Sérologies Lyme - Association Ariane

ERYTHEMA MIGRANS
 EM initial : au lieu de la piqûre de tique
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Souvenir d ’une piqûre : 31%
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Incubation : 3 à 32 jours
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Diamètre : médiane = 15 cm ( 3 à 68 cm )
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Evolution centrifuge avec éclaircissement central
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50% des cas : EM secondaires
Mini-erythema migrans
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Weber Dermatology 2006
Smaller than 5 cm in diameter
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
Serology for early Lyme disease
( erythema migrans )
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Wormser N Engl J Med 2006 ; Wormser Clin Vaccine Immunol
2008 ; Lieber M’bomeyo Presse Med 2003 ; Assous Med Mal Infect
2007
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Positive serology in only 20 to 50% of the cases
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Seroconversion within 2 weeks
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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 !
Lyme serology
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Aguero-Rosenfeld Infect Dis Clin N Am 2008
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ELISA : whole sonicate of in vitro cultured strain B31 of B. burgdorferi
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Guidelines : two-tier testing
 First step : ELISA
 If positive, confirmation by Western blot (IgG and IgM)
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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)
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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)
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C6 peptide antibody assay : could replace the Western blot ?
Only one step ?
ELISA serology : different sensitivities
according to the test
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Marangoni J Med Microbiol 2005
3 different commercial ELISA tests
Discrepant results
Sensitivity for the same sera : 36,8% to 70,5% !
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De Marteno Med Mal Infect 2007
Comparison of 14 ELISA kits for the diagnosis of
neuroborreliosis
Sensitivity varies from 20,9 to 97,7% !
Diagnostic tests of Lyme disease
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Aguero-Rosenfeld Infect Dis Clin N Am
2008
Accuracy of the tests impossible to
measure
Because of the absence of a gold
standard +++
ELISA sensitivity is measured on healthy
controls !!! (in each region)
European concerted action on Lyme borreliosis (EUCALB)
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In a geographic area : the labs should test at least 100 controls
among blood donors
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The cut-off of the test is chosen to avoid more than 5% of blood
donors positive
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Consequence : for the same patient with the same symptoms :
serology is positive in Paris or Lille but negative in Strasbourg or
Limoges !
Negative serology in confirmed cases of
Lyme disease
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Wallet Jpn J Infect Dis 2008
Bilateral panuveitis
Serology : ELISA positive , Western blot negative
CSF : normal
PCR for Borrelia burgdorferi in CSF : positive
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Holl-Wieden Rheumatol Int 2007
Juvenile idiopathic arthritis for 5 years
Seronegative
Positive PCR for Borrelia burgdorferi in synovial fluid
Cured with antibiotics
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Dietrich Cornea 2008
Crystallin keratopathy, Seronegative
Positive PCR in explanted cornea
Immune complexes as a cause
of false-negative serology
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Reported 20 years ago in the N Engl J Med
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Brunner Clin Vaccine Immunol 2006
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Sequestration of antibodies in immune complexes
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Free antibodies at a level under the threshold of the
serology test
Different species of Borrelia
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Rudenko FEMS Microbiol Letter 2009 ; Bouattour Arch Inst Pasteur Tunis
2004 ; Lopes de Carvalho Clin Rheumatol 2008
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Borrelia burgdorferi sensu lato
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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
Serology often does not
Borrelia japonica
cross react :
Borrelia sinica
false negative results
Borrelia tanukii
Commercial tests :
Borrelia turdi
Borrelia carolinensis
isolate B31 (1982)
Scotland : improvement of sensitivity
of Western blot
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Mavin J Clin Microbiol 2007 ; Mavin J
Clin Pathol 2009
Using local Scottish strains :
 Borrelia burgdorferi sensu stricto
 and Borrelia afzelii
Sensitivity of serology and PCR
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Chmielewska Ann Agric Environ Med 2006
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180 patients with a clinical diagnosis of Lyme disease
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Presence of antibodies
 ELISA
IgM 61,7%
IgG 53,9%
 Western blot
IgM 62,2%
IgG 59,4%
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Positive PCR : 11,1%
Serology, PCR, culture
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Assous Med Mal Infect 2007 ; Aguero-Rosenfeld Infect Dis Clin N Am
2008 ; Remy Med Mal Infect 2007
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Sensitivity :
Serology

Erythema migrans
Early complicated forms
Neuroborreliosis
Arthritis
Acrodermatitis
chronica atrophicans
20 to 50%
70 to 90%
42 to 90%
60 to 100%
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33 to 86%
PCR
Culture
skin 50 to 64%
50 to 88%
blood 10 to 18%
CSF 5 to 38%
10 to 30%
syn.fluid 37 to 97%
60 to 90%
20 to 60%
Lyme neuro-borreliosis
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Blanc Neurology 2007
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Intrathecal antibody index (IAI) necessary to diagnose neuroborreliosis
in Europe, not in the USA !
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Sensitivity of IAI : 55 to 80%
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Recognize the lack of gold standard : propose pragmatic criteria
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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
Lyme neuro-borreliosis in children
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Tveitnes Scand J Infect Dis 2009
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3 groups
A : cranial neuropathy
B : cranial neuropathy and other neurological symptoms
C : neurological symptoms ( without cranial neuropathy )
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Isolated cranial neuropathy : lower WBC and protein levels in CSF,
lower frequency of positive antibodies in serum and in CSF
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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
Lyme neuro-borreliosis (LNB) in children
Bennet Infection 2008 (Sweden)
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Diagnosis of LNB
 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
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Intrathecal Borrelia antibody production : 71% of children (this
intrathecal antibody production is less frequent in the USA than in Europe)
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Positive antibody in serum : 23% of children !
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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 !
False positive and false negative
tests
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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 »
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A patient with chronic symptoms and a positive Lyme serology
may be sent to a psychiatrist
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A patient with chronic symptoms and a negative Lyme
serology is always sent to a psychiatrist
Evolution of antibodies after antibiotic
treament
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IgM : decrease and become usually negative (< 3
years)
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IgG :
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C6 ELISA test : 91% of patients : decrease of at least 4
dilutions or the test becomes negative
negative in 43% of patients at 5 years
may remain positive at 10 years
( during a follow-up period of 6 to 12 months )
MALADIE DE LYME CHRONIQUE et
syndromes chroniques apparentés
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Pourquoi les Borrelia échappent
- au système immunitaire ?
1) variation antigénique
2) les tubules des cellules eucaryotes dérivent des spirochètes
(Sagan et Margulis)
-
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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)
MALADIE DE LYME CHRONIQUE OU
SYNDROMES APPARENTES
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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)
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Complexes Ag-Ac
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Nouvelles espèce de Borrelia décrites (Sahel, Pérou,
Europe)
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Beaucoup de sous-espèces de B. burgdorferi
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Seuils artificiels des tests
 Co-infections
– Bartonella
- Erhlichia
– Rickettsia
- Coxiella
– Brucella
– Mycoplasmes
- Chlamydia
– HHV-6
- Babesia +++
Documented co-infections
after tick-bite
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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
Ehrlichiose et anaplasmose
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Ehrlichiose monocytique américaine (USA)
Ehrlichia chaffeensis
Infecte les monocytes
Tique Amblyomma americanum
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Anaplasmose (Europe)
Anaplasma phagocytophilum
Infecte les polynucléaires
Tique Ixodes (idem Lyme)
Syndrome pseudo-grippal + lymphopénie
Sérologie.
Traitement : cyclines
Brazil : Lyme-like syndrome
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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
PERSISTENCE OF BORRELIA BURGDORFERI AFTER
ANTIBIOTIC TREATMENT
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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.
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Hunfled et al, AAC 2005 : Posiive cutaneous biopsy after antibiotic
treatment