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Laboratory Diagnosis of Lyme Borreliosis Anti-Borrelia

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Laboratory Diagnosis of Lyme Borreliosis Anti-Borrelia Linköping University Medical Dissertations No. 1222 Laboratory Diagnosis of Lyme Borreliosis Anti-Borrelia Antibodies and the Chemokine CXCL13 Ivar Tjernberg Department of Clinical Chemistry, Kalmar County Hospital, Kalmar Divisions of Infectious Diseases and Clinical Immunology, Department of Clinical and Experimental Medicine Faculty of Health Sciences Linköping University, Sweden Linköping 2011 Ivar Tjernberg, 2011 Cover illustration: Gunnar Tjernberg, displaying a Lyme borreliosis spirochete with flagella and an IgG antibody. Published articles have been reprinted with the permission of the copyright holder. Printed in Sweden by LiU-Tryck, Linköping, Sweden, 2011 ISBN 978-91-7393-256-1 ISSN 0345-0082 Acti labores jucundi Contents CONTENTS ABSTRACT ................................................................................................................... 1 SAMMANFATTNING PÅ SVENSKA......................................................................... 3 LIST OF PAPERS .......................................................................................................... 5 ABBREVIATIONS ........................................................................................................ 7 INTRODUCTION .......................................................................................................... 9 Lyme borreliosis ................................................................................................ 9 Historical notes.................................................................................................. 9 Hard ticks are vectors of Lyme borreliosis ..................................................... 10 The spirochete complex Borrelia burgdorferi sensu lato................................ 11 Epidemiology of Lyme borreliosis in Europe.................................................. 13 Pathogenesis of Lyme borreliosis.................................................................... 15 Clinical characteristics of Lyme borreliosis ................................................... 17 Swedish treatment recommendations for Lyme borreliosis............................. 23 Prognosis of Lyme borreliosis......................................................................... 24 Comparative thoughts on Lyme borreliosis and syphilis ................................ 26 Laboratory tests and diagnosis of Lyme borreliosis ....................................... 27 INITIATION OF THE STUDY ................................................................................... 35 AIMS OF THE STUDY ............................................................................................... 37 PATIENTS AND METHODS ..................................................................................... 39 Definitions used in this thesis .......................................................................... 39 Patients and controls (papers I–IV) ................................................................ 41 Laboratory methods......................................................................................... 44 Statistics........................................................................................................... 50 Ethics ............................................................................................................... 51 RESULTS AND DISCUSSION................................................................................... 53 Erythema migrans (papers I and III)............................................................... 53 Lyme neuroborreliosis (papers II and IV)....................................................... 59 Acrodermatitis chronica atrophicans and Lyme arthritis (paper I)................ 69 Contents Blood donors (papers I and III)....................................................................... 70 Conditions with potentially cross-reacting antibodies (paper I)..................... 72 Two-tiered testing and Western blot results (previously unpublished) ........... 73 General discussion .......................................................................................... 76 CONCLUDING REMARKS........................................................................................ 80 PROPOSED DIAGNOSTIC ROUTINES.................................................................... 81 ACKNOWLEDGEMENTS.......................................................................................... 83 REFERENCES ............................................................................................................. 85 APPENDIXES............................................................................................................ 103 Papers I–IV.................................................................................................... 103 Abstract ABSTRACT Lyme borreliosis (LB), the most common tick-borne disease in Europe and North America, is caused by spirochetes of the Borrelia burgdorferi sensu lato complex. The spirochetes can invade several different organs, thereby causing many different symptoms and signs. Diagnosis of LB relies on patient history, physical examination, and detection of anti-Borrelia antibodies. However, anti- Borrelia antibodies are not always detectable, and they commonly persist even after LB is successfully treated or spontaneously healed. The aim of my work was to study diagnostic aspects on clinical cases of LB and control subjects in an area endemic to LB, with a focus on newly developed anti- Borrelia antibody tests. A total of 617 patients with symptoms and/or signs consistent with LB, as well as 255 control subjects, were studied. The diagnostic panel included the following new LB tests: Immunetics Quick ELISA C6 Borrelia assay kit (C6), invariable region 6 peptide antibody assays (IR6), Liaison Borrelia CLIA (Li) and the chemokine CXCL13. Results were compared with the older Virotech Borrelia burgdorferi ELISA (VT) and with a Western blot method, the Virotech Borrelia Ecoline IgG/IgM Line Immunoblot (WB EL), when appropriate. In general, no significant differences were noted between the C6, VT and Li tests regarding serosensitivity in various LB manifestations. However, the seropositivity rate was lower for the C6 test compared with the VT and Li tests 2–3 and 6 months after diagnosis of erythema migrans (EM), indicating normalization of antibody levels. In addition, EM patients reporting a previous LB episode had a C6 seropositivity rate similar to that of patients without a previous LB episode, and seroprevalence in healthy blood donors was lower in the C6 test than the VT and Li tests. Taken together, these results support the recommendation of the serum C6 test as a Borrelia serological test due to its ability to reflect ongoing or recent infection. Although the majority of EM patients at presentation showed concordant serological responses to IR6 peptides representing the three main Borrelia species and the C6 peptide, there were also clinical EM cases that were C6- negative and could be detected mainly by a seroresponse to a B. burgdorferi sensu stricto-derived IR6 peptide. Thus, an antibody test combining antigens could be of value in the serodiagnosis of LB in Europe. The serosensitivity of the C6 test in cases of Lyme neuroborreliosis (LNB) was shown to be associated with symptom duration. A serosensitivity rate of 93% was found in LNB patients ≥ 12 years of age with a symptom duration of more 1 Abstract than 30 days. Therefore, a negative C6 test in serum in such a patient argues against an LNB diagnosis. The presence of chemokine CXCL13 in cerebrospinal fluid was confirmed to be a reliable marker of LNB. CXCL13 differentiated LNB from other conditions and also indicated a high probability of LNB in children with short symptom duration where anti-Borrelia antibodies were still lacking in the cerebrospinal fluid. A two-tiered approach (C6 test in combination with WB EL) showed no significant improvement in specificity over the C6 test alone. However, WB EL may be useful in diagnosing suspected cases of acrodermatitis chronicum atrophicans and Lyme arthritis, usually displaying multiple IgG bands. In conclusion, although the serodiagnosis of LB remains to be settled, this thesis provides some practical tools regarding the use and interpretation of Borrelia serology including proposed diagnostic routines. 2 Sammanfattning på svenska SAMMANFATTNING PÅ SVENSKA Borrelios är den vanligaste fästingburna infektionen i Europa och Nordamerika. Infektionen orsakas av spiralformade bakterier med samlingsnamnet Borrelia burgdorferi sensu lato. Dessa bakterier kan invadera många olika organ och därmed ge upphov till många olika symptom. Vid lokal hudinfektion, så kallat erythema migrans, baseras diagnostiken på sjukhistoria och typiska fynd vid kroppsundersökning. Vid övriga tillstånd utgör påvisning av borreliaantikroppar ett komplement för att ställa korrekt diagnos. Borreliaantikroppar kan dock inte alltid detekteras, särskilt tidigt i förloppet. Borreliaantikroppar kan dessutom ofta påvisas under lång tid även efter framgångsrikt behandlad eller spontanläkt infektion och det kan också förekomma falskt positiva reaktioner vid andra sjukdomstillstånd. Syftet med avhandlingsarbetet var att undersöka nyttan av nya borreliatester hos patienter med olika former av borrelios och olika kontrollgrupper i ett område med hög förekomst av borrelios. Totalt studerades 617 patienter med symtom förenliga med borrelios samt 255 kontrollpersoner. Följande borreliatester användes: De antikroppsdetekterande
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