (instead Department of Epidemiology) 284

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