ERYTHEMA MIGRANS. EM initial : au lieu de la piqûre de tique. Diamètre : médiane = 15 cm ( 3 à 68 cm )

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1 ERYTHEMA MIGRANS EM initial : au lieu de la piqûre de tique Souvenir d une piqûre : 31% Incubation : 3 à 32 jours Diamètre : médiane = 15 cm ( 3 à 68 cm ) Evolution centrifuge avec éclaircissement central 50% des cas : EM secondaires

2 Mini-erythema migrans Weber Dermatology 2006 Smaller than 5 cm in diameter

3 Dans la vraie vie La maladie de Lyme n est pas toujours caractéristique : la «grande simulatrice» C est une maladie fréquente universelle. Problème de la sérologie qui a une sensibilité faible Conséquence : largement sous-diagnostiquée

4 Serology for early Lyme disease ( erythema migrans ) Wormser N Engl J Med 2006 ; Wormser Clin Vaccine Immunol 2008 ; Lieber M bomeyo Presse Med 2003 ; Assous Med Mal Infect 2007 Positive serology in only 20 to 50% of the cases Seroconversion within 2 weeks Serology may remain negative if early antibiotic treatment Diagnosis is clinical at this stage +++ Enquiry among GPs in Alsace in 2003 : 50% thought a positive serology is required!

5 Lyme serology Aguero-Rosenfeld Infect Dis Clin N Am 2008 ELISA : whole sonicate of in vitro cultured strain B31 of B. burgdorferi Guidelines : two-tier testing First step : ELISA If positive, confirmation by Western blot (IgG and IgM) IgM Western blot test : not to be done after 4 weeks of evolution. Positive IgM Western blot : at least 2 of 3 significant bands (41, 39 kda, OspC) Positive IgG Western blot : American recommendation : at least 5 of 10 significant bands (these criteria are not relevant in Europe, but lack of standardization of Western blot in Europe : seven different rules, variability according to the Borrelia species) C6 peptide antibody assay : could replace the Western blot? Only one step?

6 ELISA serology : different sensitivities according to the test Marangoni J Med Microbiol different commercial ELISA tests Discrepant results Sensitivity for the same sera : 36,8% to 70,5%! De Marteno Med Mal Infect 2007 Comparison of 14 ELISA kits for the diagnosis of neuroborreliosis Sensitivity varies from 20,9 to 97,7%!

7 Diagnostic tests of Lyme disease Aguero-Rosenfeld Infect Dis Clin N Am 2008 Accuracy of the tests impossible to measure Because of the absence of a gold standard +++

8 ELISA sensitivity is measured on healthy controls!!! (in each region) European concerted action on Lyme borreliosis (EUCALB) In a geographic area : the labs should test at least 100 controls among blood donors The cut-off of the test is chosen to avoid more than 5% of blood donors positive Consequence : for the same patient with the same symptoms : serology is positive in Paris or Lille but negative in Strasbourg or Limoges!

9 Negative serology in confirmed cases of Lyme disease Wallet Jpn J Infect Dis 2008 Bilateral panuveitis Serology : ELISA positive, Western blot negative CSF : normal PCR for Borrelia burgdorferi in CSF : positive Holl-Wieden Rheumatol Int 2007 Juvenile idiopathic arthritis for 5 years Seronegative Positive PCR for Borrelia burgdorferi in synovial fluid Cured with antibiotics Dietrich Cornea 2008 Crystallin keratopathy, Seronegative Positive PCR in explanted cornea

10 Immune complexes as a cause of false-negative serology Reported 20 years ago in the N Engl J Med Brunner Clin Vaccine Immunol 2006 Sequestration of antibodies in immune complexes Free antibodies at a level under the threshold of the serology test

11 Different species of Borrelia Rudenko FEMS Microbiol Letter 2009 ; Bouattour Arch Inst Pasteur Tunis 2004 ; Lopes de Carvalho Clin Rheumatol 2008 Borrelia burgdorferi sensu lato Borrelia burgdorferi sensu stricto (USA, Europe, North Africa) Borrelia afzelii (Europe, Asia) Borrelia garinii (several serotypes) (Europe, Asia, North Africa) Borrelia valaisiana Borrelia lusitaniae (Portugal, Italy, North Africa) : vasculitis Borrelia spielmanii (Netherlands, Germany, Hungary, Slovenia) Borrelia bisettii (Slovenia, Czechia, North America) Borrelia andersonii Borrelia californiensis Borrelia japonica Borrelia sinica Borrelia tanukii Borrelia turdi Borrelia carolinensis Serology often does not cross react : false negative results Commercial tests : isolate B31 (1982)

12 Scotland : improvement of sensitivity of Western blot Mavin J Clin Microbiol 2007 ; Mavin J Clin Pathol 2009 Using local Scottish strains : Borrelia burgdorferi sensu stricto and Borrelia afzelii

13 Sensitivity of serology and PCR Chmielewska Ann Agric Environ Med patients with a clinical diagnosis of Lyme disease Presence of antibodies ELISA IgM 61,7% IgG 53,9% Western blot IgM 62,2% IgG 59,4% Positive PCR : 11,1%

14 Serology, PCR, culture Assous Med Mal Infect 2007 ; Aguero-Rosenfeld Infect Dis Clin N Am 2008 ; Remy Med Mal Infect 2007 Sensitivity : Serology PCR Culture Erythema migrans 20 to 50% skin 50 to 64% 50 to 88% Early complicated forms 70 to 90% blood 10 to 18% Neuroborreliosis 42 to 90% CSF 5 to 38% 10 to 30% Arthritis 60 to 100% syn.fluid 37 to 97% Acrodermatitis chronica atrophicans 33 to 86% 60 to 90% 20 to 60%

15 Lyme neuro-borreliosis Blanc Neurology 2007 Intrathecal antibody index (IAI) necessary to diagnose neuroborreliosis in Europe, not in the USA! Sensitivity of IAI : 55 to 80% Recognize the lack of gold standard : propose pragmatic criteria Diagnosis requires 4 of 5 items : No past history of neuroborreliosis Positive CSF antibodies by ELISA Positive intrathecal antibody index Favorable outcome after specific antibiotic treatment No differential diagnosis

16 Lyme neuro-borreliosis in children Tveitnes Scand J Infect Dis groups A : cranial neuropathy B : cranial neuropathy and other neurological symptoms C : neurological symptoms ( without cranial neuropathy ) Isolated cranial neuropathy : lower WBC and protein levels in CSF, lower frequency of positive antibodies in serum and in CSF Proportion of children with antibodies in serum and CSF and of positive antibody index : related to duration of symptoms Antibody index positive in 51% of children if symptoms < 7 days in 80% of children if symptoms > 7 days

17 Lyme neuro-borreliosis (LNB) in children Diagnosis of LNB Bennet Infection 2008 (Sweden) probable > 75% mononuclear cells in CSF and antibodies in serum or CSF possible > 75% mononuclear cells in CSF, no antibodies but a compatible clinical presentation and response to antibiotic therapy Intrathecal Borrelia antibody production : 71% of children (this intrathecal antibody production is less frequent in the USA than in Europe) Positive antibody in serum : 23% of children! Anti-Borrelia IgM in serum found in children with a different (?) diagnosis : «Viral meningitis» (15%) Various neurological symptoms with normal CSF (25%) Considered as «false-positive» by the authors. No criteria to exclude Lyme disease, except the authors conviction!

18 False positive and false negative tests A patient with acute or subacute symptoms and a negative Lyme serology may be considered as a patient with a «viral» or «idiopathic» syndrome, or with an «atypical auto-immune disease» A patient with chronic symptoms and a positive Lyme serology may be sent to a psychiatrist A patient with chronic symptoms and a negative Lyme serology is always sent to a psychiatrist

19 Evolution of antibodies after antibiotic treament IgM : decrease and become usually negative (< 3 years) IgG : negative in 43% of patients at 5 years may remain positive at 10 years C6 ELISA test : 91% of patients : decrease of at least 4 dilutions or the test becomes negative ( during a follow-up period of 6 to 12 months )

20 MALADIE DE LYME CHRONIQUE et syndromes chroniques apparentés Pourquoi les Borrelia échappent - au système immunitaire? 1) variation antigénique 2) les tubules des cellules eucaryotes dérivent des spirochètes (Sagan et Margulis) - aux antibiothérapies prolongées? : - phénomènes de cycles avec formes mobiles et formes kystique ( Brorson APMIS 1998, Brorson Int Microbiol 2002 ) Rôle vraisemblable des co-infections du terrain génétique, modulant la réponse immunitaire (familles à risque)

21 MALADIE DE LYME CHRONIQUE OU SYNDROMES APPARENTES Maladie de Lyme séronégative? Publié depuis plus de 15 ans (y compris dans le N Engl J Med, y compris récemment dans un grand essai thérapeutique) Co-infections Complexes Ag-Ac Nouvelles espèce de Borrelia décrites (Sahel, Pérou, Europe) Beaucoup de sous-espèces de B. burgdorferi Seuils artificiels des tests Bartonella - Erhlichia Rickettsia - Coxiella Brucella Mycoplasmes - Chlamydia HHV-6 - Babesia +++

22 Documented co-infections after tick-bite Wormser N Engl J Med 2006 Early Lyme disease : 2 to 12% of patients have also human granulocytic anaplasmosis 2 to 40% of patients have also babesiosis

23 Ehrlichiose et anaplasmose Ehrlichiose monocytique américaine (USA) Ehrlichia chaffeensis Infecte les monocytes Tique Amblyomma americanum Anaplasmose (Europe) Anaplasma phagocytophilum Infecte les polynucléaires Tique Ixodes (idem Lyme) Syndrome pseudo-grippal + lymphopénie Sérologie. Traitement : cyclines

24 Brazil : Lyme-like syndrome Mantovani Braz J Med Biol Res 2007 Tick-borne disease (Tick : Amblyomma) Dark field microscope : mobile spirochetes (uncultivable) Not belonging to the genera : Borrelia, Leptospira or Treponema

25 PERSISTENCE OF BORRELIA BURGDORFERI AFTER ANTIBIOTIC TREATMENT ANIMAL MODELS Mouse : - Persistence of spirochetes DNA within macrophages after antibiotic treatment Dog : - After high dose of amoxicillin or doxycycline during 30 days : Borrelia burgdorferi DNA was detectable 500 days later Monkey: Neurologic and cardiac diseases were associated with persistent infection HUMAN STUDIES - Phillips et al, Infection 1998 : B. burgdorferi bacteriemia (microscopy by electronic immunofluorescence of antibobies) : positive in 91% of 47 patients in failure after oral and/or IV antibiotic treatment. Hunfled et al, AAC 2005 : Posiive cutaneous biopsy after antibiotic treatment

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