Chlamydia pneumoniae

Transcription

1 Chlamydia peumoiae Assessmet of the microimmuoiluorescece test for atibody detectio ad prevalece studies i adult patiets with respiratory tract ifectio A Ph.D - thesis by Mette Beedse

2 Chlamydia peumoiae Assessmet of the microimmuofluorescece test for atibody detectio ad prevalece studies i adult patiets with respiratory tract ifectio by Mette Beedse Departmet of Respiratory Ifectios, Meigitis ad STls Divisio of Microbiology States Serum Istitut Artillerivej 5, 2300 Copehage S Demark! '

3 Preface The studies i the preset dissertatio were carried out at the Neisseria Uit, Departmet of Respiratory Ifectios, Meigitis ad STis, Divisio of Microbiology, States Serum Istitut, Copehage, Demark, durig the years The objects of the studies were to assess the performace of commercially available microimmuofluorescece (MIF) tests compared to a referece method for the detectio of Chlamydia peumoia atibodies, to describe the prevalece of C. peumoiae ad other atypical respiratory tract ifectios i adult patiets with symptoms of acute ifectio ad i patiets with chroic pulmoary diseases ad to evaluate whether C. peumoiae is associated with acute exacerbatios of the diseases; fially, to assess the applicability of serological criteria for the diagosis of acute ad chroic C. peumoiae ifectios. The thesis is divided ito five chapters. Chapter oe is a itroductio to C. peumoiae based o literature studies. Chapter two describes the objects of the thesis, ad chapter three describes the study of the performaces of MIF tests ad further three mior laboratory studies. Chapter four describes the cliical studies ad fially, chapter five is a geeral coclu5io of the thesis ad a discussio of the curret kowledge of C. peumoiae ifectios i Demark. This dissertatio was preseted to the Faculty of Health Scieces, Uiversity of Copehage, Demark as a Ph.D.-thesis ad accepted to be defeded o 7 February Mette Beedse, M.D. Copehage, Jauary

4 PREFACE o 1 CONTENTS o 2 ABBREVIATIONS CHAPTER 1: CHLAMYDIA PNEUMONIAE... '7 History... 7 Biology... 8 Outer membrae structures... 9 Cliical diseases Respiratory tract diseases Cardiovascular diseases Other diseases Treatmet Acute ifectios Itervetio studies Diagostic methods Samplig Culture Polymerase Chai Reactio Direct Fluorescet Atibody test Serological methods Chlamydia Complemet Fixatio test Microimmuofluorescece test Epidemiology Trasmissio Seroepidemiological populatio studies Serological ad microbiological studies CHAPTER 2: AIMS OF THE STUDY

5 CHAPTER 3: LABORATORY STUDIES Itroductio The performace of three Chlamydia peumoiae microimmuofluorescece tests Material ad methods Results Detectio rates of C. peumoiae atibodies Edpoit titre levels Discrepat results Patiets with C. peumoiae ifectio Patiets with M. peumoiae ifectio Patiets with C. psittaci ifectio Cross-reactig atibodies Compariso of results for group I with the NAP Study Coclusio lgm atibody titre level ad duratio of icubatio of atige with serum Reproducibility of test results by the WRF MIF assay Correlatio betwee results of the CF test ad the WRF MIF assay for the detectio of C. peumoiae atibodies Discussio

6 CHAPTER 4: CLINICAL STUDIES Itroductio Objective Methods Study A: The prevalece of Chlamydia peumoiae i patiets With commuity-acquired acute respiratory tract ifectio Study populatio...,...., Results Patiets The questioaire survey Aetiological diagosis Atibody profiles Cotrols Coclusio Study B: Prevalece of Cblamydiapeumoiaei hospitalised patiets ad patiets see at a outpatiet cliic Study populatio Results Study populatio COPD The questioaire survey Classificatio accordig to cliical ad para cliical fidigs Microbiological results Serological evidece of C. peumoiae ifectios Atibody profiles C. peumoiae IgG atibody titres ad serum IgG C. peumoiae IgG atibody titres ad CRP Coclusio Discussio

7 CHAPTER 5: GENERAL DISCUSSION Epidemiology..., Seroepidemiological populatio studies Prevalece of acute C. peumoiae ifectio Microbiological studies Combied microbiological ad serological studies e Possible chroic C. peumoiae ifectio Patiets with reduced lug fuctio Patiets with cardiovascular diseases The diagosis of C. peumoiae ifectio Culture PCR CF test MIF assays Applicability of serological criteria Serological criteria of acute ifectio Serological criteria of chroic ifectio Coclusio SUMMARY Eglish Daish REFERENCES ACKNOWLEDGEMENTS TABLES FIGURES APPENDICES Appedix oe: Materials ad procedures of the MIF tests Appedix two: Questioaires

8 List of abbreviatios. Bp CF test CI COPD CRP DFA test EB EIA FEV 1 GP Hsp lg lg A lgg IgM LAB LPS MIF MOMP MRL NAA NAP Study OmcA OmcB PBMC PCR Pmp RB RF RNA rrna TWAR Base pair Complemet fixatio test 95% Cofidece iterval Chroic obstructive pulmoary disease C-reactive protei Direct Fluorescet Atibody test Elemetary body Ezyme immuoassays Forced expiratory volume i oe secod Geeral Practitioer Heat shock protei Immuoglobuli Immuoglobuli A Immuoglobuli G Immuoglobuli M Labsystems Lipopolysaccharide Microimmuofluorescece test Major outer membrae protei MRL Diagostics Nucleic acid amplificatio Nordic Atypical Peumoia Study Outer membrae complex A Outer membrae complex B Peripheral blood moouclear cells Polymerase chai reactio Polymorphic membrae proteis Reticulate body Rheumatoid factor Riboucleic acid Ribosomal RNA TW-183/ AR-39-like orgaisms (the origial ame for C. peumoiae) VC WRF Vital capacity Washigto Research Foudatio 6

9 CHAPTER ONE: CHLAMYDIA PNEUMONIAE History of Chlamydia peumoiae I 1965 Chlamydia (C.) peumoiae was isolated for the first time from the eye of a child durig a field trial testig a trachoma vaccie i Taiwa. The isolate was amed TW-183 ad cosidered a uidetifiable immuotype of C. trachomatis 57 I the microimmuofluorescece test (MIF) was developed by Wag ad Graysto for immuotypig of C. trachomatis isolates 163 ; it was further developed ito a tool for the diagosig C. trachomatis serovar-specific ifectios 164 TW-183 remaied utypeable, but whe icluded as atige i the MIF test a larger percetage of sera from 191 persos observed for the prevalece of trachoma had atibodies towards this atige tha towards C. trachomatis serovars 61 Durig the 1970s the iterest i the role of C. trachomatis i respiratory tract ifectios icreased. Serological evidece suggested that peumoia i childre < six moths of age could be caused by C. trachomatis 6 Several groups studied the possible coectio betwee C. trachomatis ad respiratory ifectios i adults. I 1978 Saikku came to the Uiversity of Washigto i Seattle to ivestigate sera from several respiratory tract ifectio outbreaks i Filad. The sera had show a positive result by the Chlamydia CF test ad he wished to test them with the ewly developed MIF test for the detectio of serotype-specific C. trachomatis atibodies. No sigificat levels of atibodies agaist ay of the C. trachomatis serovars were foud, but whe usig TW-183 as atige, IgG atibody titre rises ad IgM atibodies was demostrated implyig that a TW-183 orgaism had caused oe of the outbreaks 131 Subsequetly the group i Seattle coducted a study i studets who came to the medical cliic at the Uiversity with symptoms of acute respiratory tract ifectios. From the 39th studet a isolate (AR-39) immuologically similar of TW- 183 was foud. The orgaism was called TW- 183/AR-39-like orgaism or TWAR. To study the epidemiology of ad cliical diseases caused by TW AR, serum collectios from various places were aalysed by the MIF test. Mordhorst at States Serum Istitut i Demark provided oe of the most comprehesive collectios of sera from patiets suspected of havig psittacosis i All sera had positive test results for chlamydia! atibodies by the CF test. The results by the MIF test showed that 52-57% had serological evidece of ifectio caused by TWAR 63 7

10 I 1988 Saikku published the first study i which high levels of TWAR IgG ad IgA atibodies were correlated to the risk of havig a cardiac episode or chroic heart disease 128 The year after the orgaism was officially amed C. peumoiae ad cosidered a commo cause of acute respiratory ifectios 64 Biology Taxoomy. The order Chlamydiales has oe family, Chlamydiaceae ad oe geus, Chlamydia which cosists of four species: C. trachomatis, C. psittaci ad C. peumoiae that are huma pathoges ad the rumiat pathoge C. pecorum64;114. This classificatio is based o pheotypic, morphologic, geetic ad serologic criteria. I 1999 a ew taxoomic order was suggested based o phylogeetic aalyses of the 16S ad 23S rrna gees 45 The major criterio i this classificatio is the divergece of rrna. The Chlamydiales is divided ito four families: Chlamydiaceae, Parachlamydiaceae, Simkaiaceae ad a fourth, curretly uamed family. Chlamydiaceae is divided ito two geera: Chlamydia, which amog others cotais C. trachomatis, ad Chlamydiaphila i which C. peumoiae ad C. psittaci are icluded. So far the order of Chlamydiales cotais 14 species, icludig three species that were previously cosidered to be C. psittaci hosted i differet aimals. Curretly, the chlamydial society is debatig about which classificatio should be the official classificatio 44 ; 133 I this thesis the origial classificatio is used. Characteristics. The geus Chlamydia is a heterogeeous group of small bacteria with similarities i morphology, itracellular developmetal cycle ad atigeic properties. Lackig some of the ezymes eeded to be metabolic idepedet orgaisms, they are obligate itracellular parasites 94 ; 141 Sequeces for the whole geome of a umber of Chlamydia strais have become available sice 1998, icludig sequeces for C. peumoiae AR39 ad C. trachomatis serotypes B, D ad L2 85 ; 126 ; 142 Withi the geus Chlamydia, the geome of C. peumoiae has a 1,230,230 base pair (bp) chromosome codig for up to 1,073 proteis ad that of C. trachomatis has a 1,042,519 bp chromosome codig for up to 894 proteis 85 About 200 of these proteis are uique to C. peumoiae. 8

11 Life cycle. Commo to all Chlamydia species is the biphasic life cycle comprisig of a smaller extracellular, metabolic iert, ifectious form, the elemetary body (EB), ad a larger itracellular, metabolic active form, the reticulate body (RB) 78 EBs are small, roud or occasioally pear shaped, electro-dese structures approximately 0.3 micros i diameter. The EB is the oly ifectious stage of the chlamydia! developmetal cycle. It fuctios as a tough body whose purpose is to permit chlamydia! survival i the eviromet outside the host cell. The EBs attach to susceptible host cells ad are iteralised by a ivagiatio of the host plasma membrae to evelop the EB i a tightly associated vesicle 67 Withi the vesicle, they trasform ito RBs. RBs are the stage of the chlamydia! developmetal cycle resposible for itracellular replicatio. Typically, reticulate bodies have a diameter of 1 micro ad they are o ifectious. Reticulate bodies are metabolically active, so their cytoplasm is rich i ribosomes, which are required for protei sythesis 67 The RBs replicate withi a membrae-boud vacuole, the chlamydia! iclusio. The iclusio elarges as the umbers of progey icreases. Gradually the RBs revert to the EB form ad after app. 72 hours the life cycle is completed. The egressio of the mature, ifectious EBs occurs by lysis of the host cells 94 Outer membrae structures. Aalyses of the chlamydia! outer membrae complex has show that Chlamydia species have LPS, a major outer membrae protei (MOMP), ad two cysteierich proteis: outer membrae complex B (OmcB) ad outer membrae complex A (OmcA) 30 ' 78 I additio, C. peumoiae, C. psittaci ad C. trachomatis cotai proteis belogig to a family of proteis amed polymorphic membrae proteis 141 ad heat shock proteis 78 Lipopolysaccharide (LPS): LPS are highly immuogeic glycolipids. All species of Chlamydiae have commo LPS group atige exposed o the surface 32 However, the LPS may have more tha a geus-specific epitope 142 Surface exposure of LPS may be greater o RBs tha o EBs 15 ' 78 Chlamydia! LPS show commo epitopes with LPS from some of the eteric bacteria 40 The major outer membrae protei (MOMP): The protei preset i the largest quatity i EBs ad RBs is MOMP ecoded by the ompa gee. I all species, the 9

12 MOMP cosists of four variable segmets I domais (termed VS or VD 1 to VD 4). These variable segmets ted to be surf ace exposed ad the MOMP is both structurally ad immuologically the domiat protei i the chlamydial outer membrae complex except i C. peumoiae 78. The typig of C. trachomatis is based o the serological differetiatio of atigeic epitopes o MOMP ito 19 huma C. trachomatis serovars (A to K, Ba, Da, Ia, Ja, L1 to L3, ad L2a) 31 ; 78. I cotrast, the C. peumoiae MOMP is homogeous, is ot a major immuoge ad may ot be surface exposed 89. Cysteie rich proteis of the chlamydial outer membrae complex. Apart from MOMP, the major proteis i the chlamydial outer membrae complex are the two cysteie rich proteis, OmcA ad OmcB, preset i greatest amouts i the elemetary body 78. OmcB (=omp2) is a large 60 KDa protei. The OmcB is highly immuogeic, carries geus specific epitopes, but is thought ot to be surface exposed 78. The protei is a major target i the humoral respose i ifectios caused by C. trachomatis ad may likewise be so for ifectios caused by C. peumoiae 115 ' mcA (=omp3) is a lipoprotei with a molecular weight of 9 KDa, ot exposed at the chlamydia! surface 67 ad may ot be immuogeic. Residues 1-88 of the C. trachomatis gee are betwee 55-59% homologous with the equivalet regio of the correspodig C. psittaci ad C. peumoiae gees1a,101. Polymorphic membrae proteis (pmps). Each of the three Chlamydia species has i its geome a family of distatly related Pmp gees 30. Compariso of the C. trachomatis ad C. peumoiae pmp gee sequeces show a large amout of heterogeeity amog members of the family 142 It is ot clear whether the variability of pmp gee sequece is due to pressure from the host immue system, or is itrisic. At least some of the proteis are located a t the surface of the ifectious chlamydia! elemetary body 89. The surface exposed pmps, OMP4 (98.9 kda) ad OMP5 (97.2 kda). was described i C. trachomatis ad C. peumoiae i 1998 ad ' 142 Epitopes of OMP4 ad possible the other pmps ecoded by the C. peumoiae gee family are likely to be the target for immue respose i mice experimetally ifected C. peumoiae ifectios

13 , Heat shock proteis (hsp) are a family of closely related proteis, widely distributed i virtually all orgaisms, icludig Chlamydia ad humas. Hsp show remarkable structural similarity of the order of 40% or more. Chlamydiae are o exceptio, with the mai chlamydia! heat shock proteis showig homology with huma mitochodrial proteis cplo, hsp60 or hsp70. Hsp 70 has bee show to be located i outer membrae complexes of chlamydia! EBs, though they might ot ormally be accessible to atibodies 78. Hsp60s are highly immuogeic 166 ad might be ivolved i autoimmue resposes agaist the huma hsps 166. Cliical diseases. Respiratory tract diseases. Ifectios of the respiratory tract are the most commo recogized acute C. peumoiae ifectios. No set of symptoms or sigs is uique to acute C. peumoiae ifectios, however a subacute oset is commo There may be a biphasic patter i the cliical maifestatios, with resolutio of pharygitis prior to developmet of brochitis or peumoia Geerally, the symptoms are mild to moderate severe ad most cases do ot require hospitalisatio. Despite appropriate atibiotic therapy, complete recovery is slow; prologed illess ad relapses are commo The period from oset to cliic visit is loger tha for most other acute respiratory ifectios ad may be more tha 14 days147;14s. Upper respiratory tract ifectios. C. peumoiae has bee isolated from patiets with pharygitis, larygitis, otitis ad siusitis Sore throat, plugged ears, swolle throat, cough, headache ad fatigue may be preset aloe or i combiatio 60 : 74 : 79, Lower respiratory tract ifectios. Peumoia ad brochitis are the two most frequetly recogized acute illesses associated with C. peumoiae. The peumoia patiets ofte have sore throat ad hoarseess followed by a persistet cough. The body temperature may be slightly icreased ad seldom goes higher tha C. The leukocyte cout is ofte ormal but sedimetatio rate might be elevated. A chest radiograph usually demostrates a sigle sub lobular ifiltrate though lobular ad bilateral ivolvemet may be see, especially i the severe cases34:94:1os. 11

14 Asthma. I the last decade C. peumoiae has bee associated with asthma 35 ; 68 ; 69. It has bee suggested that C. peumoiae ifectio could be a iitiator of adultoset asthma 72 Some studies have show that C. peumoiae causes asthma exacerbatios69'109 ad that treatmet with macrolide may improve the course of asthma 18 ; 71. However, results from other studies do ot support the hypothesis that C. peumoiae is a cause of adult-oset asthma 33 ; 98. Chroic Obstructive Lug Disease. As C. peumoiae is ivolved i acute ad protracted respiratory tract ifectios, it seems reasoable that the orgaism could be ivolved i the pathogeesis of COPD. I theory C. peumoiae ca play a role i iitiatio, developmet or exacerbatios of COPD. High seroprevalece of C. peumoiae IgG atibodies i COPD patiets has bee demostr{:l.ted 69 ad a associatio betwee C. peumoiae ad acute exacerbatios foud 20. I oe study oly small differeces i serum IgG atibodies betwee patiets ad cotrols were foud, however the differeces i serum IgA prevaleces were sigificat 159. Other respiratory tract diseases. O the basis of seroepidemiological studies sarcoidosis ad lug cacer have bee suggested as beig associated with C. peumoiae99;125. Cardiovascular diseases. Sice the correlatio betwee ischaemic heart diseases ad elevated levels of C. peumoiae IgG ad IgA atibody titres was show by Saikku i , extesive research has bee coducted i the attempt to establish, whether C. peumoiae is ivolved i the developmet of atherosclerosis ad acute cardiovascular evets. Several studies have cofirmed the associatio of acute ad chroic heart diseases with a raised level of C. peumoiae atibody titres Further some studies have idetified C. peumoiae i atheromatous tissues by electro microscopy, immuocytochemistry or PCR 95 ; 139 ' 145. Culture of C. peumoiae from atherosclerotic plaques has bee reported 101. However, as these data do ot establish a causal relatioship betwee the C. peumoiae ad oset or progressio of atherosclerosis, it is possible, that C. peumoiae is o more tha a iocet bystader withi atherosclerotic plaques. Other diseases. Some have reported that reactive arthritis ca be caused by C. peumoiae, however it is less commo tha arthritis caused by C. trachomatis 25 12

15 Several reports have associated C. peumoiae with eurological diseases such as Alzheimer's disease, multiple sclerosis ad giat-cell arteritis Treatmet Data o treatmet of C. peumoiae ifectio are limited, thus most recommedatios are primary fouded o empirical observatios ad experieces from the treatmet of C. trachomatis ifectio. However, sice C. peumoiae is a itracellular orgaism, atimicrobial agets must be able to peetrate cells to be active agaist the bacteria. Macrolides, tetracyclies, chlorampheicol, quioloes ad rifampici have demostrated atimicrobial activity itracellularly for C. trachomatis or other itracellular pathoges 55 Acute ifectios. Treatmet with azithromyci oce daily for five days have bee show to have a satisfactory therapeutic outcome similar to those of amoxicilli/clavulaate or erythromyci give three times a day for 10 days for treatmet of commuity-acquired peumoia 77 Eradicatio rates of % for C. peumoiae have bee demostrated with levofloxaci, moxifloxaci, clarithromyci, erytromyci ad azithromyzi i patiets with peumoiae 75 Itervetio studies. The last few years several itervetio studies have bee coducted to study the associatio betwee cardiovascular diseases ad C. peumoiae. Regimes used were amog other: oral roxithromyci 150 mg/ for 30 days (The ROXI Trial) 66 ; azithromyci 500 mg/d for 3 days, the 500 mg weekly for three moths (The ACADEMIC Study) 3 ; azithromyci 600 mg/week for 1 year (ACES) 81 ad chlarithromyci 500 mg/d for 14 days (CLARICOR) 76 So far oly few studies are cocluded ad there is yet o geeral recommedatios as to the beefit of treatig patiets with cardiovascular diseases with atibiotics. Oe of the largest studies, WIZARD (Weekly Itervetio with Zithromax for Atherosclerosis ad Related Disorders) had icluded subject with a history of myocardial ifectio ad the presece of C. peumoiae IgG atibodies of;?: 16, They were give either azithromyci 600 mg 4 times a day for 3 days the 600 mg oce weekly for the ext 11 weeks or a placebo 41 Earlier this year it was aouced that, just before the compay was scheduled to report the results, the study was termiated o the grouds that azithromyci i this study had failed to sigificatly reduce heart disease. 13

16 Diagostic methods A laboratory diagosis of C. peumoiae ifectio ca be fouded o the detectio of the bacteria or by serological methods 6 Samplig. Sice the C. peumoiae is a itracellular bacterium it is of major importace for the detectio of the orgaism, that the sample cotais epithelial cells. Specimes obtaied for detectio of C. peumoiae tract ifectio by use of culture iclude swabs of the asopharyx, oropharyc, sputum specimes, brocheoalveolar lavage specimes ad tissue biopsy specimes 40. C. peumoiae DNA has bee detected by PCR i cliical samples ragig from respiratory specimes to samples of vascular tissue, serum ad peripheral blood moouclear cells4o; 139. There are oly few papers published o the optimal samplig site for laboratory diagosis of acute, respiratory, C. peumoiae ifectios. Oe study compared specimes collected simultaeously ad foud that a positive diagosis was more frequetly obtaied with sputum samples tha with throat ad asopharygeal swabs 22. Culture. C. peumoiae is cultured i eukariotic host cells. Origially the culture was performed i embryoated yolk sacs but durig the 1980s a method for i vitro cell culture was developed 92. The HeLa 229 cell-lie was used for the culture of C. trachomatis i several laboratories, thus it was used i the first attempts of culturig C. peumoiae 60 Later it was foud that Hep-2 cells ad the HL cells had a higher sesitivity for the culture of C. peumoiae ad are thus recommeded48;91;93. The advatage of culture is the specificity, which by use of a appropriate idetificatio method is 100% while the sesitivity is depedet o the stadard of the laboratory procedures ad of the suitability of the specimes received for testig. C. peumoiae is cosidered to be difficult to grow ad oly few laboratories perform culture. A major problem is that C. peumoiae is easily iactivated durig trasport. A 50% -80% loss of ifectivity of strais stored at room temperature i hours was documeted 48 ; 102. To ascertai the best possible result of culture, the sample must be forwarded i Chlamydia trasport 14

17 medium cotaiig fetal calf serum, the trasport time shall be as short as possible ad preferable the sample should be kept refrigerated durig trasport Polymerase chai reactio (PCR).. PCR is the ucleic acid amplificatio (NAA) techique used for the diagosis of acute C. peumoiae ifectio. There is yet o commercial NAA assay available for routie diagostics but several protocols, differet targets ad primers have bee published for the detectio of C. peumoiae DNA. The targets most widely used are the major outer membrae proteicodig gee ad the 16S rrna gees 26 ; 52 ' 151 The ested PCR methods i geeral have a higher sesitivity tha the oe-step-based PCR methods but also a higher risk of false positive results 22, 16 Though guidelies have bee developed to miimize this risk 97, comparative studies show divergig iter-laboratory results 4 ; 24 ; 97 The advatage of PCR i the diagosis of a acute C. peumoiae ifectio is the rapidity. The test ca be performed i oe to two days. The risk however, is false positive result due to cotamiatio or false egative results due to ihibitio. Thus all positive results should be cofirmed by retestig the specime with a differet set of primers 23 Direct Fluorescet Atibody (DFA) Test. Direct detectio of elemetary bodies of C. peumoiae i smears is possible by usig commercially available, fluoresceilabelled, moocloal atibodies specific for C. peumoiae 111 The sesitivity is estimated to be 20%-60% compared to culture. The specificity is said to be 95%- 99% but depeds o the expertise of the microscopist, as the readig of results is subjective. The oe beefit of the DFA test is that the result is available withi miu 9 Serological methods. Various ezyme immuoassays (EIA) for the detectio of C. peumoiae atibodies are commercially available. They use either LPS-extracted EBs or sythetic peptides uique to C. peumoiae as atige for the detectio of Chlamydia atibodies 152 Some are geus-specific others claim to be speciesspecific but assessmet of their diagostic performaces is limited ; 122 ; 123 ; 152 I cotrast to the MIF test, the EIA has a objective readout of results ad it is easier to hadle a large umber of samples. For this reaso there is ogoig research to attempt to idetify a highly immuogeic target species-specific atige. How- 15

18 ever, the CF test ad the MIF test are still the serological methods most widely used i serological testig for C. peumoiae ifectio. Complemet fixatio test. The test is based upo the ability of the atigeatibody complex to bid complemet ad thereby prevet lysis of erythrocytes. The idicator system is sheep erythrocytes sesitised with rabbit atiserum to sheep erythrocytes. The complemet source is fresh guiea pig serum. The complemet of the sera examied is iactivated by heat. The test is less techically demadig tha the MIF test ad is widely used. The atige used i the Chlamydia CF test whe performed i our laboratory is a ether-extracted, acetoeprecipitated phosphatide atige, produced i yolk sac membraes ifected with C. trachomatis 157 Geerally the atige used for the CF test is cosidered to be LPS12;11e;1s2. Atibodies reactig i the CF test are produced early i primary ifectio. Hece a serodiagosis may be made withi oe to two weeks after ifectio; i cotrast the species-specific atibodies are see after two to six weeks. Diagostic criterio of the test is a fourfold icrease i titres 123 ; 156. A sigle high titre of ;::: 64 ca sigify acute ifectio but is ot as reliable as the former criterio 60. The sesitivity of the CF test for acute, primary C. peumoiae ifectios is by some foud to be as high as 90% 42, whereas others fid it to be 60% 119 ; 122. Microimmuofluorescece (MIF) test. I the MIF test, purified EBs fixed o slides are used as the source of atige. EBs from differet Chlamydia species ca be applied to the same slide. Thus geus-specific as well as species-specific atibodies ca be detected. After icubatio with patiet sera, fluorescei-cojugated ati-huma immuoglobulis are added to detect IgG, IgM ad IgA atibodies. Appedix 1 is a i-detail descriptio of the three MIF assays further ivestigated i the preset study. The target of the atibodies i the MIF test is atiges preset i the EBs. As described above, the MOMP epitopes are the pricipal C. trachomatis atiges, while epitopes reactig with ati-c. peumoiae atibodies may be preset o the pmps. Eve after the availability of PCR, the MIF test is cosidered the gold stadard for laboratory diagosis of acute C. peumoiae ifectio 40 ad has also bee used for measurig C. peumoiae atibodies i patiets with assumed chroic C. peumoiae ifectio 127. However, there are several techical aspects to be co- 16

19 sidered regardig the MIF test: It is time-cosumig ad requires experiece to perform; a idirect immuofluorescece test is subjective ad the quality of the microscope as well as that of the reagets used is importat. It has bee demostrated that the use of a C. peumoiae atige produced from a local isolate ca result i higher detectio rates of atibodies i sera ad higher titres i the MIF test tha whe usig a stadard 17 The questio of possible cross-reactio i the C. peumoiae MIF test is cotroversial. While some fid the test to be highly species-specific , others have foud a strog associatio betwee atibody titres to the three chlamydial species 168 Part of the explaatio for this may be that eve with a great deal of experiece with the test, geus specific atibodies still ifluece the results The specificity of the MIF assay ca be attributed to the use of purified EBs of Chlamydia species rather tha RBs that predomiately express /geus-specific epitopes 40 Apart from the problem of chlamydia! species cross-reactivity I geus-specific reactivity i the MIF test, there are also cocers, that false-positive MIF test results may be due to cross-reactig atibodies to ochlamydial atiges 87 Serological cross-reactios betwee Bartoella ad C. peumoiae species have bee demostrated 104 Termiology of serodiagostic criteria. I a primary ifectio C. peumoiae IgM atibodies detectable by the MIF test occur after about three weeks ad IgG atibodies after 6-8 weeks. I reifectio IgM atibodies may ot appear or may appear oly at low titres while the IgG titre rises quickly, ofte withi 1-2 weeks. Normally IgM atibodies begi to fall withi two moths ad disappear i four to six moths but i a few cases IgM atibodies persist for a loger period. I cotrast, IgG atibodies may be detected several years after ifectio 94 Origially, Graysto divided the results of the MIF test for C. peumoiae ito acute ad pre-existig atibody profiles 60 Acute atibodies profiles are either IgM atibody titres of ::::: 16 ad/or a four-fold chage of IgG atibody titres or a stable IgG atibody titre of ::::: 512 (table 1). A high ad stable IgG atibody level is o loger strictly regarded as a sig of acute ifectio 35 ; 4 0;l 52 but may be idicative for a acute ifectio together with cliical ad epidemiological data. Pre-existig atibody profiles are IgG atibody titres I some publicatios the preexistig atibody profile has bee subdivided ito oe for chroic ifectios (with the presece of IgA atibody titres of~ 40) ad oe for past ifectio (with IgA a- 17

20 tibody titres of~ 40) ' 129 The role of IgA atibodies i associatio with the diagosis of chroic ifectios has bee questioed 162 ad is curretly ot regarded as a validated idicator of persistig ifectio 40 Rheumatoid factor. The prevalece of circulatig rheumatoid factor (RF) is strogly correlated with age ad the presece of RF i sera cotaiig C. peumoiae IgG atibodies may give false positive C. peumoiae IgM test results. I oe study 41 of 286 patiets had C. peumoiae IgM atibody titres of::::. 16. Although oly 78 % of the patiets had detectable circulatig RF, oe of the 41 sera were show to have C. peumoiae IgM atibodies after absorptio of IgG atibodies. Sice circulatig RF at a level below the threshold of detectio by routie testig for RF ca cause false positive IgM atibody results it is ecessary to cofirm all C. peumoiae IgM atibody positive results after absorptio of IgG atibodies 154 Blockig atibodies. It has bee show that the presece of C. peumoiae IgG atibodies ca ifluece the results of IgA measurig 82. The study aalysed the effect of removig IgG atibodies before testig for IgA atibodies: i most cases o chage of IgA atibody titres were see but i 9 % of samples a sigificat icrease was detected. This icrease was correlated to high IgG atibody titres suggestig that the higher bidig affiity of IgG or a much higher cocetratio of IgG tha IgA atibodies i sera blocks IgA reactivity i the test. Epidemiology C. peumoiae ifectios seems to be both edemic ad epidemic i the wester coutries 56 ' 60 ' 94 ; 149 No evidece of seasoal periodicity i C. peumoiae ifectio had bee observed 146, but recetly a correlatio betwee C. peumoiae ifectio i cliically stable COPD patiets ad seaso has bee reported 138 Sice C. peumoiae is ot ofte isolated or detected by PCR, much of the curret iformatio o the epidemiology of C. peumoiae is derived from seroepidemiological studies usig the MIF test. Trasmissio. Humas are the oly kow reservoir of C. peumoiae ad trasmissio is believed to be from ma to ma by ifectious aerosol droplets 47 ; 131 without ay aimal reservoir 94 ; 131 C. peumoiae ifectio spreads slowly with a 18

21 icubatio time of several weeks; most ofte outbreak ivestigatios have revealed o direct chai of trasmissio betwee cases but outbreaks withi families have bee described 49 ' 88 ' 112 ' 171 Seroepidemiological populatio studies. Data from studies from may parts of the world have show a worldwide diffusio of C. peumoiae atibodies. Childre uder the age of 5 years i developed coutries seldom have C. peumoiae atibodies, i cotrast some evidece has bee reported, that seroprevalece i preschool childre i developig coutries is high 130 Seroprevalece icreases durig school age, reaches 40-50% i those years old ad cotiues to icrease at a slower rate amog older age groups 19 ' 58 ' 118 ' 153 The seroprevalece is equal i both sexes uder 15 years of age; however adult me usually show a higher seropositivity rate tha adult wome. Seroprevalece is higher i immuocompromised persos tha i immuocompetet hosts 19 The first Daish, C. peumoiae seroepidemiological study was coducted durig two time periods ( ad ) 165 Sera from a total of 1763 persos were collected by Mordhorst from the Iflueza laboratory of States Serum Istitut. The sera were aalysed for C. peumoiae atibodies by Wag ad Graysto. The purpose was to estimate the "TW AR populatio atibody prevalece" defied as IgG atibody titres of i.e. preexistig atibody profile. For this reaso all sera with C. peumoiae IgG atibody titres of ~ 512 were excluded, though it was stated that less tha 10% of the sera had IgG atibody titres of~ 512. Figure 1 shows the "TWAR populatio atibody prevalece" accordig to age groups. I total, 39% had IgG atibody titres of The prevalece ad age distributio was similar to those foud i other studies 60 ; 63 A secod study by Mordhorst icluded sera from 1114 patiets submitted to the Iflueza laboratory from 1975 to 1987 o the suspicio of omithosis aalysed by MIF, all but 22 of the sera had a positive result by CF test 113 The Graysto criteria of acute ifectio were applied to assess the icidece of acute C. peumoiae ifectio. Accordig to these criteria the icidece of C. peumoiae ifectio amog the patiets durig the whole period was 54%, icludig to time periods with a much higher icidece. However, the patiets i this study were strogly selected as the vast majority had positive results by the CF test. Furthermore IgG atibody titre of~ 512 was used as a criterio of acute ifectio. 19

22 Serological ad microbiological studies. Util recetly most epidemiological data have bee based o seroepidemiological studies. Overall, whe usig the Graysto criteria for a acute ifectio, these studies have show that C. peumoiae is a commo pathoge i respiratory tract ifectios i all age groups > the age of five. As more laboratories perform culture of C. peumoiae ad with the emergece of PCR it is ow possible to compare the serological based fidigs with data from studies i which the orgaism has bee detected. C. peumoiae ifectio i childre. Acute ifectio verified by culture ca occur i childre< five years without serocoversio i MIF The positive correlatio of C. peumoiae serology ad culture is low amog pre-school childre 21 o77. I two Daish studies the prevalece of C. peumoiae amog childre with acute respiratory tract ifectios was ivestigated by detectio of C. peumoiae i respiratory tract secretios by culture ad PCR 48. I total 691 childre were examied ad C. peumoiae was foud i < 1 %. C. peumoiae ifectio i adults. I acute respiratory ifectios i adults ad older childre there is a acceptable positive correlatio betwee IgM atibody titres of~ 16 I fourfold IgG atibody titre rise ad culture ad it seems reasoable to coclude that C. peumoiae ca be recogized as a frequet cause of upper ad lower respiratory tract ifectios. Numerous studies have published fidigs that 5-10% (-20%) of peumoia cases i adults are attributed to this orgaism2'42'56'59'148. However, the prevaleces of C. peumoiae ifectio foud i differet studies vary. I several epidemiological studies the presece of IgG atibodies of ~ 512 has bee idicative of acute ifectio but the positive correlatio betwee a sigle high serum atibody titre ad detectio of C. peumoiae by culture or PCR has bee poor. 20

23 CHAPTER TWO: AIMS OF THE STUDY. To assess the performace of two commercially available MIF tests compared to a referece method whe applied to sera from differet categories of patiets. To assess the applicability of serological criteria for the diagosis of acute ad chroic C. peumoiae ifectios. To describe the prevalece of C. peumoiae ifectio i adult patiets with acute commuity-acquired respiratory tract ifectios by use of microbiological ad serological methods. To describe the prevalece of C. peumoiae ifectio i patiets with chroic respiratory pulmoary diseases ad to evaluate whether C. peumoiae is associated with acute exacerbatios of the disease by use of microbiological ad serological methods. 21

24 22 ' CHAPTER 3: LABORATORY STUDIES. Itroductio. Today several commercially available MIF assays as well as i-house assays for the detectio of C. peumoiae atibodies are widely used 106 ' 120 ' 122 ; they are based o the same priciple as the origial test 163, but some modificatios of the procedures have bee itroduced. Whe the preset study was iitiated, oly few comparative studies o the performace of the assays had bee coducted 51 ' 120 Oe study examied the iterlaboratory variatio i MIF test results obtaied i 13 laboratories aalysig 22 sera from 10 patiets 120 The agreemet betwee a referece stadard value ad the IgM atibody results obtaied by the 13 laboratories usig 11 differet methods was 50-95%; four laboratories failed to discer false-positive IgM titres possibly because of the presece of rheumatoid factor. For IgG atibody aalyses the agreemet was 68-87%. I aother study, oe laboratory compared two MIF assays (a referece method ad a commercially available assay) ad foud a sigificat differece i IgG ad IgA atibody titre levels 51 The objective of the preset study was to assess the performace of two commercially available C. peumoiae MIF assays, oe from MRL Diagostics, Cypress, USA (MRL) ad aother from Labsystems, Helsiki, Filad (LAB). The two commercial assays (LAB ad MRL) were uder evaluatio for use i our routie laboratory. A MIF assay based o C. peumoiae atige from Washigto Research Foudatio, (ow Washigto Uiversity), Seattle, USA (WRF) was used as a referece method. The WRF assay was chose as the referece method because this assay had bee available for research purposes durig decades ad because previous Daish studies o the prevalece of C. peumoiae atibodies had bee coducted with the WRF assay 76 ; 113 ; prior to the mai study the reproducibility of C. peumoiae IgG, IgM ad IgA atibody detectio by the WRF assay was determied. I the mai study the performace of the three assays was assessed by aalysig sera from patiets with acute respiratory tract ifectio of kow aetiology obtaied at various itervals after the oset of the disease. Sera from persos erolled i the Copehage City Heart Study were icluded due to the preset iterest i the detectio of C. peumoiae atibodies i patiets with cardiovascular diseases 76 ; 128

25 Sice the MIF assays are expesive ad time cosumig to perform, resources will be saved by use of a screeig procedure prior to the aalyses by the MIF assay. I previous C. peumoiae seroepidemiological studies some authors foud a high positive correlatio betwee the detectio of atibodies by the CF test ad the MIF assay 50 ; 167, whereas others foud that positive CF test results were poor predictors of positive MIF results To examie whether the CF test was a appropriate tool for screeig sera prior to the MIF assay aalyses a study was coducted to assess the correlatio betwee positive test results by the CF test ad by the WRF MIF assay. 23

26 THE PERFORMANCE OF THREE CHLAMYDIA PNEUMONIAE MICROIMMUNOFLUORESCENCE ASSAYS. Material ad methods Sera. This study icluded sera from three groups of patiets: two with acute respiratory tract ifectio ad oe icludig patiets with possible chroic ifectio but without kow acute ifectio. Group I cosisted of 83 sera from 28 patiets erolled i the Nordic Atypical Peumoia Study (NAP Study) 116 The sera were kidly provided by J. S. Jese, the Mycoplasma Laboratory, States Serum Istitut with the permissio of P. Saikku, Uiversity of Oulu, Filad. The 28 patiets had serological evidece of ifectio with C. peumoiae (13 patiets), Mycoplasma peumoiae (11 patiets), or both agets (4 patiets). The origial MIF aalyses for C. peumoiae atibodies had bee performed usig atiges from WRF 116 Group II cosisted of 37 sera from 16 patiets with acute C. peumoiae (8 patiets) or C. psittaci (8 patiets) respiratory tract ifectio cofirmed by PCR (15 patiets) or culture {oe patiet) i our routie laboratory. Of the 15 patiets tested, 14 had developed atibodies detectable i the CF test; moreover, cliical data were available for all patiets with C. peumoiae ifectios ad for 5 of 8 patiets with C. psittaci ifectios. For the last group of patiets, epidemiological data were also available 46 Culture was performed i Hep2 ad McCoy cells; subsequet idetificatio was obtaied with geus- ad species-specific staiig methods 140 PCR was performed by a i-house method 140 Group III cosisted of 100 sera from 100 persos erolled i the Copehage City Heart Study, which is a prospective, ogoig, populatio study: 20 persos who developed acute myocardial ifarctio withi three years after the blood samples were draw ad 80 cotrols matched accordig to geder ad age at etry without subsequet myocardial ifarctio 5. MIF assays. The three MIF assays are described i brief below. For a detailed descriptio of the methods see appedlx 1. Prior to IgM ad IgA atibody testig all sera were treated with GullSORB, Gull Laboratories, Salt Lake City, USA. All aalyses were performed usig a Zeiss axioskop 20 microscope with a plaeofloar 40x/0.75 objective. The procedures followed the recommedatios of each maufacturer. 24

27 WRF assay: As atiges, purified C. peumoiae elemetary atibodies (strai AR 39) were fixed oto the slides. The cojugates used were fluorescei-labelled rabbit ati-huma IgM, IgG ad IgA (DAKO, Glostrup, Demark). The icubatio time was 30 mi for both sera ad cojugates. MRL assay. For MIF assays (IgM, IgG ad IgA) from MRL, the slides were purchased with prefixed atige dots for C. peumoiae (strai TW183), C. trachomatis (8 serotypes(d-k)j ad C. psittaci (strais 6BC ad DD34). For the detectio of IgM atibodies, the icubatio period with serum was 90 mi.; for the detectio of IgG ad IgA atibodies, it was 30 mi. The subsequet icubatio time with the cojugate {fluorescei-labelled goat ati-huma!g's (MRL)) was 30 mi. LAB assay. The slides for MIF assays (IgM ad IgG) were purchased with prefixed atige dots for C. peumoiae (strai Kajaai 6), C. trachomatis (strai LGV/ L2) ad C. psittaci (strais OF 6bC ad EAE). For the detectio of IgM atibodies, the icubatio period with serum was 180 mi., for the detectio of IgG atibodies, it was 30 mi. The subsequet icubatio time with the cojugate (fluorescei-labelled goat ati-huma Ig's (LAB)) was 30 mi. All sera were tested i serial twofold dilutios from the followig iitial dilutios ad util a edpoit was reached. Group I: 1:16 (IgM), 1:64 {IgG), 1:16 (IgA); group II: 1:16 (IgM), 1:64 (IgG); group III: 1:16 (IgG), 1:16 (IgA). A edpoit titre was defied as the highest serum dilutio with a positive test result. A overview of the experimets performed is preseted i table 2. Statistics A compariso of detectio rates obtaied by the three assays was performed with Cochra's Q test; the likelihood ratio test was used to compare the proportio of cocordat results (both assays positive or both assays egative) obtaied by the WRF ad the MRL assays versus that obtaied by the WRF ad the LAB assays. For compariso of the atibody titres Friedma's oparametric 2-way aalysis of variace was used. These aalyses were performed by SAS, versio 8 (SAS Istitute Ic.). For compariso of the prevalece of atibody titres at certai levels withi groups, x 2 or Fisher's exact test was used. The 95% cofidece itervals (CI) for detectio rates were calculated as follows: 25

28 The proportio (p) ± 2~p(I- p)/) (ormal approximatio). P < 0.05 was cosidered sigificat. I geeral, test results for all sera are used for the statistical aalyses. The results of the same aalyses whe oly icludig the first serum sample per patiet are give separately. Results Detectio rates of C. peumojae atibodies. The presece of C. peumoiae IgM ad IgG atibodies i 120 sera from 65 patiets with acute respiratory tract ifectio (groups I ad II) is illustrated i tables 3-6. No differeces betwee the WRF, the MRL ad the LAB assays were see i the detectio rates of IgM atibody titres of;?: 16, or i the detectio rates of IgG atibody titres of ;?: 64 (table 7). Cocordat results (both assays positive or both assays egative) were demostrated i ~ 92% of the sera for IgM atibodies ad i~ 76% of the sera for IgG atibodies by the MRL ad the LAB assays versus the WRF assay (table 8). No sigificat differeces i the percetage of cocordat results were see (IgM: P= 0.16, IgG: P=0.43). Similar results were obtaied whe oly oe sample per patiet was icluded (tables 9-10). For IgA atibodies, the results of the MRL assay versus the WRF assay were cocordat i 80% of the sera (66 of 83), (data ot show). Whe a day-to-day variatio of± 1 dilutio step was take ito cosideratio (see page xx), the results for the MRL assay versus the WRF assay were cocordat i 98, 88 ad 88% of the sera for IgM, IgG ad IgA atibodies ad for the LAB assay versus the WRF assay i 98 ad 87% of the sera for IgM ad IgG atibodies. For the sera from the patiets i group III, agreemet i detectio rates by the MRL assay versus the WRF assay was foud i 92 % of the sera (90 of 98) for IgG atibodies ad i 91 % of the sera (91 of 100) for IgA atibodies (table 9-10). Whe the day-to-day variatio was take ito cosideratio, the correspodig results were 97 ad 95% of the sera, respectively. Edpoit titre levels. Table 11 shows the geometric mea titres foud by each of the three MIF assays of all sera with a atibody level cosidered sigificat for the diagosis of C. peumoiae, i.e. IgM atibody titres of~ 16, IgG atibody titres of ~ 64 ad IgA atibody titres of ;?: 32. The geometric mea of IgM atibody 26

29 titres obtaied by the LAB assay was about three times as high as those obtaied by the WRF ad the MRL assay. The differece i geometric mea titres for IgM atibodies obtaied by the three assays was sigificat. This differece was due to the divergig results by the LAB assay. Comparig the geometric mea titres for IgG atibodies obtaied by the three assays i sera from group I ad II, the overall differece demostrated was due to a lower lgg atibody level by the WRF assay ad a higher level by the LAB assay. Similar geometric mea titres for IgM ad IgG atibodies were obtaied whe oly oe sample per patiet was icluded (table 11). With the WRF assay 2 of 120 sera (1.7%) had C. peumoiae IgG atibody titres of ~ 512 as opposed to 9 of 120 sera (7.5%) with the MRL assay ad 7 of 120 sera (5.8%) with the LAB assay (P=0.10, Fisher's exact test), (table 5-6). However, i oly five sera were IgG atibody titres of ~ 512 demostrated by both the MRL ad the LAB assay. The differece i mea edpoit titres of sera positive by two assays ca also be expressed i dilutio steps. Results by the MRL assay compared to those by the WRF assay were 0.4 dilutio steps lower for IgM atibodies (groups I+II); 0.4 dilutio steps higher for IgG atibodies (groups I+II+III) ad 0.3 dilutio steps higher for IgA atibodies (groups I+III). For the LAB assay compared to the WRF assay the mea titres for groups I+II were 1.7 dilutio steps higher for IgM ad 0.4 dilutio steps higher for IgG atibodies. I coclusio, the determiatios of edpoit titres were reproducible with less tha oe dilutio step differece for all three methods, except that the mea IgM atibody titre foud by the LAB assay was almost two dilutio steps higher tha that, foud by the other two methods. Discrepat results: The MRL ad the WRF assay results were discrepat (positive versus egative) i 9 of 120 (8%) of sera tested for C. peumoiae IgM atibodies (table 3). Two of the three sera with a positive result for IgM atibodies by the MRL assay but ot by the WRF assay came from a patiet with C. psittaci ifectio verified by PCR aalysis. The third serum, from a group I patiet was foud egative by both the WRF ad the LAB assay but subsequet sera from the same patiet were foud positive for IgM atibodies by all three assays. The six sera with a egative result for C. peumoiae IgM atibodies by the MRL assay but with positive results by the WRF assay came from two group I patiets (three sera from each) ad all six sera were also positive for IgM atibodies by the LAB assay. 27

30 The LAB ad the WRF assay results were discrepat i 5 of 120 (4 %) sera tested for IgM atibodies (table 4). The five IgM results were all foud i the first or the last of three cosecutive sera, the two correspodet sera of which were positive for C. peumoiae IgM atibodies by both assays. For these five sera the results by the WRF assay were cocordat with those by the MRL assay. For the majority of sera with discrepat IgG atibody results the discrepacy was betwee sera with IgG atibody titres of < 64 i oe assay ad IgG atibody titres of 64 to 256 i the other assay (tables 5-6). Patiets with C. peumoiae ifectio. A total of 66 sera from 25 patiets with either serological evidece of C. peumoiae ifectio (group I, = 17) or microbiological evidece of C. peumoiae ifectio (group II, = 8) were icluded. A total of 51 sera origiated from these 17 patiets i group I. For IgM atibodies the agreemet i detectio rates was 98% of the sera (50 of 51) for the LAB versus the WRF assay ad 88% of the sera (45 of 51) for the MRL versus the WRF assay, while accordace i edpoit titre levels, defied as the WRF assay titre± 1 dilutio step, were 78% (40 of 51) ad 73% (37 of 51), respectively. For the IgG atibody detectio rate the agreemet was 90% (46 of 51) for both the LAB versus the WRF assay ad the MRL versus the WRF assay, while accordace i edpoit titre levels was foud i 84% (43 of 51) of sera by both assays. For the IgA atibodies the agreemet i detectio rates for the MRL versus the WRF assay was 86% (44 of 51) ad the accordace i edpoit titre levels was 75% (38 of 51). Table 12 shows the proportio of patiets with C. peumoiae ifectio i group I ad II fulfillig each of the Graysto criteria of acute ifectio (table 1). Amog the patiets i group I, IgM atibody titres of 2: 16 were detected by all three assays i sera from 6 of 8 patiets, i sera from the two last patiets IgM atibodies were detected by the LAB ad the WRF assay but ot by the MRL assay. A fourfold rise of IgG atibody titres was detected i oe patiet by all three assays, i further four patiets was the criterio fulfilled by oe or two of the assays. IgG atibody titres of 2: 512 were see i 5 patiets, though oly i oe of these by all three assays. Amog the patiets i group II, IgM atibody titres of 2: 16 were detected by all three assays i sera from 7 of 8 patiets (11 of 15 sera), (table 12). A fourfold rise of IgG atibody titres was detected i oe patiet by all three assays ad i further 28

31 oe patiet by the LAB assay. IgG atibody titres of ~ 512 were ot detected by ay of the assays. For group II, table 13 shows the serological results by all three methods for each patiet with cofirmed C. peumoiae ifectio. The accordace i IgM atibody detectio rates for the 15 sera from 8 patiets were 87% (13 of 15) for the LAB versus the WRF assay ad 100% (15 of 15) for the MRL versus the WRF assay, while accordace i edpoit titre levels defied as the WRF assay titre ± 1 dilutio step, were 47% (7 of 15) ad 67% (10 of 15). For IgG atibodies the accordace i detectio rates were 53% (8 of 15) for the LAB versus the WRF assay ad 47% (7 of 15) for the MRL versus the WRF assay, while accordace i edpoit titre levels were foud i 40% (6 of 15) of sera by the LAB assay ad 47% (7 of 15) by the MRL assay, respectively. I coclusio, the agreemet i detectio rates of IgM atibodies was betwee 87 ad 100% by the two commercial assays compared to the WRF assay ad the accordace i detectio rates of IgG atibodies was 90% i sera from group I ad about 50% i sera from group II. The accordace i edpoit titre levels betwee both assays ad the WRF assay was somewhat lower tha the accordace i detectio rates. The agreemet betwee all three methods i fulfillig the criteria of serological diagosis was high for the detectio of IgM atibody titres of~ 16 (13 of 15) but low for the detectio of IgG atibody titres of~ 512 (1 of 5) ad for the detectio of a fourfold rise of IgG atibody titres (1 of 5). Patiets with M peumojaeifectio. The agreemet of C. peumoiae IgG atibody test results was 91 % by the MRL versus the WRF assay ad 87% by the LAB versus the WRF assay i the 32 sera from 11 patiets icluded i group I. By the MRL ad the WRF assay, the test results for C. peumoiae IgA atibodies were i accordace i 29 of 32 sera. I oe of the sera was C. peumoiae IgM atibodies, fourfold rise of IgG atibody titres or IgG atibody titres of~ 512 detected by ay of the assays. IgG atibody titres of 64 to 256 were detected i 46 to 57% of sera by the three assays. 29

32 II Patiets with C. psittaciifectio. A total of 22 sera from 8 patiets with C. psittaci ifectio were aalysed (table 14). Overall, four of eight patiets had C. psittaci IgM atibodies ad/or fourfold rise of IgG atibody titres by the LAB assay. By the MRL assay oe of the eight patiets had IgM atibodies towards all three species ad further oe had a fourfold rise of IgG atibody titres. C. peumoiae IgM atibodies were detected i four sera from two patiets by the MRL assay, but i oe of the sera by the LAB ad the WRF assay. C. peumoiae IgG atibody titres of 64 to 256 were detected i sera from three, four ad six patiets by the WRF, the LAB ad the MRL assay, respectively. Cross-reactig atibodies: I patiets with C. peumoiae IgM atibodies, C. psittaci IgM atibodies were detected i 3 of 6 patiets i group I (5 of 15 sera) ad i 4 of 9 patiets of group II (5 of 16 sera) by the MRL assay. I 5 of the 10 sera there were less tha fourfold differece betwee C. peumoiae ad C. psittaci IgM atibody titre levels. C. trachomatis IgM atibodies were see i three patiets (6 sera), who showed positive test results for C. peumoiae as well as C. psittaci IgM atibodies. I patiets with C. peumoiae IgM atibodies, C. psittaci IgM atibodies were detected i 5 of 8 patiets i group I (13 of 21 sera) ad i 2 of 7 patiets of group II (2 of 12 sera) by the LAB assay. I 9 of the 15 sera there were less tha fourfold differece betwee C. peumoiae ad C. psittaci IgM atibody titre levels. C. trachomatis IgM atibodies were see i three patiets (4 sera) i whom C. peumoiae IgM were demostrated, while C. psittaci IgM atibodies were demostrated i oe of these patiets. Compariso of results for group I with the NAP study. The results of group I, tested with atiges from WRF i the preset study was compared to the results origially obtaied i the NAP Study 116 with the same cut off applied to both sets of results. The detectio rate of C. peumoiae IgM atibodies of ~ 16 was 24 % (CI: 19 to 29%) versus 31 % (CI: 26 to 36%) i the NAP Study ad that of IgG atibodies of~ 64 was 45% (CI: 40 to 50%) versus 69% (CI: 64 to 74%), but the detectio rate of IgA atibodies of~ 16 was 64% (CI: 59 to 69%) versus 40% (CI: 45 to 55%). The mea edpoit titres obtaied i the preset study were 2.0 ad 1.5 di- 30

33 lutio steps lower for IgM ad IgG atibodies ad 0.3 dilutio step higher for IgA atibodies tha i the NAP Study. The proportio of patiets fulfillig each of the Graysto criteria of acute C. peumoiae ifectio by at least oe of the two studies is show i table 15. The results of the two studies were i agreemet i 89% of cases regardig the presece of IgM atibodies, while agreemet i fulfilmet of the two other criteria could oly be demostrated i 30 ad 9% of cases. Coclusio The overall agreemet betwee results by the WRF assay ad by the two commercial assays was excellet for IgM atibody detectio rate (98%). The accordace i detectio rates for IgG ad IgA atibodies i sera from patiets with acute ifectios was acceptable (87 to 88%) ad i sera from group III it was excellet (95 to 97%). No sigificat differece amog the detectio rates of the three assays was see for ay of the lg classes. The reproducibility of C. peumoiae IgG ad IgA atibody edpoit titres was high, though IgG atibody titres by the WRF assay were o average 0.4 dilutio step lower tha those by the two other assays. The IgM atibody edpoit titres foud by the LAB assay were almost two dilutio steps higher tha those foud by the WRF ad the MRL assay. A compariso of the results of the MRL versus the WRF assay of the sera from group I+ II ad group III showed a tedecy towards greater variatio of edpoit titres amog the patiets with acute ifectio tha amog the group without acute ifectio (table 3-8). The agreemet betwee the three assays i the capacity to distiguish betwee acute ifectio ad o acute ifectio i patiets with C. peumoiae ifectio was high for the detectio of IgM atibody titres of ~ 16 (13 of 15 patiets), but low for the detectio of IgG atibody titres of~ 512 ad for the detectio of fourfold rise of IgG atibody titres (1 of 5 patiets respectively). Noe of the patiets with M. peumoiae ifectio had serological evidece of acute C. peumoiae ifectio, whereas two patiets with C. psittaci ifectio had C. peumoiae IgM atibodies by the MRL ad i further two patiets a fourfold 31

34 32 rise of C. peumoiae IgG atibodies were demostrated by both the MRL ad the LAB assay. Whe comparig the results by the WRF assay i our laboratory to the results obtaied i the origial study the agreemet i the ability to fulfil the criteria of acute ifectio was high for the criterio of the presece of IgM atibodies but low for the two criteria i which the level of IgG atibodies is decisive.

35 IGM ANTIBODY TITRE LEVEL AND DURATION OF INCUBATION OF ANTIGEN WITH SERUM. I the mai study the detectio rate for C. peumoiae IgM atibodies was the same by all three assays. However, the mea level of IgM atibody edpoit titres was almost two dilutio steps higher by the LAB tha by the WRF assay, but idetical for the MRL ad the WRF assay. Oe of the modificatios used i the commercial MIF assays was a chage of the duratio of icubatio of atige with serum. While Wag ad Graysto recommed a icubatio time of 30 mi., MRL Diagostics ad Labsystems recommed 90 mi. ad 180 mi respectively (appedix 1). To test to what exted the IgM atibody edpoits titres are iflueced by the differeces i icubatio time the followig study was coducted usig the referece method (the WRF assay) ad the MRL assay, which the had bee chose for use i our routie laboratory. Material ad methods Sera. The study icluded 25 selected sera from 11 patiets icluded i group I ad II i the mai study. Three sera had had a egative IgM atibody result by both the WRF ad the MRL assay; each of these three sera was the first of three cosecutive sera of which the two latter sera had had IgM atibody positive results by both assays. Further oe serum had had a egative result by the WRF but ot by the MRL assay. All other sera had had positive IgM atibody result by both assays. MIF assays. All sera were tested for C. peumoiae IgM atibodies i serial twofold dilutios begiig at 1:32. By each MIF assay the dilutios of sera were tested i duplicate: oe dilutio row with a icubatio time for sera ad atige of 30 mi. ad the other with a icubatio time of 90 mi. Otherwise, the procedures followed the recommedatios of each maufacturer (appedix 1). Statistics. The correlatio betwee the icubatio time ad the C. peumoiae IgM atibody titre level was assessed for the WRF ad for the MRL assays with a 33

36 Sig Test. The results of the aalyses whe usig oly oe serum from each patiet are give separately. Results Of the 25 sera tested for C. peumoiae IgM atibodies four sera had a egative test result by both assays icludig the three sera that origially had had a egative test result ad a serum that had had a weak positive result (32 ad 16) by the two assays. Further oe serum was egative by the WRF assay with both icubatio periods; this serum had origially had a egative test result. The aalyses were coducted o the basis of the result from 20 sera tested by the WRF ad 21 sera tested by the MRL assay. Tables 16 ad 17 preset overviews of the results by the WRF ad the MRL assays, respectively. The WRF assay. The geometric mea titre of the 20 sera was 256 whe the icubatio time was 30 mi. ad 431 with 90 mi.'s icubatio, (P=0.0002); that equals a 0.75 dilutio step rise of IgM atibody titre with a chage from 30 to 90 mi. of icubatio time; 11 sera had a rise of 1 dilutio step, 2 had a rise of 2 dilutios steps ad the last 7 did ot show ay chage i C. peumoiae IgM atibody titres. The geometric mea titres for the 11 sera (1 serum per patiet) were 309 ad 481, (P=0.03) correspodig to a 0.64 dilutio step differece. The MRL assay. The geometric mea titre of the 21 sera was 868 whe the icubatio time was 30 mi. ad 1592 with a 90 mi.'s icubatio, (P<0.0001); that equals a 1.0 dilutio step rise of IgM atibody titre with a chage from 30 to 90 mi. of icubatio time. For 16 sera a oe-dilutio step chage was see, 3 sera had a rise of two dilutio steps ad the last 2 sera did ot show ay chage i C. peumoiae IgM atibody titres. The geometric mea titres for the 11 sera were 1237 ad 2806, (P=0.001) correspodig to 1.18 dilutio steps differece. Coclusio A positive correlatio betwee the duratio of icubatio time (30 ad 90 mi.) ad C. peumoiae IgM atibody titres was demostrated usig both methods. A icrease of titres was see regardless of titre levels. Whether this fidig implies that the detectio rate of C. peumoiae IgM atibodies icreases with i- 34

37 creasig icubatio time will have to be assessed i aother study, though i this study oe of the sera origially tested by the WRF assay with a icubatio time of 30 mi. with a egative test result had a positive test result with a 90 mi. icubatio time. The geometric mea titres for IgM foud by the MRL assay both at 30 mi ad 90 mi. were 3-4 times higher tha those foud by the WRF assay. I the mai study o differece betwee the geometric mea titres of IgM atibodies by the two assays was see. A limitatio of this study was, that the results were based o sigle determiatios usig oly oe batch of reagets from each assay. 35

38 REPRODUCIBILITY OF TEST RESULTS BY THE WRF MIF ASSAY I order to determie the reproducibility, a study of the itra- ad iter-day variatio for C. peumoiae atibody titres obtaied by the MIF referece method (WRF) was coducted. Sera Two sera were tested for C. peumoiae IgG, IgM ad IgA atibodies i triplicate o four experimetal days by the MIF assay. Thus for each atibody class 24 observatios were made. The experimets were carried through by the same perso who performed the aalyses of the mai study. The sera were aalysed i serial, twofold dilutio steps startig at 1:16. Statistics To determie the reproducibility of ed-poit titres for each serum for each day a two-way aalysis of variace was performed. The iter-day variatio was estimated with a F-test. The variatio of reproducibility was estimated for each lg class ad for the three classes together. Fially a 95% cofidece iterval (CI) of the titres was calculated. All aalyses were performed by SAS, SAS Istitute Ic. Results The variatio of reproducibility was estimated to 0.41 dilutio step for IgM, 0.48 for IgG, ad 0.48 for IgA atibody titres. Overall the day-to-day variatio of MIF edpoit titres was 0.46 dilutio step with 66 degrees of freedom. The CI was 2 x 0.46 = 0.92 ::::: 1 dilutio step. Coclusio ad commets This study showed, that the MIF assay results varied less tha oe dilutio step. The result of this study is i accordace with results from aother laboratory experieced i C. peumoiae MIF aalyses

39 CORRELATION BETWEEN RESULTS OF THE CF TEST AND THE WRF MIF ASSAY FOR THE DETECTION OF C. PNEUMONIAE ANTIBODIES. I the early era several studies o the seroepidemiology of C. peumoiae ifectios were performed usig collectios of CF atibody positive sera. Sice a high proportio of these sera were show to have detectable IgG atibodies by the MIF assay, the CF test was cosidered a appropriate tool of screeig sera before further testig by the more costly MIF assay To test whether this procedure was appropriate, a study of the correlatio betwee the presece of CF atibodies ad IgG atibodies detected by the MIF test was carried through. The reaso for choosig results for IgG ad ot IgM atibodies was that while approximately 30% of sera received i our routie laboratory had detectable C. peumoiae IgG atibodies, IgM atibodies were foud i less tha 1 % of sera (upublished data). Materials ad methods Sera. We coducted a study of 1008 cosecutive sera submitted to our routie laboratory for the CF test ad/or the MIF test from hospitals ad GPs from all over the coutry. CF test. For the CF test, Chlamydia atige from States Serum Istitut (Copehage, Demark) was used 157 All sera were tested i serial threefold dilutios begiig at 1: Titres of< 12 were cosidered egative, titres 12 to 108 weakly positive ad titres of> 108 strogly positive. MIF assay. The WRF assay was used as the referece method i this study. All sera were tested for C. peumoiae IgG atibodies i serial, twofold dilutio rows startig at a iitial dilutio of 1:64. Titres of< 64 were cosidered egative, titres 64 to 256 weakly positive ad titres of~ 512 strogly positive. Results Of the 1008 sera, 231 had positive results by the CF test ad/or the MIF assay (table 18).

40 38 Of the 157 sera with a positive CF test result 42 (27%) had detectable C. peumoiae IgG atibodies. Of these 157 sera, 27 had CF atibody titres of> 108, oe of these had C. peumoiae IgG atibodies of~ 512. A positive CF test result was foud i 42 of 116 sera (36%) with a positive C. peumoiae IgG atibody result by the MIF assay. Thus, the sesitivity of the CF test to predict the presece of C. peumoiae IgG atibodies detectable by the MIF assay was 42 of 116 (36%), while 42 of 157 (27%) of the CF test positive sera were foud with the MIF test. Four sera had a positive result of C. peumoiae IgG titre of~ 512, three of these were egative ad oe was weakly positive by the CF test. Coclusio ad commets I the preset study, the predictive value of a positive CF test result for the presece of C. peumoiae IgG atibodies was 36%, idicatig that the CF test is ot a suitable tool for screeig sera prior to testig for C. peumoiae IgG atibodies. Foseca et al compared results by the CF test ad the MIF assay for 120 sera (40 sigle ad 40 pairs) from patiets with a history of acute respiratory tract ifectios ad a CF test result of >16 i the acute samples 50 I 77% of all sera ad i 54 % of acute sera were detected C. peumoiae IgG atibodies of ~ 16. C. peumoiae IgG atibodies of ~ 64 were detected i 65% of all sera. Thus Foseca et al foud a higher positive correlatio betwee positive results by the two assays tha did the preset study. This could be due to differeces betwee the study populatios of the two studies. Persso ad Boma 122 compared the performace of five serological tests for the diagosis of acute C. peumoiae ifectios by aalysig paired sera from 240 patiets with symptoms of acute respiratory tract ifectio. Amog the five tests was a CF test usig the same atige as the oe used i the studies of the preset thesis ad the MIF test from Labsystems. I that study the sesitivity of the CF test to predict a sigificat reactio by the MIF assay was 69% (66 of 95 cases). I 90% of cases positive by the CF test (66 of 73 cases) the MIF test was also positive. The iclusio criteria of the Persso study were paired sera ad a history of respiratory tract ifectio, whereas we studied cosecutive sera received i our routie laboratory without ay cliical iformatio about the patiets. Further Persso

41 studied the performace of the tests by assessig the degree of agreemet betwee the tests i detectig sigificat reactios ad/or sigificat levels of atibodies for the diagosis of C. peumoiae ifectio, whereas i the preset study the performace of the MIF test ad the CF test was assessed by comparig the degree of agreemet i detectig IgG atibodies ad atibodies detectable by the CF test, respectively. I the studies by Foseca et al 50 ad Persso ad Boma 122 as well as i the preset study a proportio of sera positive by the CF-test had egative results by the MIF assay. The chlamydia! atibodies detected by the CF test persist for a limited period of time, whereas the atibodies detected by the MIF assay emerge ad disappear later tha the before metioed atibodies 162 Therefore, it is reasoably to expect a early phase with serocoversio detected oly by the CF test, a itermediate phase with detectable atibodies by both assays ad a late phase with atibodies oly detectable by the MIF assay. I coclusio, while the CF test is ot suitable as a tool for screeig sera before further testig by the MIF assay, it is a excellet supplemet for the diagosis of early phase chlamydia! ifectio. 39

42 40 DISCUSSION We coducted a study o the performace of three MIF assays (the WRF, the MRL ad the LAB assay) for the detectio of C. peumoiae IgG, IgM ad IgA atibodies9. The agreemet i detectio rates was high for all three methods ad all three Ig classes. The reproducibility of C. peumoiae IgG atibody edpoit titres was high; although IgG atibody titres by the WRF assay were o average 0.4 dilutio step lower tha those by the two other assays. However, this differece is withi the day-to-day variatio of oe dilutio step. Similarly, aother recet study comparig IgG atibody titres obtaied by the WRF assay ad the LAB assay foud a isigificat differece of oe dilutio step higher titre levels by the latter assay106. The IgM atibody edpoit titres foud by the LAB assay were almost two dilutio steps higher tha those foud by the two other methods. Labsystem recommeds that whe testig for IgM atibodies the icubatio time for sera with the atige should be 180 mi.; that is, six times as log as origially recommeded by Wag 161 ; 162. Results of the experimet described earlier i this chapter suggest, that a prologed icubatio time does affect the IgM atibody titre levels, thus differeces i IgM atibody titre levels obtaied by differet assays could be due i part to the differeces i icubatio times. It is iterestig however, that the prologed icubatio time did ot seem to affect the detectio rate of C. peumoiae IgM atibodies sice the percetages of sera with IgM atibodies were the same by the same assays usig differet icubatio periods. For the MRL assay the mea IgM atibody edpoit titres was 0.4 dilutio step lower tha those foud by the WRF assay; ad 0.4 ad 0.3 dilutio step higher for IgG ad IgA atibodies, respectively. Freidak et al foud the mea titres for IgG atibodies ad IgA atibodies by the MRL assay to be 2.5 ad 3.0 dilutio steps higher tha those obtaied by the WRF assay 51. Freidak et al used ati-huma Igs from Medac i the WRF assay whereas we used ati-huma Igs from Dako. Though oly mior differeces amog IgG atibody levels detected by the three assays were foud, a differece betwee the proportios of sera with atibody titres of~ 512 was see. IgG atibody titres of~ 512 were demostrated by the WRF assay i 1.6% of sera ad by the MRL assay i 7.5% of sera. Amog sera studied by Freidak 2% had titres of~ 512 with the WRF assay, compared to 30% with the MRL assay.

43 It has bee demostrated that the use of a C. peumoiae atige produced from a local isolate ca result i higher detectio rates of atibodies i sera ad higher titres i the MIF assay tha whe usig a stadard atige 17 The three assays studied each use a differet C. peumoiae strai as the atige. The WRF atiges are whole iactivated (formali fixed) orgaisms fixed oto the slides with acetoe; the two commercial assays do ot iform o the details of the pretreatmet. However, the detectio rates ad edpoit titres by these three assays were similar, with IgM atibody edpoit titres foud by the LAB assay as the oly otable exceptio. I the iterlaboratory study by Peelig et al 120 the agreemet amog the participatig laboratories for IgM titres was 50 to 95%. I the preset study the agreemet amog the three assays of detectig IgM atibodies i sera from the eight persos with microbiological verified C. peumoiae ifectio was 87 to 100%. Cosiderig the techical complexity of the MIF test ad the subjective ature of readig the titres, it is hardly surprisig that the iterlaboratory variatio of that study is higher tha the iterassay variatio of the preset study. Recetly Peelig has preseted a follow-up study i which 11 laboratories aalysed 10 sera before ad after a two-days hads-o workshop 121 Of the eleve laboratories, ie showed a certai improvemet i their % agreemet with the referece stadard after the workshop. Before the workshop 6 of 11 laboratories had % agreemet with the referece laboratory, after the workshop this figure had. rise to 9 of 11 laboratories. The agreemet betwee the serological ad the microbiological diagoses for the patiets with cofirmed C. peumoiae ifectio was high, i cotrast to that for the patiets with C. psittaci ifectio. For most of the patiets with psittacosis, sera were obtaied early i the course of the disease, ad it is possible that if sera had bee obtaied later, C. psittaci atibodies would have bee detected. Furthermore, there is a greater diversity betwee C. psittaci strais tha betwee C. peumoiae strais ad it is possible that the C. psittaci strais used i the LAB ad the MRL assay did ot react with C. psittaci atibodies from Daish patiets with ifectios caused by local strais. 41

44 The detectio rates of C. peumoiae IgG ad IgM atibodies as well as the atibody titre levels, were low amog sera from group I of the mai study compared to the results i the NAP Study; the detectio rate of C. peumoiae IgA atibody as well as the atibody titre level was higher i the preset study, tha i the NAP study. Although the same atige for the MIF assay was used i the two studies, other differeces i the test procedures occurred, e.g. the use of differet cojugates ad iterpersoal differece i the readig of slides might explai some of the variatios of the results obtaied. Furthermore, i the preset study the sera had bee absorbed before the IgA atibody aalyses; removal of IgG has bee show to raise IgA titres i sera with a high level of IgG atibodies 82 Fially the patiets i the NAP Study were recruited betwee 1990 ad 1993 ad the sera were aalysed shortly thereafter, whereas i our study the sera were tested after storage at -20 C for up to eight years. Though the edpoit titre levels foud by the NAP study were higher tha i the preset study, the agreemet i fulfillig the criterio of IgM of ~ 16 was high while the agreemet i fulfillig the two other criteria of acute ifectio were much lower. I both of the latter criteria, the levels of IgG atibodies are importat, thus with higher IgG atibody values i geeral it is to be expected that more sera fulfil the criteria. The statistical aalyses used i this chapter imply idepedece betwee samples, a coditio that is ot etirely met by the preset study as 120 sera origiated from 44 patiets (group I ad II). However, the assessmet of the performace of diagostic tools should reflect the cliical situatios i which the tools are used. For the sero-diagosis of a acute ifectio, it is importat to have more tha oe serum sample per patiet, as the diagosis is based o the emergece, rise or disappearace of differet classes of atibodies. Therefore, it is ot possible to select oe sigle sample period i the course of ifectio represetative for the whole course. The coflict betwee the biological reality ad the demads of the statistical aalyses most commoly used caot be solved easily. I papers published withi the serological field the authors have igored, that the samples icluded are ot completely idepedet I coclusio, the three MIF assays ivestigated showed about the same ability to detect C. peumoiae IgG, IgM ad IgA atibodies but some variatio was foud i the atibody levels demostrated. I the light of the Graysto criteria for the se- 42

45 rological diagosis of acute C. peumoiae ifectio, the differece i average edpoit titre levels of IgM atibodies is of o mayor cocer, as the emphasis of the criteria rests o the presece of IgM atibodies, ot o the level. I cotrast the differece i IgG atibody edpoit titres by differet assays presets a problem as log as a high level of IgG titres (~512) is used as a isolated criterio for the sero-diagosis of acute C. peumoiae ifectio. 43

46 44 CHAPTER 4: CLINICAL STUDIES. Itroductio Sice detectio of C. peumoiae by culture or by PCR is difficult ad util recetly mastered oly by few laboratories, the diagosis of C. peumoiae ifectio has bee fouded o serological criteria. Detectio of C. peumoiae IgM, IgG ad IgA atibodies by the MIF assay is still the gold stadard for detectio of past or preset C. peumoiae ifectio 40 ; 150 ; 162 however, the iterpretatio of test results is still cotroversial. Graysto classified the test results for IgM ad IgG atibodies as acute ad preexistig atibody profiles (table 1) 60 Acute atibody profiles were four-fold titre rise ad/or IgM titres of~ 16 ad/or IgG titres of~ 512. For acute ifectios a distictio betwee primary ifectio ad reifectio might be possible. I the latter case the IgM respose might be weak or o-existet. The sigificace of IgG atibody titres of~ 512 as a isolated criterio for a acute C. peumoiae ifectio has bee questioed 35 ;ao ad i the preset two studies this criterio is assessed ad discussed separately. Serologic criteria of reifectio used i the preset studies are fourfold rise of IgG atibody titre ad IgM titres of < 16. Preexistig or persistet atibodies ca be due to either previous or chroic ifectios. The profile for previous ifectio was defied as IgG atibody titres of< 512 ad IgM atibodies of < 16. Cliical criteria of chroic ifectio have ot bee established ad there is o clear distictio betwee the serological profiles associated with previous ad chroic ifectio. The role of IgA atibodies is ot clear. Some believe that IgA atibodies have o idepedet sigificace 162, while others cosider IgA atibodies a marker of chroic ifectio 128 ; 158 Fouded o results from studies o the role of C. peumoiae i patiets with asthma or COPD, a serological profile of chroic C. peumoiae ifectio has bee proposed: IgG titres of ~ 128 ad IgA titres of~ 40 without the presece of IgM atibodies 70. Objective The objective was to describe the prevalece of C. peumoiae ad other "atypical respiratory tract pathoges" 73, amely Mycoplasma (M.) peumoiae, Bordetella (B.) pertussis ad Legioella (L) peumophila i two groups of adult patiets: A) patiets with acute respiratory tract ifectios ad healthy cotrols see by a

47 Geeral Practitioer (GP) ad B) patiets with respiratory tract ifectios ad COPD patiets with or without exacerbatio or peumoia admitted to a departmet of iteral medicie or see at the associated out-patiets cliic for pulmoary diseases. Further a assessmet of the applicability of serological criteria for acute ad chroic C. peumoiae ifectio was made. Methods The two studies A ad B were coducted usig the same pael of microbiological ad serological aalyses for the diagoses of C. peumoiae, M. peumoiae, B. pertussis ad L. peumophila ifectios. Microbiology: Sputum samples ad asopharygeal swabs were examied for Chlamydia species by culture i Hep2 cells ad McCoy cells followed by immuospecific staiig as previously described 48 Furthermore all samples were aalysed by i-house PCR's for the detectio of Chlamydia species (C. peumoiae, C. psittaci ad C. trachomatis), M. peumoiae, L. peumophilla ad B. pertussis140. I the study B sputum samples were examied for respiratory tract pathoges by microscopy ad culture at the local departmet of cliical microbiology; urie samples were aalysed for peumococcal atiges by the Streptococcus Uit, States Serum Istitut 140 All other microbiological aalyses were performed i the routie laboratories of our departmet 140 Serology: Sera were aalysed for geus-specific chlamydial atibodies by a complemet fixatio (CF) test with chlamydial atige from States Serum Istitut 157 usig three-fold dilutio steps from a iitial dilutio of 1:12 140, ad for C. peumoiae IgM, IgG ad IgA atibodies by a microimmuofluorescece (MIF) assay from MRL Diagostics (Cypress, USA) usig two-fold dilutio steps from a iitial dilutio of 1:16. All MIF aalyses were coducted by the same techicia. Readig of the MIF test was performed idepedetly by the techicia ad by the author of the preset thesis. Discrepat results were rare ad a differece of more tha oe dilutio step was ot see. Sera were aalysed for M. peumoiae ad Legioella atibodies by a M. peumoiae CF test ad a Legioella atibody i' test

48 46 The serological criteria for acute ifectios were as follows: Chlamydia CF test: four-fold titre rise or titre of ~ 108; M. peumoiae CF test: four-fold titre rise or titre of ~ 256; Legioella atibody test: four-fold titre rise or titre of ~ 256; C. peumoiae MIF assay: see table 1. Quatitatio of serum IgG cocetratio was performed by the Departmet of Cliical biochemistry, SSI usig rateephelometry o the first serum sample from each patiet 135 Statistics. Statistical aalyses were coducted by use of SAS versio 8 (SAS Istitute Ic, Cary, NC, USA) ad R versio (Statistical program for health sciece researches, freeware). Prior to the studies, the optimal umber of participats for a estimate of the prevalece of C. peumoiae ifectio i each study group was calculated: With 80% power, a sigificace level of 5% ad a estimated prevalece of 5 % amog patiets with acute respiratory tract ifectios ad of 15 % amog the patiets with chroic pulmoary disease, the umber of patiets i each category should be 160. The categories beig patiets ad cotrols i study A ad patiets with peumoia, patiets with ifectious exacerbatio ad patiets without peumoia or ifectious exacerbatio i study B. For compariso betwee groups, x 2 or Fisher's exact test was used. Logistic regressio aalyses were used to examie whether the levels of C. peumoiae IgG ad IgA atibodies were associated with selected cliical ad paracliical parameters. Associatio betwee treatmet with systemic steroids ad the level of serum IgG cocetratio was assessed with a t-test. The associatio betwee the levels of C. peumoiae IgG ad serum IgG cocetratio was assessed by Spearma's rak correlatio test. P < 0.05 was cosidered sigificat. 95% cofidece itervals (CI} for prevaleces were calculated as: The proportio (p) ± 2 ~ p(i- p) I (ormal approximatio). The studies were approved by the Scietific Ethical Committee for Copehage ad Frederiksberg muicipalities. The study of patiets see by GP's was further approved by Multipraksisudvalget ad supported by the Muicipal Health Service, which covered the cost of extra cosultatios due to the study. Writte iformed coset was obtaied from all participats.

49 STUDY A: THE PREVALENCE OF CHLAMYDIA PNEUMONIAE IN PATIENTS WITH COMMUNITY-ACQUIRED ACUTE RESPIRATORY TRACT INFECTION Study populatio. Patiets ~ 18 years of age with symptoms of acute respiratory tract ifectio cosultig 20 GPs i the Muicipality of Copehage from November 1999 through September 2000 were erolled. Cotrols were persos seeig the GP without havig symptoms of respiratory tract diseases, matched accordig to geder ad age. Accordig to the protocol the patiets should be see three times: day 0, 14 ad 36. O each occasio blood samples, asopharygeal swabs ad if possible sputum were collected, set by mail to States Serum Istitut ad received at the laboratory the ext day. O day 0 ad day 14 structured questioaires were completed by the GP, icludig questios regardig symptoms, cliical diagosis ad atibiotic treatmet (appedix 2). Examiatio of cotrols followed the same procedures as those for patiets o day 0, but follow-up visits were ot icluded. Results Patiets. A total of 112 patiets were erolled i the study (44 me, media age 37 years ad 68 wome, media age 46 years) ad 45 cotrols (18 me, media age 41 years ad 27 wome, media age 45 years). A total of 69 % of the participats were recruited durig the first three moths of the study. Of the 112 patiets 64 (57%) were see three times, 20 (18%) were see twice ad 28 (25%) failed to retur for follow-up visits. The umber of patiets ad especially the umber of cotrols erolled by each Practitioer was lower tha expected, thus the optimal umber of subjects was ot erolled. The questioaire survey. The patiets were icluded i the survey as log as the ame ad the ID of the patiet was give. Table 19 shows selected data from the first questioaire survey comprisig 94 (84%) out of 112 patiets icluded. At the first visit the domiatig symptoms ad sigs reported were cough, fever, dyspoea, catarrhal ad hoarseess. The most commo cliical diagoses icluded brochitis acuta, o-specified respiratory tract ifectio, peumoia, upper respiratory tract ifectio, catarrhalia, iflueza ad asthma. Withi 8 weeks prior to 47

50 48 erolmet 14 of 90 patiets reported havig received atibiotics: peicilli (7), macrolides (3) ad sulfamethizol (1); for the last three patiets o iformatio about choice of atibiotics was give. Atibiotic therapy was iitiated amog 26 of 90 patiets: peicilli (9) ad macrolides (17). Table 20 shows selected data from the 77 (69%) of 112 questioaires that were received from the secod visit. At the secod visit 43 of 77 patiets (56%) reported that they were ot completely recovered. Six of 75 patiets were treated with local steroids. Oe patiet with ospecified respiratory tract ifectio was admitted to hospital. Aetiological diagosis. Table 21 shows results of microbiological ad serological aalyses for six patiets i whom a aetiological diagosis was made. Two patiets had C. peumoiae, two had M. peumoiae ad two had B. pertussis ifectios. The two patiets with C. peumoiae ifectio had IgM atibodies demostrated by the MIF test, both at levels of;::: 1024 i all three sera ad both had, as the oly two patiets i the study, a fourfold rise i C. peumoiae IgG atibody titres. Oe had sigificat icrease i titres by the CF test ad C. peumoiae was demostrated by culture as well as by PCR. From the other patiet C. peumoiae was ot isolated; this patiet had bee treated with peicilli four weeks prior to erolmet. I two patiets M. peumoiae ad i aother two patiets B. pertussis were detected by PCR. Oe of the patiets with a positive B. pertussis result by PCR had a fourfold titre rise i the M. peumoiae CF test from titre of::;; 16 to titre 64 idicatig a acute M. peumoiae ifectio. No other patiets had serological test results idicatig a acute M. peumoiae ifectio. Noe of the patiets had serological evidece of Legioella ifectio. The two patiets with M. peumoiae ifectio verified by PCR did ot retur for follow up visits, thus it is ot kow whether they developed atibodies detectable by the M. peumoiae CF test later durig the course of illess. Table 22 describes the cliical features of the six patiets. Noe of the six patiets were hospitalised, however the four patiets with C. peumoiae or B. pertussis ifectio all reported at day 14 that they had ot yet recovered. Overall, about 50% of those aswerig the secod questioaire reported to be recovered at day 14. Two patiets, oe with C. peumoiae ifectio ad oe with B. pertussis ifectio had see their GP about four weeks prior to erolmet i this study with symptoms of respiratory tract ifectio, idicatig that the symptoms of the pre-

51 set ifectio may have lasted for more tha six weeks. Oe of the two patiets with C. peumoiae ifectio reported symptoms of acute arthralgia durig the course of illess. Atibody profiles. IgM atibodies were see i 2 of 112 patiets (2%). Overall, the prevalece of IgG atibody titres of~ 16 was 59% (CI: 50-68%) ad the prevalece of IgG atibody titres of~ 64 was 40% (CI: 31-49%), (table 23). The distributio curve accordig to IgG atibody titres showed a peak at titre of 64, (figure 2). A logistic regressio aalysis i which, risk factors suspected of ifluecig the IgG atibody titres served as idepedet variables ad the presece of C. peumoiae IgG atibodies of ~ 64 as the depedet variables. The followig idepedet variables were selected: Beig patiet or cotrol, age, sex ad serum IgG cocetratio. Age ad serum IgG cocetratio were coded as cotiues variables; sex ad beig patiet or cotrol were coded as dichotomous categorical variables. No sigificat correlatio betwee the presece of C. peumoiae IgG atibodies of~ 64 ad ay of these variables was foud. The prevalece of IgA atibody titres of ;::: 16 was 34% (CI: 25-43%) ad the prevalece of IgA atibody titres of~ 32 was 21 % (CI: 13-29%), (table 23). The distributio curve accordig to IgA atibody titres showed a peak at titre 64 (figure 3). A similar logistic regressio aalysis as the oe above was performed with the presece of IgA atibody titres~ 16 as the depedet variable ad same idepedet variables as above. The oly sigificat associatio foud was betwee the presece of IgA atibodies ad advaced age (P = correspodig to Odds Ratio of per year (CI: )). Amog the 106 patiets i whom a aetiological diagosis were ot achieved C. peumoiae IgM atibodies were ot demostrated ad i sera from the 82 patiets from whom more tha oe serum were obtaied o sigificat chages i C. peumoiae IgG or IgA atibody titres were foud. 10 patiets (9%) had IgG atibody titre of~ 512 at their first ad at all subsequet visits ad thus fulfilled oe of the Graysto criteria of acute ifectio (table 1); oe of the remaiig patiets had developed IgG atibody titres of;::: 512 at the follow-up visits. Of the 10 patiets 4 were me, media age 45 years old (27-67 years old) ad 6 were wome, media age 45 years old (25-75 years old). The patiets with IgG atibody titres of ;::: 512 reported to have had symptoms of acute respiratory tract ifectio 4-21 days j 49

52 (mea 8 days), whereas the patiets with IgG atibody titres of :::;; 256 had had symptoms for 1-60 days (mea 9 days). The criterio of previous ifectio was fulfilled i 53 (50%) ad the criterio of chroic ifectio was fulfilled i 17 patiets (16%). Of the latter 17 patiets 7 had IgG atibody titres of~ 512 ad thus fulfilled the Graysto criteria of acute ifectio ad the remaiig 10 patiets fulfilled the Graysto criterio of previous ifectio. Cotrols. Of the 45 persos recruited as cotrols, 16 (36%) had o physical complaits. Withi the latter group the most commo reasos for seeig the GP were to accompay childre or to receive health checks/certificates. The reaso for the cosultatio for six cotrols was pai i head, shoulder or back. The remaiig cotrols had a variety of o- respiratory tract symptoms. Oe patiet had had atibiotic treatmet (tetracyclie) withi 8 weeks prior to erolmet but oe received prescriptios of atibiotics i coectio with the cosultatio, at which they were erolled. Noe of the cotrols had 11 atypical respiratory tract pathoges 11 demostrated by culture or PCR. The cotrols either had chlamydia! CF titres idicative of acute ifectios or C. peumoiae IgM atibodies, or serological evidece of ifectios with M. peumoiae ad Legioella. The prevalece of C. peumoiae IgG atibody titres of ~ 16 was 62% (Cl: 48-76%) ad 31 % had titre of~ 64 (CI: 17-45%), (figure 2 ad table 23). The prevalece of C. peumoiae IgA atibody titres of ~ 16 was 36% (CI: 22-50%) ad 15% (Cl: 8-22%) for lga atibody titres of~ 32 (figure 3). Oe cotrol (2%) had C. peumoiae IgG atibodies at 512, idicatig a acute ifectio accordig to the Graysto criteria. This cotrol was a healthy woma accompayig her child to the GP. Previous ifectio as defied by the Graysto criteria was see i 27 cotrols (60%). The criterio of chroic ifectio was fulfilled i 8 cotrols (18%); amog these oe had IgG atibody titre at 512 ad the remaiig 7 fulfilled the criterio of previous ifectio. Noe of the eight cotrols fulfillig the criterio of chroic ifectio reported to have cardio-pulroary diseases. 50

53 Coclusio I the preset study of adults presetig themselves to the GP with symptoms of acute respiratory tract ifectios, six patiets obtaied a aetiological diagosis: Two patiets had C. peumoiae ifectio (2%), two had B. pertussis ifectio (2%) ad two had M. peumoiae ifectio (2%). The two patiets with acute C. peumoiae ifectio both had high-level IgM atibody titres ad fourfold rise of IgG atibody titres. Both patiets had developed C. peumoiae IgM atibodies at the first visit to the GP ad both had fever ad coughs ad oe of them reported to have arthralgia. Further 10 patiets (9%) ad 1 cotrol (2%) had C. peumoiae IgG atibody titres of~ 512 ad thus met oe of the Graysto criteria for acute C. peumoiae ifectio 60 ; the observed differece betwee patiets ad cotrols was ot sigificat (P=0.18, Fishers test). However, it is strikig that these patiets did either show ay chage of atibody titres durig the 36 days of observatio or had ay positive test results by culture or PCR, although they reported to have had respiratory tract symptoms o the average eight days before erolmet i the study ad thus should be i a early phase of acute ifectio. No sigificat differeces betwee patiets ad cotrols i the prevaleces or the distributio of IgG or IgA atibodies were observed. No correlatio betwee C. peumoiae IgG atibody titres of ~ 64 ad age, sex or serum IgG cocetratio was see, i cotrast IgA atibody titres of~ 16 were correlated to advaced age, but to oe of the other parameters. The overall prevalece of C. peumoiae IgG atibody titres of ;;::: 16 for patiets ad cotrols together was 59%, icludig 37% with titres of;;::: 64; 7% had IgG atibody titre of~ 512 ad 25% fulfilled the criteria of chroic ifectio show i table 1. No sigificat differeces were see betwee the percetages of patiets ad of cotrols fulfillig the criteria of chroic C. peumoiae ifectio or betwee the prevaleces of C. peumoiae IgG atibodies. 51

54 STUDY B: PREVALENCE OF CHLAMY.DIA PNEUMONIAE IN HOSPITALISED PATIENTS AND PATIENTS SEEN AT AN OUTPATIENT CLINIC Study populatio From October 1998 through December 1999 patiets were erolled either from the out-patiets' cliic for respiratory diseases or withi 48 hours after admittace to the Departmet of Iteral Medicie, Bispebjerg Hospital, Copehage, Demark. Iclusio criteria were COPD or symptoms of acute respiratory tract if ectio. The patiets were see three times: day 0, 14 ad 36. O day 0 white blood cell cout ad CRP were measured, sputum was collected for culture of respiratory tract pathoges, ad a chest radiograph was performed. From the hospitalised patiets a urie sample was collected. Day 0 ad day 36 the lug fuctio was measured ad a questioaire was filled out (appedix 2). The questioaire icluded equiries regardig smokig, treatmet with glucocorticoids, treatmet with atibiotics withi the last eight weeks prior to erolmet i the study ad withi the study period. Each time blood samples, asopharygeal swabs ad if possible, sputum specimes were collected. Each patiet could oly participate i the study oce. Defiitio ad classificatio of the severity of COPD followed the recommedatios from Europea Respiratory Society 137 : Patiets had COPD if the ratio: FEV 1 /VC was < 0.88 of predicted i me or was < 0.89 of predicted i wome (i.e. >1.64 residual stadard deviatio below predicted value). The severity of COPD was assessed by the FEV 1 i relatio to referece values: FEV 1 x 100/predicted FEV 1 : mild COPD;::::: 70%, moderate COPD 50-69% ad severe COPD < 50%. I order to miimize the risk of classifyig the patiets o the basis of a opermaet reductio i lug fuctio due to acute ifectio, the highest FEV 1 value of the two lug fuctio measuremets (i most cases the secod) was used for the classificatio. The patiets were also classified accordig to the cliical ad paracliical fidigs idicative for the presece or absece of actual ifectio: Group I, peumoia verified by a chest radiograph. Group II, ifectious exacerbatio i.e. the patiets 52 J

55 fulfilled at least two out of three criteria: Fever > 38 C, white blood cell cout > /L or CRP > 10 mg/l. Group III comprised patiets that did ot fulfil the criteria set up for group I ad II (without ifectio). Results Study populatio. A total of 210 patiets were erolled i the study: '76 me, years old (media 68 years) ad 134 wome, years old (media '70 years). Of the 210 patiets 11'7 were recruited from the out-patiet cliic ad 93 were recruited from the departmet of iteral medicie. The first questioaire was completed for all 210 patiets ad the secod was filled i for 199 patiets. Blood samples ad asopharygeal swabs were collected from 210, 175 ad 164 patiets from day 0, 14 ad 36, respectively. The first moths of the study-period the daily erolmet met the expectatios, but thereafter may of the eligible patiets admitted to the departmet or see at the out-patiet cliic were already erolled i the project. COPD. Out of 210 patiets 159 ('76%) fulfilled the criterio for COPD. Amog these 11 % had mild, 20% had moderate ad 69% had severe COPD (figure 4). Of the remaiig 51 patiets, 85% had a FEV 1 below '70% of expected FEV1, idicatig that, though ot fulfillig the criterio of COPD, they had a reduced lug fuctio (figure 5). The cliical diagoses for these 51 patiets were stable COPD (16 patiets), exacerbatio of COPD (15 patiets) ad other (20 patiets). Thus the majority of patiets icluded i the study had COPD or reduced lug fuctio without obstructio at a level sufficiet for the diagosis of COPD. The patiets without COPD or other uderlyig diseases were ofte discharged from the departmet so early that erolmet i the project was ot possible. The questioaire survey. Table 24 shows selected data from the first questioaire survey from 93 hospitalised patiets ad 11 '7 patiets from the outpatiet cliic icluded i the study. The hospitalised patiets had a higher frequecy of symptoms related to ifectios of the respiratory tract tha did the patiets erolled from the outpatiet cliic. The majority of the former patiets had either exacerbatio of their COPD or peumoiae, whereas most of the patiets from the outpatiet cliic had COPD without exacerbatio. Atibiotic treatmet withi the eight weeks prior to erolmet had bee received by 61ad44%, respectively. A 53

56 higher proportio of the hospitalised patiets was curret smokers ad had cardiovascular ad other chroically diseases compared to the patiets from the outpatiet cliic. Table 25 presets selected data from the secod questioaire survey for the 91 patiets admitted to the departmet of iteral medicie ad the 108 patiets see at the outpatiet cliic for whom the questioaire was filled out. About oe-third i both groups received treatmet with systemic steroids durig the six weeks study period, whereas atibiotics were received by oe third ad oe fifth, respectively of the groups. Classificatio accordig to cliical ad paracliical fidigs. The umber of patiets i each group accordig to cliical ad paracliical fidigs was group I (peumoia): 39 (19%), group II (ifectious exacerbatio): 52 (25%) ad group III (without ifectio): 119 (57%), (table 26). I group I ad II a sigificatly higher proportio of the patiets had fever, leucocytosis ad elevated CRP tha i group III. However, these three parameters were used i the classificatio of ifectious exacerbatio. No sigificat differeces were foud betwee the three groups accordig to systemic treatmet of glycocorticoids, smokig withi the last year or severe COPD. Microbiological results. "Atypical respiratory tract pathoges" were foud i three patiets (table 26).. I a patiet with peumoia, M. peumoiae ifectio was diagosed by a positive PCR result ad by the followig results i the M. peumoiae CF test: titre 2048, titre 4096 ad titre 1024 for the three cosecutive samples respectively. No other patiets had serological evidece of M. peumoiae ifectio. Oe patiet had psittacosis verified by culture ad PCR. The followig serological test results were obtaied: MIF assay: egative for C. psittaci atibodies i all three samples, C. peumoiae IgG atibodies at 512, 512 ad 1024 respectively, egative for C. peumoiae IgM atibodies. CF test: egative, titre 108 ad titre 36 respectively, which is cosistet with ifectio caused by C. psittaci as well as C. peumoiae. The cliical diagosis for this patiet was febrile exacerbatio of COPD. I a patiet without sigs of acute ifectio B. pertussis was detected by PCR. Noe had microbiological or serological evidece of L. peumophila ifectio. From 76 patiets microscopy ad culture of sputum were performed; 22 samples showed growth of potetially pathogeic bacteria: Haemophillus ifluezae (10), 54

57 Streptococcus peumoiae (10) ad Moraxella catarrhalis (2). The bacteria were foud i 5 of 39 patiets with peumoia, i 8 of 52 patiets with ifectious exacerbatio ad i 9 of 119 patiets with o ifectio (P=0.35). Out of the te patiets with S. peumoiae demostrated i sputum, oly two had had urie aalysed for peumococcal atige. Of these two patiets oe had a positive test result. Overall peumococcal atige was detected i further 2 of 40 urie samples tested. Serological evidece of C. peumoiae ifectio. Noe of the patiets had IgM atibodies. Except i oe patiet o sigificat differeces betwee titre levels of IgG or IgA atibodies were see i the 1., 2., ad 3. blood samples. Oe patiet had a rise of IgA titres from 16 to 64 ad IgG titres at 256 ad 512. The patiet with psittacosis was the oly oe with sigificat rise of titres i the CF test. All calculatios described below were based o results from the first blood sample. Atibody profiles. Overall, the prevalece of IgG atibody titres of~ 16 was 70% (CI: 64-76%) ad the prevalece of IgG atibody titres of~ 64 was 62% (CI: 55-69%), (table 23). The distributio curve accordig to IgG atibody titres showed a peak at 256 (figure 6). The prevalece of IgA atibody titres of~ 16 was 51 % (CI: 44-58%) ad 43 % (CI: 36-50%) had IgA atibody titres ~ 32 (table 23), with the distributio curve accordig to IgA atibody titres showig a peak at 32 (figure 7). No patiets had C. peumoiae IgM atibodies or a fourfold rise of IgG atibody titres. Of the 210 patiets 43 (20%) had C. peumoiae IgG atibody titres of ~ 512 ad thus fulfilled the criteria of acute C. peumoiae ifectio 58 ; 60 No sigificat differeces were see betwee the three groups (table 27) though IgG atibody titres of~ 512 were foud i 13% of patiets i group III ad i 28% of patiets i group I (P=0.25). I total, 28% of the me ad 16% of the wome had IgG atibody titres of~ 512 (P=0.13). For the 27 patiets with test results idicatig a aetiological diagosis other tha C. peumoiae ifectio (by demostratio of possible pathogeic bacteria i sputum, peumococcal atige i urie or "atypical respiratory tract pathoge" i asopharygeal swabs) 10 (37%) had C. peumoiae IgG atibody titres of~ 512; of the 183 patiets without a aetiological diagosis 33 (18%) had IgG atibody titres of ~ 512. The differece betwee the 55

58 prevaleces of IgG of~ 512 amog the patiets with a aetiological diagosis ad the patiets without ad aetiological diagosis was ot sigificat (P=0.13). IgG atibody titres idicatig previous ifectio (table 1) was see i sera from 106 patiets (50%). No sigificat differeces were demostrated betwee the three groups (table 27). The criterio of chroic ifectio (IgG of~ 128 ad IgA of~ 32) was met by 77/210 (37%) of the patiets. However, 37 (48%) of the 77 patiets had IgG atibody titre of~ 512, thus they would be categorized as acute ifectio accordig to oe of the Graysto criteria. The remaiig 40 patiets would be categorized as previous ifectio accordig to the Graysto criteria. There were o sigificat differeces i the percetages of patiets from the three groups fulfillig the criteria of chroic ifectio (table 27) Table 28 shows the results of a logistic regressio aalysis i which, risk factors suspected of ifluecig the IgG atibody titres served as idepedet variables ad three levels of IgG atibodies (1. IgG :::; 64, 2. IgG , 3. IgG ~ 512) as the depedet variables. The followig idepedet variables were selected: +/ COPD, degree of COPD, outpatiet/hospitalised patiet, peumoia (group!)/ifectious exacerbatio (group II)/without ifectio (group III), age, sex, smokig (+/- smokig withi the last year), atibiotics withi last eight weeks, treatmet with systemic steroids, serum IgG cocetratio, leucocytosis, +/- elevated level of CRP. Age, degree of COPD ad serum IgG cocetratio were coded as cotiues variables. The remaiig variables were coded as dichotomous categorical variables. Age ad serum IgG cocetratio showed a positive correlatio with the level of IgG atibodies ad treatmet with systemic steroids showed a egative correlatio. No sigificat correlatio was foud betwee treatmet with systemic steroids ad the level of serum IgG cocetratio (P=0.115, (t-test)). A similar logistic regressio aalysis as the oe above was performed with the presece of IgA atibody titres of~ 16 as the depedet variable ad same idepedet variables as above. Noe of the parameters showed a sigificat correlatio with the presece of IgA atibodies. C p11eumo11iae IgG atibody titres ad serum IgG cocetratio. The correlatio betwee the amout of total serum IgG ad C. peumoiae IgG atibody titre levels was also assessed by Spearma's rak correlatio coefficiet (figure 8) ad 56

59 a sigificat positive correlatio was foud (P < 0.001), idicatig that a high level of specific IgG atibodies was associated with a high level of total IgG. C pemoiae IgG atibody titres ad CRP. The amout of CRP was assessed accordig to C. peumoiae IgG titre levels (figure 9) ad a o-sigificat positive correlatio was foud (P = 0.08, Spearma's rak correlatio coefficiet), idicatig that a high level of C. peumoiae IgG atibodies might be associated with a ogoig ifectio. There was o sigificat correlatio betwee the cocetratio of CRP ad serum IgG cocetratio. I I I i! I Coclusio The majority of the 210 patiets ivestigated had severe reductio of their lug fuctio. Oe fifth of the patiets had peumoia, oe fourth had ifectious exacerbatio, while the remaiig patiets did ot fulfil the criteria set up for curret ifectio. Noe of the patiets had microbiological or serological evidece of acute C. peumoiae ifectio, except that 20% had a isolated C. peumoiae IgG atibody titre of~ 512. I three patiets other atypical respiratory tract pathoges were detected. The prevaleces of C. peumoiae IgM, IgG ad IgA atibody titres of~ 16 were zero, 72 ad 52%, respectively. Half of the patiets fulfillig the criteria of chroic ifectio had IgG atibody titres ~ 512. No correlatio betwee atibody level ad lug fuctio was see ad o sigificat differeces i the prevaleces of atibodies or i the percetages of patiets fulfillig the criteria of chroic ifectio amog patiets with ad those without ifectio was foud. A correlatio was foud betwee C. peumoiae IgG atibody level ad the followig variables: serum IgG cocetratio, treatmet with steroid ad age, idicatig that a high level of specific IgG atibodies was associated with a high level of serum IgG ad advaced age, while treatmet with systemic steroids ted to lower the level of specific IgG atibodies. 57

60 DISCUSSION The aims of the two cliical studies were to assess the applicability of the serological criteria for the diagosis of acute ad chroic C. peumoiae ifectios ad to describe the prevalece of C. peumoiae ifectios i two differet groups of adult patiets: Patiets who had cotracted a commuity-acquired acute respiratory tract ifectio ad patiets with a chroic respiratory tract disease who might be proe to have a chroic C. peumoiae ifectio. The results of the two studies are summarized i table 23. C. peumoiae as a cause of acute respiratory tract ifectio. I study A of patiets seeig the GP the prevalece of C. peumoiae ifectio was 2% whe usig microbiological methods ad/or the presece of IgM atibodies ad/or fourfold rise of IgG atibody titres as criteria. I a Dutch study of 557 patiets who cotacted a GP with symptoms of acute respiratory tract ifectio i 1994/1995, asopharygeal swabs were examied by PCR 105 The prevaleces of C. peumoiae ad M. peumoiae ifectios were 1.1 % ad 1.3% respectively, thus about the same as foud i the preset study. A Fiish study of 304 adults with commuity-acquired peumoia seeig a GP revealed a prevalece of C. peumoiae ifectio of 10 % by usig the same serological criteria as i the preset studies 84 The iclusio criteria i study A were adults seeig a GP with ay kids of symptoms of acute respiratory tract ifectio. It is possible that the prevalece of C. peumoiae ifectio had bee higher if the iclusio criteria had bee restricted to patiets with lower respiratory tract ifectio. I a Italia multiceter study of 613 hospitalised childre with lower respiratory tract ifectio 87 (14.1 %) had C. peumoiae ifectio 124 Serological evidece of acute C. peumoiae ifectio was foud i 52 childre, however oe were show to have IgM atibodies by the MIF test, 36 had a fourfold rise of IgG titres ad 16 had IgG atibody titres of~ 512. Of the 52 childre 13 had a positive PCR test result but it is ot stated which of the serological criteria these childre fulfilled. Thus up to 31 % (16/52) of the childre with serologic evidece of C. peumoiae ifectio ad up to 18% (16/87) of all the childre with C. peumoiae ifectio had bee give this diagosis oly because they had IgG atibody titres of ~ 512. I study A the prevalece of acute C. peumoiae ifectio i adult pa- 58

61 tiets would rise from 2 to 11 % if the patiets with isolated IgG atibody titres of ~ 512 were icluded. I some prevalece studies usig IgG atibody titres of~ 512 as a isolated criterio of acute ifectio, the deductio of the percetage of patiets oly fulfillig this criterio is ot possible; thus the compariso betwee results foud by these studies 43 ; 116 ad results foud by studies i which the criterio is ot used 84 is hampered. Further, i the Italia study C. peumoiae DNA was detected i 36 childre without ay serological evidece of acute ifectio. I a total of 8% (49) of the childre C. peumoiae DNA was detected by PCR 124 Durig the prevalece of C. peumoiae i Demark amog childre with acute respiratory ifectios was ivestigated i two studies by detectio of C. peumoiae i respiratory tract secretios by culture ad PCR 48 I the first study 427 childre (age 0-11 years) with symptoms of respiratory ifectios were erolled at a departmet of paediatrics. I the secod study 264 patiets ad cotrols (media age 3 years; 3 moths - 24 years) were erolled at their Geeral Practitioer, i both studies the prevalece of C. peumoiae was < 1% 48 I the preset studies, oe of the patiets showed a fourfold rise of IgG atibodies ad oe had a specime tested positive for C. peumoiae by PCR ad/or culture without the simultaeous detectio of high levels of IgM atibodies. Chroic respiratory tract disease. The majority of the patiets i study B had either severe COPD or severely reduced lug fuctio without havig COPD, thus the failure to fid a correlatio betwee the level of obstructio ad C. peumoiae IgG atibodies might be due to lack of statistical power caused by the low umber of patiets with mild ad moderate lug disease; however, the results are supported by other studies 138 ; 144, i cotrast oe study demostrated a positive correlatio betwee IgG atibody titre ad the severity of COPD 158 I the preset study defiitio ad classificatio of the severity of COPD followed the recommedatios from Europea Respiratory Society 137 ; however recetly the Global Iitiative for Chroic Obstructive Lug Disease (GOLD) has published their recommedatios for defiitio ad classificatio of the severity of COPD ( The latter recommedatios would have bee used i the preset study, had they bee lauched earlier. 59

62 60 I study B oe of the patiets had acute C. peumoiae ifectio whe the IgG titres of~ 512 as a isolated criterio was omitted. This result is i agreemet with other studies that have cocluded that acute C. peumoiae ifectio is rare i COPD patiets However, 20% of the patiets had C. peumoiae IgG atibody titres of~ 512 as opposed to 10% of the patiets from study A ad 2% of the cotrols (P=0.006), (table 23). The questio is whether the high level of C. peumoiae IgG is a sig of acute ifectio or whether it might be due to a raised atibody level i geeral i patiets with chroic diseases. With o sigificat differeces betwee C. peumoiae IgG atibody titre levels amog the two groups with acute ifectios ad the group with o ifectio as defied by cliical ad paracliical parameters ad with o further evidece of acute C. peumoiae ifectio, it seemed ulikely that theses patiets actually had C. peumoiae ifectio. Furthermore, IgG atibody titres of~ 512 as a criterio of acute ifectio have previously bee questioed by several authors Still, the level of CRP ad the C. peumoiae IgG atibody levels showed a tedecy towards a positive correlatio, however this was ot statistical sigificat. Whe classifyig the patiets accordig to the cliical ad paracliical fidigs previously idicated, possible cofoudig factors should be cosidered. Treatmet with glucocorticoids teds to icrease the white cell cout ad lower temperature ad the level of CRP. Further such classificatio based o symptoms ad paracliical fidigs of ifectio might ot be the ideal tool to idetify groups of patiets with C. peumoiae ifectio. Miyashita et al have described the cliical presetatio of patiets with commuity-acquired peumoia caused by C. peumoiae 108 For the 40 patiets, i whom C. peumoiae was the oly pathoge foud to cause the ifectio(= culture positive or fourfold rise i atibody titre level), the mea WBC ad mea CRP level was just borderlie elevated ad the mea temperature was 37.9 ± 1.0 C. However, elevated WBC couts have previously bee reported i associatio with C. peumoiae peumoia 86 I study B there was a highly sigificat positive correlatio betwee C. peumoiae IgG atibody titre levels ad the amout of total IgG, idicatig that patiets with chroic ifectios (or frequet ifectios) such as COPD patiets have raised levels of specific atibodies as a result of icreased total IgG. We have demostrated the same positive correlatio i a small study of patiets with edocarditis,

63 i whom a aetiological diagosis other tha C. peumoiae was demostrated i the majority of the patiets 11 It could be argued that the reaso for ot detectig the bacteria was that COPD patiets are ofte treated with atibiotics ad glucocorticoids. However, the patiets are i geeral oly treated with atibiotics whe they have a exacerbatio of their symptoms, that is cosidered to be caused by a ifectio ad eve i that case, it is possible to detect the bacteria by PCR aalyses days to weeks after the oset of the treatmet. I cotrast may patiets with severe COPD are treated costatly with glucocorticoids. I vitro experimets have show that glucocorticoids ehace the growth of C. peumoiae 160, so this treatmet should ot hider the isolatio of the bacteria from the patiets. Preexistig atibody profiles. The serological criterio of chroic C. peumoiae ifectio (table 1) was fulfilled by 16%, 37% ad 18% of the study A patiets, study B patiets ad cotrols respectively (table 23). This differece was highly sigificat (P= 0.004). Whe excludig the sera with IgG atibody titres of ;::: 512, the criterio of chroic ifectio was o loger sigificat (P= 0.12). Sice there was o differece betwee the three groups i the prevaleces of IgG atibodies, the mai differece i the atibody profiles was the raised level of IgG atibodies i the study B patiets, i.e. i patiets with frequet or chroic ifectios. Whether the presece of C. peumoiae IgA atibodies ca be used as a marker of chroic ifectio, is questioable. Although it is supported by some groups 128 ' 158 others do ot believe there is ay evidece to support this hypothesis 162 A major problem is the lack of cliical defiitios or sigs of chroic C. peumoiae ifectio, that could help i distiguishig the patiets suspected of chroic ifectio from those with previous ifectio. I study B there was o differeces i the prevalece or i the level of IgA atibodies betwee the three groups (table 27), however the study B patiets had a higher prevalece of IgA tha the patiets from study A, though o differece was see i titre levels (figure 3 ad 7). This is i cotrast to aother study of patiets with either peumoia, severe or mild COPD, i whom oly small differeces i C. peumoiae IgG atibody levels were foud, whereas the differeces i serum IgA atibody levels were highly sigificat 158 Oe study examied sera from 219 patiets with COPD admitted to a departmet of iteral medicie ad 100 cotrols 100 ad foud a highly sigificat differece betwee patiets ad 61

64 cotrols i the prevaleces of C. peumoiae IgG ad IgA atibodies, but like the preset study the atibody prevaleces were ot correlated to the severity of airway obstructio. I that study IgG ad the IgA atibodies were aalysed i twofold titratios begiig at 1:64 ad 1:32, respectively, whereas i the preset studies A ad B the iitial dilutio step applied was 1:16 for both atibody classes. I the preset studies, the differece i the prevaleces of IgG atibody titres ~ 64 ad IgA atibody titres ~ 32 amog patiets from study A, B ad the cotrols were sigificat (table 23). This example illustrates a problem whe prevaleces i study groups with differet distributio curves are compared. The prevalece foud is iflueced by the iitial dilutio step chose, especially, for groups i which the majority of subjects has low level seropositivity as ofte see i cotrol groups, whereas the prevalece amog groups of patiets with a high level seropositivity as see amog the patiets i study B is less iflueced by a high iitial dilutio step. Therefore, what is see as a differece i prevalece might just be a differece i atibody titre levels. If Acute ifectio with other "atypical respiratory tract pathoges". M. peumoiae were foud i two patiets (2%) i study A. Although a M. peumoiae epidemic occurred i Demark durig the first moths of study B 83 oly oe patiet i this study where show to have M. peumoiae (<1 %), idicatig that this ifectio is rare i older patiets with chroic respiratory diseases. B. pertussis were likewise demostrated i two patiets (2%) i study A ad i oe patiet (<1 %) i study B. The umber of participats was too low to estimate the actual prevaleces of these pathoges i patiets cosultig the GP. However, it is oteworthy that whoopig cough is a illess to be cosidered amog adults. The patiet i study B had COPD i a stable phase ad acute ifectio was ot suspected. That B. pertussis ca be foud amog adult patiets has bee cofirmed by other studies 28 ; 170 Recetly, a Daish study of 201 adult patiets with cough for more tha three weeks, showed that 7% had B. pertussis ifectio verified by culture or PCR 14 C. psittaci was demostrated i oe patiet i study B ad i oe of the patiets i study A. Orithosis is a rare disease i Demark with about 30 cases reported per year. However, it is to be suspected that cases with a milder course of the dis- 62

65 ease ted to be overlooked, thus the true icidece might be higher tha that reported29. Noe of the patiets had serological or microbiological sigs of Legioella ifectio. The cocer about cotamiatio of pharygeal swabs or sputum with L. peumophila from tap water showed to be ufouded as the bacteria was ot detected by PCR i ay of the over samples from the 367 patiets participatig i the two studies. Coclusio. The assumptio that 5 ad 15% of the patiets i the two studies respectively, should have a acute C. peumoiae ifectio was ot met ad the umber of subjects erolled i the two studies was much lower tha plaed. However, the material is still sufficietly large to coclude that i Demark, acute C. peumoiae ifectio is ucommo amog patiets with symptoms of acute respiratory ifectio ad very rare amog patiets with chroic pulmoary disease. However, if the criterio of C. peumoiae IgG of ~ 512 was used as a marker of acute ifectio, 20% of the study B patiets ad 10% of the GP patiets would have had a acute C. peumoiae ifectio. Usig the serological criterio of chroic ifectio, 37% ad 16% of the patiets, respectively would have had chroic C. peumoiae ifectio but lackig ay other evidece of C. peumoiae ifectio the diagosis seemed ulikely; especially i the patiets ad cotrols seeig the GP. Microbiological results were i agreemet with serological results as the bacteria was ot isolated from ay patiets that did ot have a atibody profile idicatig acute C. peumoiae ifectio.

66 CHAPTER FIVE: GENERAL DISCUSSION Epidemiology Durig the last decade several Daish studies o the prevalece of either C. peumoiae atibodies ad of acute C. peumoiae ifectio has bee performed. Except for the two early studies by Mordhorst et al 113 ; 165 all serological aalyses for the detectio of C. peumoiae atibodies i the Daish studies metioed i this chapter were coducted i the Neisseria Uit, SSI ad if o other iformatio is give by the WRF assay. The results are comparable sice o iterassay or iterlaboratory variatio of test results has to be cosidered. Seroepidemiological populatio studies. I a Daish study of 106 healthy blood doors about 40 years old (± 10 years) the prevalece of C. peumoiae IgG atibody titres of 2: 64 was 40% ad C. peumoiae IgG atibody titres of 2: 512 were foud i 1 % 13 No sigificat chage i C. peumoiae atibody titre levels betwee the two visits was see whe the doors were retested four moths later. This study alog with study described i chapter oe 165 showed that the prevalece of C. peumoiae IgG atibodies i the Daish populatio is high, which is cosistet with fidigs from other coutries 19 ' 165 That a high prevalece would be associated with frequet C. peumoiae ifectios seems to be a reasoable workig hypothesis. Thus it should be possible to ecircle groups of patiets i whom the icidece of C. peumoiae ifectio is high. Prevalece oi acute C peumoiae ifectio. Seroepidemiological studies have idicated, that a epidemic of C. peumoiae ifectios occurred i Demark i Mordhorst et al, ad Farholt ad Hase each described a family outbreak of C. peumoiae ifectio but beyod this, maily sporadic cases have bee reported 49 ; 112 Microbiological studies. Durig the prevalece of C. peumoiae amog childre with acute respiratory ifectios was ivestigated i two studies by detectio of C. peumoiae i respiratory tract secretios by culture ad PCR 48 I total 691 childre were examied ad C. peumoiae was foud i< 1 %. 64

67 Combied microbiological ad serological studies. The preset studies A ad B: As oly two patiets i study A ad oe of the study B patiets were show to have acute C. peumoiae ifectio, we coclude that the icidece of acute C. peumoiae ifectio was low amog Daish adult patiets with acute or chroic respiratory tract ifectios. I group II of the mai laboratory study of comparig the performace of differet MIF assays, eight patiets with respiratory tract ifectio had C. peumoiae ifectio verified by a positive PCR result. I additio two patiets from the study A had acute C. peumoiae ifectio (table 13 ad 21). It is iterestig that ie out of te patiets had C. peumoiae IgM atibodies at the first cotact with the GP that elicited takig of specimes for C. peumoiae aalyses. Sice detectable IgM atibodies emerge after about two weeks, it is a idicatio of symptoms of C. peumoiae ifectios beig slow i developig. This is i cotrast to C. psittaci ifectios, that develop more rapidly, these patiets are ofte C. psittaci IgM atibody egative whe they first seek medical assistace 10 I a study of 201 patiets with cough for more tha three weeks the prevalece of C. peumoiae ifectio was show to be 3 of 201 (1.5%) 13 All three were cofirmed by PCR ad were show to have C. peumoiae IgM atibodies by MIF. Further six patiets had persistet IgG atibody titres of~ 512, oe of these had a positive PCR result. I a study of 67 cosecutive patiets with commuity-acquired peumoia admitted to a hospital durig brochoalveolar lavage was performed o all patiets as well as blood samples were obtaied 90 The obtaied liquid was subsequetly examied by PCR for the detectio of Chlamydia species. C. peumoiae DNA was ot detected i ay of the specimes but oe patiet had C. psittaci DNA Sera were aalysed by Chlamydia CF test ad by MIF test. Noe of the patiets had serological evidece of C. peumoiae ifectio by the MIF test but two patiets had a sigificat rise of CF titre icludig the patiet with orithosis. I aggregate, the three Daish studies, icludig the preset, usig a combiatio of microbiological ad serological methods for the assessmet of the prevalece of acute C. peumoiae ifectio demostrated a very low prevalece (0-2 % ) of 65

68 acute ifectio. Further, a high correlatio betwee the detectio of C. peumoiae DNA by PCR ad the detectio of C. peumoiae IgM by MIF test was foud. Possible chroic C. peumoiae ifectio. Patiets with reduced lug fuctio (COPD). I the secod cliical study the prevalece of C. peumoiae ifectios amog hospitalised patiets ad outpatiets, the majority of patiets icluded had COPD or reduced lug fuctio, was assessed. COPD is a lifelog disease with sigificat morbidity. Smokig is the pricipal factor i the developmet of COPD, however as most smokers do ot develop the disease there might be additioal factors participatig i the developmet of pulmoary obstructio 136 ' 160 Though widely studied, the role of recurret ifectios i the aetiopathogeesis of COPD has remaied cotroversial. The emphasis of these studies has bee o opportuistic pathoges such as Stre ptococcus peumoiae ad Haemophilus ifluezae but o clear role has bee foud for these bacteria i the pathogeesis of COPD 136 C. peumoiae differs from the agets covetioally associated with exacerbatios i that it is a itracellular orgaism that does ot belog to the ormal asopharygeal flora. C. peumoiae ifectios i COPD patiets might amplify smokig- associated iflammatio ad worse irreversible obstructio. Theoretically, oe could distiguish betwee acute C. peumoiae ifectios causig exacerbatios of COPD ad chroic ifectios ivolved i the pathogeesis of the disease. Cosiderable data suggest that C. peumoiae might be associated with chroic diseases 20 ' 128 ' 159 ad serological evidece of C. peumoiae ifectio have bee reported i 7-63% of patiets with COPD It is well established that chroic or recurrig C. trachomatis ifectios ca cause scarrig of eyes (trachoma) ad of the Falloppia tubes (salpigitis) leadig to blidess 169 or ifertility 132 ; therefore it seems plausible that C. peumoiae might be ivolved i similar processes i the lugs. It has previously bee reported that 4 % of exacerbatios may be associated with C. peumoiae ifectio 20 However, amog the 210 study B patiets icludig 81 with acute ifectio oe had serological sigs of acute C. peumoiae ifectio ad i oe of the patiets were C. peumoiae detected by microbiological methods. 66

69 67 Patiets with cardiovascular diseases. Sice Saikku i 1988 demostrated serological evidece of a associatio of C. peumoiae with chroic coroary heart disease ad acute myocardial ifarctio 128, several studies have cofirmed this associatio37. We ivestigated the prevalece of C. peumoiae atibodies amog 830 patiets with ischaemic heart disease (IHD) selected from a larger study 76 ad matched accordig to age ad geder to the study B patiets (upublished data). IgG atibody titres of ~ 512 was measured i 7% of the 830 IHD patiets (figure 10) i cotrast to 20% of the patiets i study B (P< 0.001), although the same percetage (70-72%) showed the presece of IgG atibody titres of~ 16. There was o differece i IgA atibody level betwee the two groups (figure 11). The criterio of chroic ifectio was met i 141/631 (22%) of the IHD patiets i cotrast to 77/210 (37%) i the preset study (P=0.003). Figure 10 shows the distributio of C. peumoiae IgG atibodies i the patiets ad cotrols of the two preset studies ad the IHD study. Although the prevalece of IgG atibodies i the study B patiets were equal to that of the IHD patiets ad oly slightly higher tha the GPpatiets, the distributio curves accordig to IgG atibody titres showed a peak at 256, 128 ad 64 respectively for the three groups idicatig that the level of IgG atibodies is raised i patiets with chroic or recurret ifectios. This was supported by the sigificat correlatio betwee specific C. peumoiae IgG atibodies ad total serum IgG cocetratio. I cotrast, there were o sigificat differeces i IgA atibody edpoit titres amog the study populatios as discussed i chapter 4. The IHD study was coducted durig the same period ad i the same area of the coutry as the two preset studies, thus it is ulikely that the differeces were due to local outbreaks of C. peumoiae ifectio. Aother study of patiets with cardiovascular diseases showed poor correlatio betwee the detectio of C. peumoiae DNA i coroary artery segmets aalysed by PCR ad the detectio of C. peumoiae IgG atibodies by MIF 103 These fidigs are supported by a study of serum immuoglobuli levels i patiets with tuberculosis as a prototype of chroic ifectio ad Klebsiella ifectio represetig acute ifectio 134 The study demostrated sigificatly elevated levels of IgG amog the TB-patiets compared to the Klebsiella patiets i cotrast, o such differece was foud i the IgA levels cocludig that these fidigs reflected the polycloal hypergarraglobuliaemia foud i chroic ifectios.

70 I a meta-aalyses that pooled 15 prospective studies, Daesh et al foud o overall relatioship betwee C. peumoiae IgG seroprevalece ad subsequet cardiovascular evets after adjustig for age, geder, smokig ad socio-ecoomic status 39. Circulatig C. peumoiae DNA has bee foud i peripheral blood moouclear cells (PBMC) of patiets with cardiovascular diseases 139 By a metaaalysis of uadjusted casecotrol studies a pooled prevalece of 14.3 % was foud i cardiovascular disease patiets versus 8.5% i cotrols correspodig to a odds ratio of 2.03 (CI: , P < 0.001). However, whe the authors added a ew study ot yet published that adjusted for smokig ad seaso the overall associatio was o loger sigificat (OR= 1.6, CI: , P=0.22). As described i chapter oe, C. peumoiae has bee isolated from atherosclerotic plaques, but o correlatio betwee C. peumoiae serology ad the detectio of DNA i plaques has bee foud 139 The questio of whether C. peumoiae cotributes to atherosclerotic iitiatio or progressio or to cardiovascular evets remais so far uaswered. The diagosis of C. peumoiae ifectio The high prevalece of C. peumoiae ifectio reported i studies exclusively based o serodiagostic methods have bee questioed i studies based o both serological ad microbiological methods. To assess the rate of C. peumoiae ifectio both sesitive ad specific diagostic methods are eeded. So far, the descriptio of acute C. peumoiae ifectios has bee hampered by a lack of diagostic methods that fulfil these eeds. Culture. While culture with appropriate idetificatio methods per defiitio is specific, the sesitivity depeds o may factors, icludig suitable trasport coditios for specimes to the laboratory ad a skilled hadlig i the laboratory 48 A practical obstacle i obtaiig specimes for culture is that special Chlamydia trasportmedia cotaiig fetal calfserum is compulsory ad is ot always available at the cliical departmets. Further oly few laboratories have a sesitive culture method sice cell lies used for the culture of C. trachomatis are ot optimal for the culture of C. peumoiae 48 PCR. As the PCR aalyses do ot deped o live microorgaism, they are less sesitive tha culture to variatios i the "pre-laboratory" hadlig of specime. 68

71 69 Some PCR methods, especially amog the ested PCR assays, have a high sesitivity24. However, the aalysis has a risk of yieldig false positive results due to lack of specificity or cotamiatio of samples. Although may i-house PCR methods are i use, either a stadardised method or commercially available assays for routie diagostics exist. Earlier this year experiece with a commercial PCR assay has bee published, though so far it is oly recommeded for scietific purposes 27. The specime collectio depeds o the type of cliical coditio. Suitable specimes for the detectio of C. peumoiae DNA iclude sputum 22, swabs of asopharyx or throat2 2, brochoalveolar lavage 36 ad tissue 145. Lately several groups have reported o the detectio of C. peumoiae DNA i peripheral blood moouclear cells (PBMC) maily i patiets with cardiovascular disease but also i patiets with COPD ad blood doors 24. It remais uclear which cells withi the PBMC layer cotai C. peumoiae. The two studies of patiets with COPD observed prevaleces C. peumoiae DNA i PBMC of 24 % ad 48%, respectively 139. I oe of these two studies were icluded cotrols. I a study 15 idetical sets with 50 samples each icludig 23 egative cotrol samples were set to each of 15 laboratories performig PCR aalyses. The samples cosidered positive cotaied DNA from cultured C. peumoiae ad PBMC from previously PCR positive patiets with cardiovascular disease. The levels of sesitivity of the differet PCR methods raged widely ad 5 of 17 laboratories reported false positive results 24. The DNA has also bee detected i atherosclerotic plaques however, there is o clear correlatio betwee detectio of DNA i plaques ad i PBMC or betwee DNA i plaques ad the presece of C. peumoiae IgG by MIF test2 4 ; 139. I oe study the performaces of 16 PCR methods i 9 cetres i detectig C. peumoiae i carotid vessels was compared 4. The coclusios were that there was o cosistet patter of positive results amog the various laboratories, ad there was o correlatio betwee the detectio rates ad the sesitivity of the assay used. Without a thorough validatio of these methods by the compariso of PCR results with those of a sesitive culture system ad at least oe other validated PCR method targetig a differet gee or a differet sequece of the same gee, the PCR results will be less covicig.

72 70 CF test. With the geus-specific CF test a large umber of sera ca be hadled with a miimum of mapower ad at a low cost. The results of the test ca be iflueced by the preparatio of the atige used, ad by the procedures of the aalysis. I the early era of the study of C. peumoiae ifectios, i several of the major studies of the epidemiology of acute C. peumoiae ifectios, the CF atige from SSI was used ad geerally a high correlatio betwee the CF test results ad the presece of C. peumoiae IgM by the MIF test was see 62 ' 63 ' 113 ' 131 ' 147. This correlatio is also see i papers published withi the recet years 90 ' 122. Previously i this thesis it was demostrated that the test was ot suited for screeig sera prior to IgG atibody aalyses by the MIF test. I coclusio, the method is a excellet supplemet for the diagosis of early phase chlamydia! ifectio. MIF assays. Amog the differet serological methods the MIF test, though ot perfect, is still cosidered the method of choice for the diagosis of C. peumoiae ifectios 40. Reported differeces i the prevaleces of C. peumoiae ifectios i various geographic areas at differet periods of time could be due to problems cocerig the performace of the MIF test 43 ' 147 I the preset study it was demostrated that the reproducibility of detectio rates betwee WRF as referece method ad the two commercial assays (MRL ad LAB) was excellet for the detectio of IgM atibodies, for IgG ad IgA atibodies i patiets without acute ifectios ad acceptable for IgG ad IgA atibodies i patiets with acute respiratory tract ifectios. Other factors tha the quality of the assays used ca ifluece o results obtaied. Apart from the udeiable subjectivity i the readig of MIF slides ad differeces i laboratory procedures, the results ca be iflueced by the cojugates used; especially the ati-iga cojugates are reported to be of varyig quality 162. Prior to IgA ad IgM atibody testig it is recommeded that IgG should be removed40. Those laboratories that did ot remove IgG might have reported false positive IgM atibody results due to circulatig rheumatoid factor. Sice the presece of IgM atibodies is cosidered a sig of acute ifectio, it is possible that some studies have reported prevaleces of acute C. peumoiae ifectio that have bee iflated due to the iclusio of patiets with false positive IgM atibody results.

73 I aggregate, culture of C. peumoiae is a specific method, however the sesitivity vary betwee laboratories; it is expesive, requires careful pre-laboratory hadlig of specime ad is ot assessable everywhere. I cotrast the PCR method is more robust to pre-laboratory hadlig of specime, some PCR assays ca be very sesitive, but the specificity vary betwee laboratories ad is difficult to assess without a very sesitive ad well defied gold stadard, which curretly does ot exist. Of the two serological methods described, the CF test is i our laboratory a good ad sesitive method for the diagosis of early phase chlamydia} ifectio but it caot distiguish betwee ifectios caused by the differet species. The MIF test is a resource demadig method. Cosiderable iterlaboratory variatios of test results have bee demostrated, however we have show that withi the same laboratory oly small differeces i test results obtaied by differet assays occur. I our hads it is a good ad reliable method for the diagosis of acute C. peumoiae ifectio ad for the detectio of C. peumoiae IgG ad IgA atibodies. Applicability of serological criteria. The serological criteria of acute ad chroic C. peumoiae ifectio were also discussed i chapter four. Serological criteria of acute ifectio. The Graysto criteria of acute ifectio are based o three criteria (table 1). The validity of the criteria of detectio of IgM atibodies of:::: 16 ad of four-fold rise of IgG titres are supported by the frequet fulfilmet of these criteria simultaeous with the detectio of C. peumoiae by culture or PCR. I our laboratory we very seldom fid specimes positive for C. peumoiae by culture or PCR without fidig the above criteria fulfilled if adequate serum samples are obtaied. I cotrast, we see samples fulfillig the serological criteria without beig able to detect the orgaism by PCR or culture. The third criterio of acute ifectio IgG atibodies of~ 512 is dubious. Amog the patiets icluded i the preset studies i chapter three ad four, oly oe of te patiets with cofirmed C. peumoiae ifectio developed IgG atibody titres of ~ 512, while 20% of the study B patiets had IgG atibodies of~ 512 at a costat level through the six week observatio period. I cotrast oly 2% of the cotrols

74 72 ad 10% of the patiets with acute respiratory tract ifectio fulfilled this criteria. Amog the study B patiets it was ot possible to detect ay differeces i the levels of IgG atibody titres betwee patiets with acute ifectio ad patiets without acute ifectio. Moreover as the bacteria was ot detected i ay of these patiets, it seems ulikely that the patiets had acute C. peumoiae ifectio ad thus the criterio of IgG atibodies of~ 512 has ot bee useful i the preset studies ad if ever used as a isolated criterio of acute ifectio, it should be iterpreted with cautio. If reifectio is defied as a rapid rise of C. peumoiae IgG atibodies ad/or the detectio of the bacteria with microbiological methods without the simultaeous presece of IgM atibodies we did ot detect ay proof of such coditio. O a purely theoretical basis, it ca be speculated that some of the patiets with a high, stable level of IgG atibodies had a reifectio ad that blood samples were draw to late i the course of ifectio to detect the chage i titre levels. Serological criteria of chroic ifectio. (table 1). A major problem is the lack of cliical criteria for defiig a chroic C. peumoiae ifectio. Amog the study groups of chapter four the criterio was fulfilled by 16%, 37% ad 18% respectively of the GP-patiets with acute ifectio, study B patiets ad cotrols. Apart from the issue of the lack of detectio of the bacteria discussed above, it is ulikely that 18% of cotrols ad 16% of patiets with acute ifectio of whom oe have cliically detected, chroic cardio-pulmoary diseases, should have chroic C. peumoiae ifectio. Amog the study B patiets it was ot possible to sigle out ay subgroup of patiets that sigificatly differed from the others i the percetages fulfillig the criterio of chroic ifectio, thus the hypothesis that 37% of the COPD patiets should have chroic C. peumoiae ifectios could ot be supported by ay cliical or paracliical data. I aggregate, for the serodiagosis of acute C. peumoiae ifectio the detectio of IgM atibodies or a fourfold rise of IgG atibody titres has show to be trustworthy criteria, i cotrast IgG atibody titres of~ 512 is either a sesitive or a specific criterio. We have ot bee able to cofirm the existece of specific serological criteria for reifectio. Likewise we have ot bee able to cofirm the applicability of the proposed serological criteria of chroic ifectio. We have foud a high correlatio betwee culture ad PCR results ad that positive microbiological results correspoded well with the detectio of C. peumoiae IgM.

75 73 Coclusio. I this thesis it has bee demostrated that test results of WRF as referece method ad the two commercially available assays from MRL ad LAB were equivalet whe sera from differet groups of patiets were aalysed i oe laboratory. All three methods had about the same detectio rate of atibodies ad the mea level of edpoit titres was likewise the same withi a variatio of oe dilutio step. The oe exceptio was IgM atibody titres foud by LAB, which were o the average about two dilutio steps higher tha the mea edpoit titres foud by the two other assays. The assessmet of the applicability of serological criteria for the diagosis of acute C. peumoiae ifectio cofirmed that the presece of IgM atibodies of ~ 16 was sig of acute ifectio. Moreover most of the patiets with acute C. peumoiae ifectio had developed IgM atibodies prior to seekig medical assistace, thus the presece of IgM atibodies is a useful tool for routie diagostics also whe oly oe serum sample is obtaied. Fourfold rise of C. peumoiae IgG atibody titres likewise seems to be a valid criterio for the diagosis of acute C. peumoiae ifectio, but the rise of IgG atibody titres occurs later tha IgM atibodies ca be detected. I cotrast IgG of;:::: 512 is either a sesitive or a specific criterio. We have ot bee able to cofirm the existece of specific serological criteria for reifectio. Likewise we have ot bee able to cofirm the applicability of the proposed serological criteria of chroic ifectio. The edemic occurrece of acute C. peumoiae ifectios amog adults with ad without chroic pulmoary diseases was low; combied with the results of the other Daish prevalece studies of C. peumoiae ifectios 13 ' 13 ' 48 ' 113, it ca be cocluded that so far it has ot bee possible to distiguish groups of patiets i Demark i whom C. peumoiae ifectios are frequetly occurrig. The results from the microbiological ad serological study of patiets with reduced lug fuctio did ot support the hypothesis that C. peumoiae should be ivolved i acute exacerbatios of COPD. Nevertheless, the prevalece of C. peumoiae IgG atibodies was high i all populatios studied, but the mea level of edpoit titres was elevated amog the patiets with chroic diseases. The reasos for the persistece of chlamydia! atibodies ad the elevated levels '; 'i

76 foud i certai groups are ot well uderstood. The future challege will be to fid the mechaisms for log-term persistece of C. peumoiae atibodies, to explore why some groups of patiets have elevated levels of chlamydia! atibodies ad to examie to what exted the bacteria cause chroic ifectio. 74

77 Summary The aims of the Ph.D. study were: To assess the performace of two commercially available MIF assays for the detectio of C. peumoiae atibodies compared to a referece method whe applied to sera from differet categories of patiets. To assess the applicability of the followig serological criteria recommeded for the diagosis of C. peumoiae ifectio. Acute ifectio: IgM atibody titre of ~ 16, four-fold rise of IgG atibody titres or IgG atibody titres of~ 512. Chroic ifectio: IgG atibody titres of~ 128, IgA atibody titres of~ 40 ad IgM atibody titres of ::; 8. By combied use of microbiological ad serological methods to describe the prevalece of C. peumoiae ifectio i adult patiets with acute commuityacquired respiratory tract ifectios. By combied use of microbiological ad serological methods to describe the prevalece of C. peumoiae ifectio i patiets with chroic obstructive pulmoary diseases with ad without acute exacerbatios of the disease. The results obtaied by assays from MRL Diagostica (MRL) ad Labsystems (LAB), respectively, were compared to those obtaied with a MIF assay based o C. peumoiae atige from Washigto Research Foudatio (WRF) as a referece method. I total 220 sera from three differet groups of patiets were aalysed. I: Patiets with serological evidece of acute C. peumoiae ad/or M. peumoiae ifectio. II: Patiets with C. peumoiae or C. psittaci ifectio verified by PCR. III: Persos erolled i the Copehage City Heart Study amog whom oe fifth later developed AMI. The overall agreemet betwee results by the WRF assay ad by the two commercial assays was excellet for IgM atibody detectio rate (98%). The accordace i detectio rates for IgG ad IgA atibodies i sera from patiets with acute ifectios was acceptable (87 to 88%) ad i sera from group III it was excellet (95 to 97%). No sigificat differece amog the detectio rates of the three assays was see for ay of the Ig classes. The reproducibility of C. peumoiae IgG ad IgA atibody edpoit titres was high, though IgG atibody titres by the WRF assay were o average 0.4 dilutio 75

78 76 step lower tha those by the two other assays. The IgM atibody edpoit titres foud by the LAB assay were almost two dilutio steps higher tha those foud by the WRF ad the MRL assay. The applicability of the serological criteria for the diagosis of acute C. peumoiae ifectio was assessed. The study cofirmed that C. peumoiae IgM atibodies of 2:: 16 was a sig of acute ifectio. Most patiets had developed IgM atibodies before seekig medical assistace, thus detectio of C. peumoiae IgM atibodies is a useful tool for routie diagostics. Fourfold rise of C. peumoiae IgG atibody titres likewise seems to be a valid criterio for the diagosis of C. peumoiae ifectio but the rise of IgG atibody titres occurs later durig the course of illess tha detectable IgM atibodies. The presece of C. peumoiae IgG atibody titres of:::: 512 was ot useful i the diagosis of C. peumoiae ifectio. We have ot bee able to cofirm the existece of specific serological criteria for reifectio. Likewise we have ot bee able to cofirm the applicability of the proposed serological criteria of chroic ifectio. The edemic occurrece of acute C. peumoiae ifectios amog adults with ad without chroic pulmoary diseases was low i.e. < 2%; combied with results of other Daish prevalece studies of C. peumoiae ifectios, it ca be cocluded that so far it has ot bee possible to distiguish groups of patiets i Demark i whom C. peumoiae ifectios are frequetly occurrig. The results from the microbiological ad serological study of patiets with reduced lug fuctio did ot support the hypothesis that C. peumoiae should be ivolved i acute exacerbatios of COPD. Nevertheless, the prevalece of C. peumoiae IgG atibodies was high i all populatios studied, but the mea level of edpoit titres was elevated amog the patiets with chroic diseases. A positive correlatio betwee C. peumoiae IgG atibody titres ad cocetratio of serum IgG was foud. This fidig might be cosistet with polycloal stimulatio of B-lymfocytes; i cotrast the results do ot support the occurrece of chroic C. peumoiae ifectio. The reasos for the elevated levels of IgG C. peumoiae atibodies foud i certai groups are still ot well uderstood.

79 Dask resume Formalet med ph.d. - studiet var: At vurdere ydeeve af to kommercielle MIF tests til pavisig af C. peumoiae atistoffer sammeliget med e referece metode ved at uders0ge sera fra forskellige patietgrupper. At vurdere brugbarhede af iteratioalt abefalede serologiske kriterier til diagostik af C. peumoiae ifektio. Akut ifectio: IgM atistof titer ;::: 16, fire-fold stigig i lgg atistof titer eller IgG atistof titer;::: 512. Kroisk ifektio: IgG atistof titer;::: 128, IgA atistof titer;::: 40 og IgM atistof titers; 8. Pa grudlag af bade mikrobiologiske og serologiske uders0gelser at beskrive forekomste af C. peumoiae if ektioer bladt vokse patieter med akut luftvejsifektio set i alme praksis. Pa grudlag af bade mikrobiologiske og serologiske uders0gelser at beskrive forekomste af C. peumoiae ifektioer bladt patieter med kroisk obstruktiv lugesygdom med og ude akut exacerbatio af sygdomme. For at uders0ge ydeeve af MIF teste blev resultater opaet ved assays fra heholdsvis MRL Diagostica (MRL) og Labsystems (LAB) sammeliget med resultatere opaet ved e referece metode baseret pa atige fra Washigto Research Foudatio (WRF). Der blev i alt uders0gt 220 sera fra tre forskellige patietgrupper. I: Patieter med serologisk teg pa akut C. peumoiae og/eller M. peumoiae ifektio. II: Patieter med C. peumoiae eller C. psittaci ifektio verificeret ved PCR aalyse. III: Persoer idgaet i 0sterbro-uders0gelse, bladt hvilke e femtedel seere udviklede AMI. Overesstemmelse mellem resultatere opaet ved WRF og de to kommercielle metoder var god for pavisig af IgM atistoffer (98%). Overesstemmelse i detektios rater for IgG og IgA atistoffer i sera fra patieter med akutte ifektioer var acceptabel (87 til 88 %) og i sera fra gruppe III var de god ( 95 til 97%).For ige af Ig klasseme, var der oge sigifikat forskel i detektios rater opaet ved de tre test metoder.

80 Reproducerbarhede for C. peumoiae IgG ad IgA atistof titer iveau opaet med de tre metoder i var ligeledes h0j, dog var IgG atistof titer iveauet pavist ved WRF teste geemsitligt 0.4 fortydigstri lavere ed ved de to adre metoder. IgM atistof titer iveauet pavist ved LAB teste var geemsitligt 1.7 fortydigs tri h0jere ed iveauet opaet med WRF og MRL testee. Avedelighede af de serologiske kriterier til at stille cliagose akut C. peumoiae ifektio blev vurderet. Studiet bekrceftede, at C. peumoiae IgM atistoffer ~ 16 var et teg pa akut ifektio. De fleste patieter havde udviklet C. peumoiae IgM atistoffer f.0r de s0gte lcege, det vi1 sige at pavisig af IgM atistoffer er et brugbart redskab til rutie diagostik. Fire-fold titer stigig af C. peumoiae IgG syes ligeledes at vcere et avedeligt kriterium for at stille diagose akut C. peumoiae ifektio, me IgG atistof titer stigig forekommer seere i forl0bet af sygdomme ed daelse af IgM atistoffer. Tilstedevcerelse af C. peumoiae IgG atistof titre ~ 512 viste sig ikke brugbart til at stille diagose akut C. peumoiae ifektio. Det har ikke vceret muligt at fastlcegge serologiske kriterier til pavisig af reifektioer eller at vurdere brugbarhede af de foreslaede kriterier til serologisk pavisig af kroisk ifektio. De edemiske forekomst af akut C. peumoiae ifektio bladt vokse med og ude kroisk lugesygdom var lav, i.e. <2%. Nar disse resultater sammeliges med resultater fra yere daske studier ma det kokluderes, at det edu ikke har vceret muligt at idkredse patietgrupper i Damark med h0j forekomst af C. peumoiae ifektioer. Resultateme fra det mikrobiologiske og serologiske studium af patieter med kroisk lugesygdom kue ikke bekrcefte hypotese om at C. peumoiae er associeret til akutte exacerbatioer hos KOL patieter. Forekomste af C. peumoiae IgG atistoffer var h0j i alle studiepopulatioer, me det geemsitlige titer iveau var forh0jet bladt patieter med kroiske sygdomme. Der fadtes e sigifikat positiv korrelatio mellem C. peumoiae IgG atistof titre og kocetratioe af serum IgG. Dette ka vcere foreeligt med polykloal B-lymfocyt stimulatio, hvorimod det ikke har vceret mulig at sadsyligg0re forekomste af kroisk C. peumoiae ifektio. Baggrude for at visse patietgrupper har forh0jet C. peumoiae IgG atistofiveau er ikke edeligt afklaret.

81 Referece List 1. Allegra L, Blasi F, Cetai S, Cosetii R, Deti F, Raccaelli R et al. Acute exacerbatios of asthma i adults: role of Chlamydia peumoiae ifectio. Eur.Respir.J. 1994;7: Almirall J, Morato I, Riera F, Verdaguer A, Priu R, Coll P et al. Icidece of commuityacquired peumoia ad Chlamydia peumoiae ifectio: a prospective multicetre study. Eur.Respir.J. 1993;6: Aderso JL,.Muhlestei JB. The ACADEMIC study i perspective (Azithromyci i coroary artery disease: elimiatio of myocardial ifectio with Chlamydia). J.lfect.Dis.2000.Ju; 181.Suppl.3: Apfalter P, Blasi F, Boma J, Gaydos CA, Kudi M, Maass M et al. Multiceter Compariso Trial of DNA Extractio Methods ad PCR Assays for Detectio of Chlamydia peumoiae i Edarterectomy Specimes. J.Cli.Microbiol Feb;3~2): Appleyard M, Hase AT, Schor P, Jese G, Nyboe J. The Copehage Heart Study, 0sterbrouders0gelse: a book of tables with data from the first examiatio ( ) ad a five-year follow-up ( ). Scad J Soc Medicie 1989;170: Bares RC. Laboratory diagosis of huma chlamydia! ifectios. Cli.Microbiol.Rev. 1989;2: Beaty CD, Graysto JT, Wag SP, Kuo CC, Reto CS, Marti TR. Chlamydia peumoiae, strai TWAR, ifectio i patiets with chroic obstructive pulmoary disease. Am.Rev.Respir.Dis. 1991; 144: Beem MO,.Saxo EM. Respiratory-tract coloizatio ad a distictive peumoia sydrome i ifats ifected with Chlamydia trachomatis. N.Egl.1.Med. 1977;296: Beedse M, Berthelse L, Lid I. Performace of Three Microimmuofluorescece Assays for Detectio of Chlamydia peumoiae Immuoglobuli M, G, ad A Atibodies. Cli.Diag.Lab Immuol. 2002;9: Beedse, M. ad Filskov, A. A outbreak of psittacosis amog employees at a poultry abattoir. I Saikku P, ed.: Proceedigs of the Fourth Meetig of the Europea Society for Chlamydial Research, p Beedse, M., Kjerulf, A., ad Lid, I. Elevated level of Chlamydia peumoiae IgG atibodies i patiets with edocarditis. I Saikku P, ed.: Proceedigs of the Fourth Meetig of the Europea Society for Chlamydia! Research, p Berdal BP, Fields PI, Melbye H. Chlamydia peumoiae respiratory tract ifectio: the iterpretatio of high titres i the complemet fixatio test.scad.j.ifect.dis.1991;23:

82 13. Birkebaek NH, Jese JS, Seefeldt T, Deg J, Huiche B, Aderse PL et al. Chlamydia peumoiae ifectio i adults with chroic cough compared with healthy blood doors. Eur.Respir.J.2000.Jul; 16(1): Birkebaek NH, Kristiase M, Seefeldt T, Deg J, Moller A, Hero I et al. Bordetella pertussis ad chroic cough i adults. Cli.lfect.Dis. 1999;29: Birkelud S, Ludemose AG, Christiase G. Chemical cross-likig of Chlamydia trachomatis. lfect.lmmu. 1988;56: Black CM, Fields PI, Messmer TO, Berdal BP. Detectio of Chlamydia peumoiae i cliical specimes by polymerase chai reactio usig ested primers. Eur J Cli Microbial Ifect Dis 1994;13: Black CM, Johso JE, Farshy CE, Brow TM, Berdal BP. Atigeic variatio amog strais of Chlamydia peumoiae. J.Cli.Microbiol. 1991;29: Black PN. The use of macrolides i the treatmet of asthma. Eur Respir Rev. 1998;6: Blasi F, Cosetii R, Schoeller MC, Lupo A, Allegra L. Chlamydia peumoiae seroprevalece i immuocompetet ad immuocompromised populatios i Mila. Thorax 1993;48: Blasi F, Legai D, Lombardo VM, Negretto GG, Magliao E, Pozzoli R et al. Chlamydia peumoiae ifectio i acute exacerbatios of COPD. Eur.Respir.J. 1993;6: Block S, Hedrick J, Hammerschlag MR, Cassell GH, Craft JC. Mycoplasma peumoiae ad Chlamydia peumoiae i pediatric commuity-acquired peumoia: comparative efficacy ad safety of clarithromyci vs. erythromyci ethylsucciate. Pediatr.lfect.Dis.J. 1995;14: Boma J, Allard A, Persso K, Ludborg M, Juto P, Wadell G. Rapid diagosis of respiratory Chlamydia peumoiae ifectio by ested touchdow polymerase chai reactio compared with culture ad atige detectio by EIA. J.lfect.Dis. 1997;175: Boma J, Gaydos CA. Chlamydia peumoiae: Molecular Biology Methods. I Allegra L, Blasi F, eds. Chlamydia peumoiae, pp Milao, Italy: Spriger- Verlag, 24. Boma, J., H0glid, J., Alakarppa, H., Allard, A., Blasi, F., Campbell, L.A., et al. Chlamydia peumoiae PCR methods developed i-house: a survey of itra- ad iter-laboratory variatio of sesitivity ad specificity amog 12 laboratories I Schachter J, Christiase G, Clarke IN, et al, eds. Chlamydial Ifectios. Proceedigs of the Teth Iteratioal Symposium o Huma Chlamydial Ifectios, pp Brau J, Laitko S, Trehare J, Egges U, Wu P, Distler A et al. Chlamydia peumoiae--a ew causative aget of reactive arthritis ad udifferetiated oligoarthritis. A.Rheum.Dis. 1994;53:

83 26. Campbell LA, Perez Melgosa M, Hamilto DJ, Kuo CC, Graysto JT. Detectio of Chlamydia peumoiae by polymerase chai reactio. J.Cli.Microbiol. 1992;30: Cheresky M, Smieja M, Schachter J, Summersgill J, Schidler L, Solomo Net al. Compariso of a idustry-derived LCx Chlamydia peumoiae PCR research kit to i-house assays performed i five laboratories. J.Cli.Microbiol. 2002;40: Cherry JD. Epidemiological, cliical, ad laboratory aspects of pertussis i adults. Cli.Ifect.Dis. 1999;28 Suppl 2:S Christiase, A. H., Samuelsso, S., Beedse, M., ad Filskov, A. Orithosis EPI NEWS 16/17, States Serum Istitut, Copehage, Demark. 30. Christiase G, Birkelud S. Chlamydia structure - A molecular approach to uderstad the structure of Chlamydia. I Schachter J, Christiase G, Clarke IN, et al, eds. Chlamydial Ifectios. Proceedigs of the Teth Iteratioal Symposium o Huma Chlamydia! Ifectios, pp Christiase G, Madse AS, Kudse K, Mygid P, Birkelud S. Stability of the outer membrae protei of Chlamydia peumoiae. I Stephes RS, Byre GI, Christiase G, Clarke IN, et al, eds. Proceedigs of the Nith Iteratioal Symposium o Huma Chlamydial Ifectio. 1998, pp Christiase G, Ostergaard L, Birkelud S. Molecular biology of the Chlamydia peumoiae surface. Scad.J.lfect.Dis.Suppl 1997;104:5-10.: Cook PJ, Davies P, Tuicliffe W, Ayres JG, Hoeyboure D, Wise R. Chlamydia peumoiae ad asthma. Thorax 1998;53: Cosetii R, Tarsia P, Blasi F. Chlamydia peumoiae: Cliical Characteristics of Acute Respiratory Ifectios. I Allegra L, Blasi F, eds. Chlamdydia peumoiae, pp Milao: Spriger-Verlag. 35. Cuigham AF, Johsto SL, Julious SA, Lampe FC, Ward ME. Chroic Chlamydia peumoiae ifectio ad asthma exacerbatios i childre. Eur.Respir.J. 1998;11: Dalhoff K,.Maass M. Chlamydia peumoiae peumoia i hospitalized patiets. Cliical characteristics ad diagostic value of polymerase chai reactio detectio i BAL. Chest 1996;110: Daesh J, Collis R, Peto R. Chroic ifectios ad coroary heart disease: is there a lik? Lacet 1997;350: Daesh J, Whicup P, Walker M, Leo L, Thomso A, Appleby P et al. Low grade iflammatio ad coroary heart disease: prospective study ad updated meta-aalyses. BMJ.2000.Jul.22;321(7255):

84 39. Daesh J, Whicup P, Walker M, Leo L, Thomso A, Appleby P et al. Chlamydia peumoiae IgG titres ad coroary heart disease: prospective study ad meta-aalysis. BMJ.2000.Jul.22;321 (7255): Dowell SF, Peelig RW, Boma J, Carloe GM, Fields BS, Guamer J et al. Stadardizig Chlamydia peumoiae Assays: Recommedatios from the Ceters for Disease Cotrol ad Prevetio (USA) ad the Laboratory Cetre for Disease Cotrol (Caada). Cli.Itect.Dis.2001.Aug.15;33(4): Due MW. Ratioale ad desig of a secodary prevetio trial of atibiotic use i patiets after myocardial ifarctio: the WIZARD (weekly itervetio with zithromax [azithromyci] for atherosclerosis ad its related disorders) trial. J.Ifect.Dis. 2000;181 Suppl 3:S Ekma MR, Graysto JT, Visakorpi R, Kleemola M, Kuo CC, Saikku P. A epidemic of ifectios due to Chlamydia peumoiae i military coscripts. Cli.Ifect.Dis. 1993;17: Ekma MR, Leioe M, Syrjala H, Liamaki E, Kujala P, Saikku P. Evaluatio of serological methods i the diagosis of Chlamydia peumoiae peumoia durig a epidemic i Filad. Eur.J.Cli.Microbiol.Ifect.Dis. 1993;12: Everett KD. Chlamydia ad Chlamydiales: more tha meets the eye. Vet.Microbial Jul.31;75(2): Everett KD, Bush RM, Aderse AA. Emeded descriptio of the order Chlamydiales, proposal of Parachlamydiaceae fam. ov. ad Simkaiaceae fam. ov., each cotaiig oe mootypic geus, revised taxoomy of the family Chlamydiaceae, icludig a ew geus ad five ew species, ad stadards for the idetificatio of orgaisms. It.J.Syst.Bacteriol. 1999;49 Pt 2: Faber, M., Jese, J. S., ad Lid, I. Five cases of acute severe psittecosis cofirmed by demostratio of Chlamydia psittaci-dna by PCR. I proceedigs of 3RD Nordic-Baltic Cogress o Ifectious Diseases p Falsey AR,. Walsh E. Trasmissio of Chlamydia peumoiae. J.Ifect.Dis.1993;168: Farholt, S. Chlamydia peumoiae. Assessmet of methods for direct detectio ad prevalece studies i patiets with respiratory tract ifectio Uiversity of Copehage. (Ph.D. thesis). 49. Farholt S,.Hase DS. [Familial occurrece of Chlamydia peumoiae ifectio]. Ugeskr.Laeger 1996; 158: Foseca K, Kluchka C, Aad CM. Screeig for atibody to Chlamydia peumoiae by the complemet fixatio test. Diag.Microbiol.Itect.Dis. 1994;18: Freidak HM, Vogele H, Eckert K. Evaluatio of a ew commercial microimmuofluorescece test for detectio of atibodies to Chlamydia peumoiae, Chlamydia trachomatis, ad Chlamydia psittaci. Eur.J.Cli.Microbiol.Ifect.Dis. 1997;16:

85 52. Gaydos CA, Fowler CL, Gill VJ, Eide JJ, Qui TC. Detectio of Chlamydia peumoiae by polymerase chai reactio- ezyme immuoassay i a immuocompromised populatio. Cli.lfect.Dis. 1993;17: Gaydos CA, Robli PM, Hammerschlag MR, Hyma CL, Eide JJ, Schachter Jet al. Diagostic utility of PCR-ezyme immuoassay, culture, ad serology for detectio of Chlamydia peumoiae i symptomatic ad asymptomatic patiets. J.Cli.Microbiol ;32: Gieffers J, Pohl D, Treib J, Dittma R, Stepha C, Klotz K et al. Presece of Chlamydia peumoiae DNA i the cerebral spial fluid is a commo pheomeo i a variety of eurological diseases ad ot restricted to multiple sclerosis. A.Neural. 2001;49: Grassi GG. Pharmacological ad Pharmacokietic Basis of Chlamydia peumoiae Treatmet. I Allegra L, Blasi F, eds. Chlamydia peumoiae. The Lug ad the Heart, pp Milao: Spriger-Verlag, 56. Graysto JT. Ifectios caused by Chlamydia peumoiae strai TWAR. Cli.lfect.Dis. 1992;15: Graysto JT. History of Chlamydia peumoiae (TWAR). I Allegra L, Blasi F, eds. Chlamydia peumoiae, pp 1-8. Milao, Italy: Spriger-Verlag, 58. Graysto JT. Backgroud ad curret kowledge of Chlamydia peumoiae ad atherosclerosis. J.Ifect.Dis. 2000;181Suppl3:S Graysto JT, Aldous MB, Easto A, Wag SP, Kuo CC, Campbell LA et al. Evidece that Chlamydia peumoiae causes peumoia ad brochitis. J.lfect.Dis. 1993;168: Graysto JT, Campbell LA, Kuo CC, Mordhorst CH, Saikku P, Thom D et al. A ew respiratory tract pathoge: Chlamydia peumoiae strai TWAR. J.lfect.Dis. 1990;161: Graysto JT, Gale JL, Yeh LJ, Yag CY. Pathogeesis ad immuology of trachoma. Tras.Assoc.Am.Physicias. 1972;85: Graysto JT, Kuo CC, Wag SP, Altma J. A ew Chlamydia psittaci strai, TWAR, isolated i acute respiratory tract ifectios. N.Egl.J.Med.1986;315: Graysto JT, Mordhorst C, Emu AL, Vee S, Wag SP. Coutrywide epidemics of Chlamydia peumoiae, strai TWAR, i Scadiavia, J.lfect.Dis.1989;159: Graysto JT, Wag SP, Kuo CC, Campbell LA. Chlamydia peumoiae sp.ov. for Chlamydia sp. strai TWAR. It.J.Syst.Bacteriol. 1989;39: Graysto JT, Wag SP, Kuo CC, Campbell LA. Curret kowledge o Chlamydia peumoiae, strai TWAR, a importat cause of peumoia ad other acute respiratory diseases. Eur.J.Cli.Microbiol.lfect.Dis. 1989;8: Gurfikel E. Iflammatio, ifectio, or both i atherosclerosis: the ROXIS trial i perspective. J.Ifect.Dis.2000.Ju;181.Suppl.3:S

86 Iteret ASA-ISA DMZ (subet) : (broadcast) /30 (252) /30 (252) Subet (subet) (broadcast) ISA DMZ Subet (subet) (broadcast) Ip Camera ServerRoomMoitor LAN o (subet) : (servere) , (pritere o.a.) ' (OHCP) (Serverroom moitor) 192; (lpcamera serverroom) (NAS) 192, (UPS) (ikke allokeret) (AP) (6F switch) (Via maagemet) (broadcast) NAS /29 (248) /29 (248) Storage CTU-Priter Color-Priter CHBG-Priter Ecri-Priter CTU users (DHCP) '------I Exe. Repl. Network ( /16)