Clostridium difficile diagnostika. Paul Naaber
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1 Clostridium difficile diagnostika Paul Naaber Paul Naaber
2 Sissejuhatus Grampositiivne anaeroob Moodustab eoseid Virulentsusfaktorid Toksiin A ja B olulised haiguse tekkes binary toxin tähtsus ebaselge Haigus asümptomaatiline seedetrakti kolonisatsioon: 3-5(-50?) % tervetesl täiskasvanutel haiglaväliselt sage (ca 50%) alla 2 aastastel lastel 7-50% hospitaliseeritutest antibiootikumraviga seotud kõhulahtisus (AAD) pseudomembranoosne koliit (PMC) 2
3 3 S. M. Poutanen et al. CMAJ (2004) 171: 51-58
4 Probleemi olulisus CDI juhu hind (13 uuringut, EU, USA, Kanada) esmane haigusjuht USD korduv CDI CD haiglavälise CD infektsiooni osatähtsus suureneb UK: CDAD diagnoosimine perearstide poolt suurenud 1-lt 22 juhuni /100,000 suureneb haigestumus ja komplitseeritud juhtude arv Kanada : sagedus 36-t 156 juhuni ; komplitseeritud juhtude arv 7-lt 18%-ni 4
5 Diagnostika meetodid: sissejuhatus CD produktide määramine Toksiin A ja/või B EIA - A+B» Immuunkromogeensed kiirtestid, well-type ELISA rakukultuur -toksiin B GDH (glutamaat dehüdrogenaas) kultiveerimine CCFA, Brazier s CCEYA, CDMN, chromid CD geenide määramine kommertsiaalsed real-time PCR GeneOhm (Beckton Dickinson): tcdb Xpert (Cephid): tcdb; cdta/cdtb; tcdc-deletion Prodesse ProGastro Cd: tcdb (Meridian Illumigene, Loop-Mediated Isothermal Amplification: tcda) in-house 5
6 Toxin A/B EIA (membrane) Toxin A/B EIA (well-type) GDH EIA (membrane) GDH EIA (welltype) Sensitivity % Range (Mean) (72)# (52)* (82)# (66)* Real-time PCR # 84-86* Specificity% Range (Mean) (98)# (98)* (97)# (98)* Eelised Sobib üksikuteks testideks Kiire, spetsiifiline, parem korrelatsioon kliinilise haigusega (90)# (90)# Sobib üksikuteks testideks (93)# (89)# # 97-98* kiire; kõrge NPV (95-100); sobilik skriininguks Tundlik ja spetsiifiline Puudused Madalam tundlikkus kui külvil; ei saa mikroobi tüve Madalam spetsiifilisus, positiivsed juhud tuleks kinnitada Kallim?; ei saa mikroobi tüve Külv ja tüve toksilisuse test Tundlik ja spetsiifiline; tüvi edasisteks uuringuteks Töö ja ajamahukas, vaja anaeroobse külvi süsteeme Toksiini määramine rakukultuuril Tundlik ja spetsiifiline Töö ja ajamahukas; vaja rakukultuure; ei saa mikroobi tüve # compared with cell culture cytotoxity assay; * compared with toxigenic culture 6
7 (Toksiini) testi tundlikkus sõltub tüvest 7
8 Diagnostika meetodid: soovitused külv (koos tox testiga kultuurist) +/- otsene tox test selektiivne sööde +/- alkohol sokk + tox test CD tüvest tox A+B EIA (rakukultuur) PCR (+/- külv) real-time PCR külv (epidemioloogiline vms vajadus) 2 astmelised algoritmid skriining-test positiivsete juhtude kinnitamine 8
9 CD diagnostika algoritm (näidis) Skriiningtest GDH GDH neg Vasta: CD neg GDH pos Kinnitav test: toksiin (+ külv) (Toks pos: vasta CD pos) Toksiin ja külv neg Vasta: CD neg Toksiin pos (külv pos v. neg) Vasta: CD pos Toksiin neg ja külv pos Määra toksiin CD tüvel Toksiin pos tüvi Vasta: CD pos Toksiin neg tüvi Vasta: CD neg 9
10 proovide säilitamine Proovivõtt C. difficile vegetatiivsed rakud ja eosed - külviks temp suurt ei mõjuta, hulgad stabiilsed päevi (nädalaid) CD toksiinid EIA testiks +4 C stabiilne päevi (nädalaid) -20 C ja (mitmed) külmutus/sulatus tsüklid vähendavad tundlikkust oluliselt 10
11 Näidustused EI ole näidustatud asümptomaatiliste patsientide skriining reeglina EI ravi kontrolli uuringud - reeglina EI (kuni 30% jäävad CD pos) mitte-vedela väljaheite uuringud - EI ON näidustatud kõik hospitaliseeritud patsiendid (>3d) kellel kõhulahtisus (sõltumata uuringu tellimisest)? haiglavälised patsiendi AB kõhulahtisusega? Epidemioloogilistel näidustustel AB tundlikkuse testimine tüpiseerimine 11
12 Infektsioon v kolonisatsioon? mida tundlikumad testid, seda enam avastame ka kolonisatsiooni kolonisatsioon + kõhulahtisus muust mehhanismist (patogeenist) 44 99% of AAD pole CD põhjustatud 7-50% haiglapatsientidest on CD-ga koloniseeritud seega, CD kolonisatsioon ja non-cd kõhulahtisus on võimalik kokkusattumus kas lab saab neid juhtumeid eristada? muud põletiku markerid: PMN; lactoferrin test pole spetsiifilised ega tundlikkud toksiini esinemine sooles viitab tõelisele infektsioonile? pole kindel marker 12
13 mild CDI vs. severe CDI: fecal toxin level 13
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