Indirekter immunologischer Nachweis einer Borrelien Infektion Serologische Labordiagnostik Spezifische Antikörpertiter im Serum Für die Diagnose einer Lyme Borreliose fordern die Deutsche Gesellschaft für Hygiene und Mikrobiologie DGHM ID: 932612
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Slide1
Lyme
-Borreliose: Diagnostik
Indirekter (immunologischer) Nachweis einer
Borrelien
-Infektion
Serologische Labordiagnostik (Spezifische Antikörpertiter im Serum)
Für die Diagnose einer
Lyme
-Borreliose fordern
die Deutsche Gesellschaft für Hygiene und Mikrobiologie (DGHM)
das Robert-Koch-Institut (RKI) und
die Centers
for
Disease
Control
and
Prevention
(CDC)
den serologischen Nachweis
Borrelien
-spezifischer Antikörper im Zuge einer
Zweistufendiagnostik.
Slide2Lyme
-Borreliose: Diagnostik
Indirekter (immunologischer) Nachweis einer
Borrelien
-Infektion
Serologische Labordiagnostik (Spezifische Antikörpertiter im Serum)
Slide32. Stufe: Bestätigungstest (IgM,
IgG) Für die Diagnose einer Lyme-Borreliose fordern
die Deutsche Gesellschaft für Hygiene und Mikrobiologie (DGHM) das Robert-Koch-Institut (RKI) und die Centers for Disease
Control and Prevention
(CDC) den serologischen Nachweis Borrelien-spezifischer Antikörper im Zuge einer Zweistufendiagnostik.
Lyme
-Borreliose: Diagnostik
Indirekter (immunologischer) Nachweis einer
Borrelien
-Infektion
Serologische Labordiagnostik (Spezifische Antikörpertiter im Serum)
1. Stufe: Antikörper-Suchtest (
IgM
,
IgG
)
positiv oder grenzwertig
Slide4Bestimmung von
IgM und IgG gegen Proteine aus Extrakten von
B. burgdorferi sensu strictu, B.
garinii und B. afzelii
sowie von IgG gegen rekombinantes Oberflächenprotein VlsE
breites Antigenspektrum
hohe Sensitivität
Lyme
-Borreliose: Diagnostik
Indirekter (immunologischer) Nachweis einer
Borrelien
-Infektion
Antikörper-Suchtest (
IgM
,
IgG
)
Testverfahren: ELISA (Enzym-gekoppelter Immun-Adsorptionstest)
unspezifische
Reaktionen unter
bestimmten
Bedingungen möglich
Slide5Lyme
-Borreliose: Diagnostik
Indirekter (immunologischer) Nachweis einer
Borrelien
-Infektion
Bestätigungstest (
IgM
,
IgG
)
Testverfahren: Western
Blot
Farbbanden zeigen die Anwesenheit von
Antikörpern an
Bestimmung von
IgM
und
IgG gegen definierte Oberflächenproteine aus
Borrelien (u.a. OspC und VlsE)
Kenedy
et al.
2012.
The role of
Borrelia
burgdorferi
outer surface proteins.
FEMS
Immunol
Med
Microbiol
66:1
OspA
+
OspC
+
VlsE
+
Hovius
et al.
2007.
Tick–
host
–pathogen interactions in
Lyme
borreliosis
.
Trends
Parasitol
23:434
Zeckendarm
Speicheldrüsen der Zecke
Slide6Lyme
-Borreliose: Diagnostik
Indirekter (immunologischer) Nachweis einer
Borrelien
-Infektion
Bestätigungstest (
IgM
,
IgG
)
Testverfahren: Western
Blot
Farbbanden zeigen die Anwesenheit von
Antikörpern an
Bestimmung von
IgM
und
IgG gegen definierte Oberflächenproteine aus
Borrelien (u.a. OspC und VlsE)
Schwan
und
Piesman
.
2000.
Temporal changes in outer surface proteins A and C of the Lyme disease-associated spirochete,
Borrelia
burgdorferi
, during the chain of infection in ticks and mice.
J
Clin
Microbiol
38:382
VlsE
=
(variable major protein-like sequence, expressed)
IgM
IgG
Müller
et al.
2012.
Evaluating frequency, diagnostic quality, and cost of Lyme
borreliosis
testing in Germany:
a retrospective model analysis
.
Clin
Dev
Immunol
ID595427.
Slide7Lyme
-Borreliose: Diagnostik
Indirekter (immunologischer) Nachweis einer
Borrelien
-Infektion
Bestätigungstest (
IgM
,
IgG
)
Testverfahren: Western
Blot
Farbbanden zeigen die Anwesenheit von
Antikörpern an
Bestimmung von
IgM
und
IgG gegen definierte Oberflächenproteine aus
Borrelien (u.a. OspC und VlsE)
hohe Spezifität und Sensitivität
Weiss et al. 1995.
False
positive
seroreactivity
to
Borrelia
burgdorferi
in
systemic
lupus erythematosus: the
value
of
immunoblot
analysis
.
Lupus 4:131.
In bis zu 40% der Patienten mit systemischem Lupus
erythematosus
(SLE) und anderen rheumatischen Erkrankungen wurde eine positive
Seroreaktivität
für
Borrelien
-spezifische Antikörper im ELISA nachge-wiesen. Die Spezifität der Serum-Antikörper konnte allerdings mittels Immunoblot-Technik nicht verifiziert werden.
Slide8Lyme
-Borreliose: Diagnostik
Indirekter (immunologischer) Nachweis einer
Borrelien
-Infektion
Bestätigungstest (
IgM
,
IgG
)
Testverfahren: Western
Blot
Zuverlässige Differenzierung zwischen aktiver und abgelaufener Infektion oft schwierig oder gar nicht möglich
Slide9 Farbbanden zeigen die Anwesenheit von
Antikörpern an Bestimmung von IgM und IgG gegen definierte
Oberflächenproteine aus Borrelien (u.a. OspC und VlsE
)
Lyme
-Borreliose: Diagnostik
Indirekter (immunologischer) Nachweis einer
Borrelien
-Infektion
Bestätigungstest (
IgM
,
IgG
)
Testverfahren: Western
Blot
hohe Spezifität und Sensitivität
ca. 10% (bis 40%)
seronegative
Patienten mit Borreliose-Symptomatik
Slide10Seronegative
Patienten mit Borreliose-Symptomatik
Lyme
-Borreliose: Diagnostik
Bestimmung der
Borrelien
-spezifischen Antikörper mittels ELISA in 23 Patienten mit diagnostiziertem EM und positivem
Borrelien
-Nachweis (in vitro-Kultur) in Hautbiopsien
9 Patienten (41%) waren
seronegativ
in 8 Patienten (35%) ließen sich nur
IgM
-Antikörper nachweisen
in 2 Patienten (8%) ließen sich nur
IgG
-Antikörper nachweisen
in 4 Patienten (16%) ließen sich sowohl
IgM
- als auch IgG-Antikörper
bestimmen
Slide11Seronegative
Patienten mit Borreliose-Symptomatik
Lyme
-Borreliose: Diagnostik
Bestimmung der
Borrelien
-spezifischen Antikörper mittels ELISA in 22 Patienten mit diagnostizierter chronischer kutaner Borreliose
in allen 22 Patienten (100%) ließen sich
IgG
-Antikörper nachweisen
in nur 1 Patienten (5%) ließen sich zusätzlich auch
IgM
-Antikörper
bestimmen
Slide12van
Dop et al. 2013. Seronegative Lyme neuroborreliosis in a patient using
rituximab. BMJ Case Rep 2012:007627
“Abstract We report on a patient who developed seronegative Lyme neuroborreliosis
complicating chemotherapy for chronic lymphatic leukemia. After the fifth cycle of chemotherapy (FCR: fludarabine, cyclophosphamide, rituximab
and prednisone) the 63-year-old patient developed night sweat,
arthralgia
in elbows, wrists, proximal
interphalangeal
joints (PIPs) and strong neuropathic pain in both legs, followed by
paresthesia
and
hypesthesia
in the feet, arms and face. ... Cerebrospinal fluid (CSF) analysis showed a
lymphomononuclear
pleocytosis
and an elevation of protein. A broad diagnostic work-up was negative including a negative
Borrelia IgG and IgM
ELISA. The patient did not remember recent tick bites, but after specific questioning he recollected a transient erythema on his leg developing just before the start of the last cycle of chemotherapy. As the combination of neuropathic pain and arthralgia, the transient erythema and the lymphomononuclear
pleocytosis raised the suspicion of Lyme neuroborreliosis, the patient was treated for 3 weeks with
ceftriaxone. On therapy all symptoms resolved and CRP normalized. Retrospective PCR analysis of a CSF sample confirmed the clinical diagnosis by detecting Borrelia garinii
DNA. This case demonstrates that in immunosuppressed patients borrelial serology may be negative and that additional diagnostic approaches (…) may be needed to demonstrate
borrelial infection.”
Mögliche Gründe für
Seronegativität
in Patienten mit Borreliose-Symptomatik
Lyme
-Borreliose: Diagnostik
mangelnde Sensitivität des Detektionssystems
Serumnahme
kurz nach der Infektion (< 4 Wochen nach Zeckenstich)
angeborene oder erworbene Immunsuppression beim Patienten
van
Dop
et al. 2013.
Seronegative
Lyme
neuroborreliosis
in a patient using
rituximab
.
BMJ Case Rep.
2012:007627
Harrer
et al. 2007.
Seronegative
Lyme
neuroborreliosis
in a patient on treatment for chronic
lymphytic
leukemia.
Infection 35:110.
“Abstract We report on a patient who developed seronegative Lyme neuroborreliosis complicating chemotherapy for chronic lymphatic leukemia. After the fifth cycle of chemotherapy (FCR: fludarabine, cyclophosphamide, rituximab
and prednisone) the 63-year-old patient developed night sweat, arthralgia
in elbows, wrists, proximal interphalangeal joints (PIPs) and strong neuropathic pain in both legs, followed by paresthesia and hypesthesia in the feet, arms and face. ... Cerebrospinal fluid (CSF) analysis showed a lymphomononuclear
pleocytosis and an elevation of protein. A broad diagnostic work-up was negative including a negative Borrelia IgG and IgM ELISA. The patient did not remember recent tick bites, but after specific questioning he recollected a transient
erythema
on his leg developing just before the start of the last cycle of chemotherapy. As the combination of neuropathic pain and
arthralgia
, the transient
erythema
and the
lymphomononuclear
pleocytosis
raised the suspicion of Lyme
neuroborreliosis
, the patient was treated for 3 weeks with
ceftriaxone
. On therapy all symptoms resolved and CRP normalized. Retrospective PCR analysis of a CSF sample confirmed the clinical diagnosis by detecting
Borrelia
garinii
DNA.
This case demonstrates that in
immunosuppressed
patients
borrelial
serology may be negative and that additional diagnostic approaches (…) may be needed to demonstrate
borrelial
infection
.
”
Slide13Mögliche Gründe für
Seronegativität in Patienten mit Borreliose-Symptomatik
Lyme
-Borreliose: Diagnostik
mangelnde Sensitivität des Detektionssystems
Serumnahme
kurz nach der Infektion (< 4 Wochen nach Zeckenstich)
angeborene oder erworbene Immunsuppression beim Patienten
Immunkomplexbildung
Verhinderung der Detektion von freien Antikörpern
Schutzer
et al. 1990.
Sequestration of antibody to
Borrelia
burgdorferi
in immune complexes in
seronegative Lyme disease. Lancet 335:312. Schutzer
et al. 1999. Borrelia burgdorferi
-specific immune complexes in acute Lyme disease. JAMA 282:1942. Brunner. 2001. New method for detection of Borrelia
burgdorferi antigen complexed
to antibody in
seronegative
Lyme disease.
J
Immunol
Meth 249:185.
Marques et al. 2005.
Detection of immune complexes is not independent of detection of antibodies
in Lyme disease patients and does not confirm active infection with
Borrelia burgdorferi. Clin
Diagn
Lab
Immunol
12:1036.
Slide14Mögliche Gründe für
Seronegativität in Patienten mit Borreliose-Symptomatik
Lyme
-Borreliose: Diagnostik
mangelnde Sensitivität des Detektionssystems
Serumnahme
kurz nach der Infektion (< 4 Wochen nach Zeckenstich)
angeborene oder erworbene Immunsuppression beim Patienten
Immunkomplexbildung
Verhinderung der Detektion von freien Antikörpern
Assoziation der Produktion
Borrelien
-spezifischer Antikörper mit bestimmten
HLA-Allelen
HLA-DR6/-DR7 ist assoziiert mit Antikörperproduktion
Seropositive
Patienten: 16/22 (72,7%)
Seronegative Patienten: 2/18 (11,1%) Kontrolle (gesunde Probanden): 13/26 (50,0%)
HLA-DR1 ist assoziiert mit dem Fehlen einer Antikörperproduktion Seropositive Patienten: 1/22 (4,5%) Seronegative
Patienten: 7/18 (38,9%) Kontrolle (gesunde Probanden): 3/26 (11,5%)
Wang und Hilton. 2001.
Contribution of HLA alleles in the regulation of antibody production in Lyme
disease.
Front
Biosci
6:B10
Slide15Mögliche Gründe für
Seronegativität in Patienten mit Borreliose-Symptomatik
Lyme
-Borreliose: Diagnostik
mangelnde Sensitivität des Detektionssystems
Serumnahme
kurz nach der Infektion (< 4 Wochen nach Zeckenstich)
angeborene oder erworbene Immunsuppression beim Patienten
Immunkomplexbildung
Verhinderung der Detektion von freien Antikörpern
Assoziation der Produktion
Borrelien
-spezifischer Antikörper mit bestimmten
HLA-Allelen
Frühe antibiotische Behandlung vermindert oder verhindert Antikörperbildung
Dattwyler
et al. 1988.
Seronegative
Lyme disease. Dissociation of specific T- and B-lymphocyte responses to Borrelia burgdorferi
. N Engl J Med 319:1441
Abstract
We studied 17 patients who had presented with acute Lyme disease and received prompt treatment with oral antibiotics, but in whom chronic Lyme disease subsequently developed. Although these patients had clinically active disease, none had diagnostic levels of antibodies to B.
burgdorferi
on either a standard enzyme-linked
immunosorbent
assay or
immunofluorescence
assay. On Western blot analysis, the level of immunoglobulin reactivity against B.
burgdorferi
in serum from these patients was no greater than that in serum from normal controls.
Slide16Mögliche Gründe für
Seronegativität in Patienten mit Borreliose-Symptomatik
Lyme
-Borreliose: Diagnostik
mangelnde Sensitivität des Detektionssystems
Serumnahme
kurz nach der Infektion (< 4 Wochen nach Zeckenstich)
angeborene oder erworbene Immunsuppression beim Patienten
Immunkomplexbildung
Verhinderung der Detektion von freien Antikörpern
Assoziation der Produktion
Borrelien
-spezifischer Antikörper mit bestimmten
HLA-Allelen
Frühe antibiotische Behandlung vermindert oder verhindert Antikörperbildung
Zelluläre Labordiagnostik
(Bestimmung der spezifischen T-Zell-
Reaktivität
durch Funktionstestungen)
Slide17Dattwyler et al. 1988. Seronegative Lyme disease. Dissociation of specific T- and B-lymphocyte
responses to Borrelia burgdorferi. N Engl
J Med 319:1441Abstract We studied 17 patients who had presented with acute Lyme disease and received prompt treatment with oral antibiotics, but in whom chronic Lyme disease subsequently developed. Although these patients had clinically active disease, none had diagnostic levels of antibodies to B.
burgdorferi on either a standard enzyme-linked immunosorbent assay or immunofluorescence
assay. On Western blot analysis, the level of immunoglobulin reactivity against B.
burgdorferi
in serum from these patients was no greater than that in serum from normal controls.
The patients had a vigorous T-cell proliferative response to whole B.
burgdorferi
, with a mean ( +/- SEM) stimulation index of 17.8 +/- 3.3, similar to that (15.8 +/- 3.2) in 18 patients with chronic Lyme disease who had detectable antibodies. The T-cell response of both groups was greater than that of a control
group of healthy subjects (3.1 +/- 0.5; P less than 0.001). We conclude that the presence of chronic Lyme disease cannot be excluded by the absence of antibodies against B.
burgdorferi
and that a specific T-cell
blastogenic
response to B.
burgdorferi
is evidence of infection in
seronegative patients with clinical indications of chronic Lyme disease.”
Lyme
-Borreliose: Diagnostik
Indirekter (immunologischer) Nachweis einer
Borrelien
-Infektion
Abstract
We studied 17 patients who had presented with acute Lyme disease and received prompt treatment with oral antibiotics, but in whom chronic Lyme disease subsequently developed. Although these patients had clinically active disease, none had diagnostic levels of antibodies to B.
burgdorferi
on either a standard enzyme-linked
immunosorbent
assay or
immunofluorescence
assay. On Western blot analysis, the level of immunoglobulin reactivity against B.
burgdorferi
in serum from these patients was no greater than that in serum from normal controls.
The patients had a vigorous
T-cell proliferative response
to whole B.
burgdorferi
, with a mean ( +/- SEM) stimulation index of 17.8 +/- 3.3, similar to that (15.8 +/- 3.2) in 18 patients with chronic Lyme disease who had detectable antibodies. The T-cell response of both groups was greater than that of a control
group of healthy subjects (3.1 +/- 0.5; P less than 0.001).
We conclude
that the presence of chronic Lyme disease cannot be excluded by the absence of antibodies against B.
burgdorferi
and
that a specific T-cell
blastogenic
response to B.
burgdorferi
is evidence of infection in
seronegative
patients with clinical indications of chronic Lyme disease
.
”
Slide18Lyme
-Borreliose: Diagnostik
Indirekter (immunologischer) Nachweis einer
Borrelien
-Infektion
Lymphozytentransformationstest
(LTT)
Prinzip: Bestimmung der Anwesenheit
/
Frequenz
Borrelien
-spezifischer
T-Lymphozyten im Blut als Indikator für eine vorausgegangene Infektion
Lymphozyten-Aktivierung
Han
et al.
2007. Nature
Reviews Microbiology 4:95
Transformation (
Blastenbildung
)
Proliferation (Zellvermehrung)
Methode: Messung der Proliferation (Zellteilung) von Gedächtnis-T-Zellen
durch Quantifizierung des Einbaus eines radioaktiv markierten
Nukleotids
(
3
H-Thymidin) in die DNA von Lymphozyten, die mit
Borrelien
-Antigenen
langzeitkultiviert (5 bis 6 Tage) werden.
Slide19Lyme
-Borreliose: Diagnostik
Indirekter (immunologischer) Nachweis einer
Borrelien
-Infektion
Lymphozytentransformationstest
(LTT)
Prinzip: Bestimmung der Anwesenheit
/
Frequenz
Borrelien
-spezifischer
T-Lymphozyten im Blut als Indikator für eine vorausgegangene Infektion
Methode: Messung der Proliferation (Zellteilung) von Gedächtnis-T-Zellen
durch Quantifizierung des Einbaus eines radioaktiv markierten
Nukleotids (
3H-Thymidin) in die DNA von Lymphozyten, die mit Borrelien-Antigenen
langzeitkultiviert (5 bis 6 Tage) werden.
Synonyme Bezeichnungen:• Lymphozyten-Aktivierungstest (LAT)
T-Zell-Proliferationstest LTT-MELISA (
ME
mory
L
ymphocyte
I
mmuno
S
timulation
A
ssay)
3HT-Memory-Test
Slide20Lyme
-Borreliose: Diagnostik
Indirekter (immunologischer) Nachweis einer
Borrelien
-Infektion
Lymphozytentransformationstest
(LTT)
Zellkultur:
Stimulation der
mononukleären
Zellen mit
Borrelien
-Antigenen für 5 bis 6 Tage
Stimulationsindex SI =
-------------------------------------------------------
Proliferation in stimulierter Kultur [
cpm
]
Proliferation in
unstimulierter
Kultur [
cpm
]
SI > Schwellenwert
Reaktion positiv
unstimuliert
Mit freundlicher Genehmigung von Dr. Verena
Raker
(Universitätsmedizin Mainz)
stimuliert
Slide21Lyme
-Borreliose: Diagnostik
Indirekter (immunologischer) Nachweis einer
Borrelien
-Infektion
Prinzip: Bestimmung der Anwesenheit
/
Frequenz
Borrelien
-spezifischer
T-Lymphozyten im Blut als Indikator für eine vorausgegangene Infektion
Methode: Messung der Proliferation (Zellteilung) von Gedächtnis-T-Zellen
durch Quantifizierung des Einbaus eines radioaktiv markierten
Nukleotids
(3
H-Thymidin) in die DNA von Lymphozyten, die mit Borrelien-Antigenen langzeitkultiviert (5 bis 6 Tage) werden.
Synonyme Bezeichnungen:
• Lymphozyten-Aktivierungstest (LAT) T-Zell-Proliferationstest
LTT-MELISA (ME
mory
L
ymphocyte
I
mmuno
S
timulation
A
ssay)
3HT-Memory-Test
Die Eignung des LTT als diagnostisches Verfahren zur Bestätigung einer
Borreliose ist labormedizinisch umstritten. Vor allen Dingen in älteren
Studien (vor 2000) werden Sensitivität und
/
oder Spezifität des LTT als
nicht ausreichend für eine valide Bewertung der Ergebnisse beurteilt.
Lymphozytentransformationstest
(LTT)
Slide22Lyme
-Borreliose: Diagnostik
Indirekter (immunologischer) Nachweis einer
Borrelien
-Infektion
Lymphozytentransformationstest
(LTT)
Dressler et al. 1991.
The T-cell proliferative assay in the diagnosis of Lyme disease.
Ann Intern Med 115:533.
OBJECTIVE:
To determine the sensitivity and specificity of the T-cell proliferative assay as a diagnostic test in Lyme disease.
DESIGN:
Cross-sectional study of patients with Lyme arthritis or chronic
neuroborreliosis
who had a history of
erythema
migrans, positive antibody responses to Borrelia burgdorferi by enzyme-linked
immunosorbent assay (ELISA), or both; patients with other diseases; and healthy subjects.PATIENTS: Forty-two of the 67 patients with active Lyme arthritis or chronic
neuroborreliosis who were seen during the study period; 16 patients with inactive late Lyme disease; 77 patients with other rheumatologic or neurologic diseases; 9 workers from the Borrelia laboratory; and 9 healthy subjects.MEASUREMENTS AND MAIN RESULTS: Nineteen of 42 patients with Lyme arthritis or chronic
neuroborreliosis and 4 of 77 patients with other diseases had positive T-cell proliferative responses to B. burgdorferi antigens. The sensitivity of the proliferative assay was 45% (95%
Cl
, 30% to 60%) and the specificity was 95% (95%
Cl
, 87% to 99%). Twelve of 27 patients with active Lyme arthritis, 7 of 15 patients with chronic
neuroborreliosis
, 4 of 16 patients with inactive Lyme disease, 4 of 9 healthy
Borrelia
laboratory workers, and 0 of 9 healthy subjects had positive responses. Three of five patients with Lyme disease who had negative or
indeterminant
antibody responses by ELISA had positive T-cell proliferative responses.
CONCLUSION:
The T-cell proliferative assay may be a helpful diagnostic test in the small subset of patients with late Lyme disease who have negative or
indeterminant
antibody responses by ELISA.OBJECTIVE: To determine the sensitivity and specificity of the T-cell proliferative assay as a diagnostic test in Lyme disease.DESIGN:
Cross-sectional study of patients with Lyme arthritis or chronic
neuroborreliosis
who had a history of
erythema
migrans
, positive antibody responses to
Borrelia
burgdorferi
by enzyme-linked
immunosorbent
assay (ELISA), or both; patients with other diseases; and healthy subjects.
PATIENTS:
Forty-two of the 67 patients with active Lyme arthritis or chronic
neuroborreliosis
who were seen during the study period; 16 patients with inactive late Lyme disease; 77 patients with other rheumatologic or neurologic diseases; 9 workers from the
Borrelia laboratory; and 9 healthy subjects.MEASUREMENTS AND MAIN RESULTS: Nineteen of 42 patients with Lyme arthritis or chronic neuroborreliosis and 4 of 77 patients with other diseases had positive T-cell proliferative responses to B. burgdorferi antigens. The sensitivity of the proliferative assay was 45% (95%
Cl
, 30% to 60%) and the specificity was 95%
(95% Cl, 87% to 99%). Twelve of 27 patients with active Lyme arthritis, 7 of 15 patients with chronic neuroborreliosis
, 4 of 16 patients with inactive Lyme disease, 4 of 9 healthy Borrelia laboratory workers, and 0 of 9 healthy subjects had positive responses. Three of five patients with Lyme disease who had negative or indeterminant antibody responses by ELISA had positive T-cell proliferative responses.CONCLUSION: The T-cell proliferative assay may be a helpful diagnostic test in the small subset of patients with late Lyme disease who have negative or indeterminant antibody responses by ELISA.OBJECTIVE: To determine the sensitivity and specificity of the T-cell proliferative assay as a diagnostic test in Lyme disease.DESIGN: Cross-sectional study of patients with Lyme arthritis or chronic neuroborreliosis who had a history of erythema migrans, positive antibody responses to
Borrelia burgdorferi
by enzyme-linked immunosorbent assay (ELISA), or both; patients with other diseases; and healthy subjects.PATIENTS: Forty-two of the 67 patients with active Lyme arthritis or chronic neuroborreliosis who were seen during the study period; 16 patients with inactive late Lyme disease; 77 patients with other rheumatologic or neurologic diseases; 9 workers from the
Borrelia laboratory; and 9 healthy subjects.MEASUREMENTS AND MAIN RESULTS: Nineteen of 42 patients with Lyme arthritis or chronic neuroborreliosis and 4 of 77 patients with other diseases had positive T-cell proliferative responses to B.
burgdorferi
antigens.
The sensitivity of the proliferative assay was 45%
(95%
Cl
, 30% to 60%) and the
specificity was 95%
(95%
Cl
, 87% to 99%). Twelve of 27 patients with active Lyme arthritis, 7 of 15 patients with chronic
neuroborreliosis
, 4 of 16 patients with inactive Lyme disease, 4 of 9 healthy
Borrelia
laboratory workers, and 0 of 9 healthy subjects had positive responses.
Three of five patients with Lyme disease who had negative or
indeterminant
antibody responses by ELISA had positive T-cell proliferative responses.
CONCLUSION:
The T-cell proliferative assay may be a helpful diagnostic test in the small subset of patients with late Lyme disease who have negative or
indeterminant
antibody responses by ELISA.
Slide23Lyme
-Borreliose: Diagnostik
Indirekter (immunologischer) Nachweis einer
Borrelien
-Infektion
Lymphozytentransformationstest
(LTT)
Buechner
et al. 1995.
Lymphoproliferative
responses to
Borrelia
burgdorferi
in patients with
erythema
migrans, acrodermatitis
chronica atrophicans, lymphadenosis
benigna cutis, and morphea.
Arch Dermatol 131:673.BACKGROUND AND DESIGN:
... We studied the lymphoproliferative response of peripheral blood mononuclear cells to B burgdorferi antigen from 99 patients (25 with
erythema
migrans
, 16 with
acrodermatitis
chronica
atrophicans
, 13 with
lymphadenosis
benigna
cutis, and 45 with localized scleroderma) and 21 control subjects. ...RESULTS: The 21 healthy seronegative controls had an SI of 3.3 +/- 2.0 (mean +/- SD). Compared with that of control subjects, the SIs were significantly elevated in patients with erythema migrans (9.8 +/- 9.1), acrodermatitis
chronica
atrophicans
(11.8 +/- 8.2), and
lymphadenosis
benigna
cutis (7.2 +/- 6.2). ... Elevated titers of antibodies to B
burgdorferi
were present in six (24%) of 25 patients with
erythema
migrans
, five (38%) of 13 patients with
lymphadenosis
benigna cutis, and 13 (29%) of 45 patients with localized scleroderma. All 16 patients with
acrodermatitis chronica atrophicans had markedly elevated antibody titers.CONCLUSIONS: Our findings show that a significant lymphoproliferative response to B burgdorferi
occurs in the majority of patients with
cutaneous manifestations of Lyme
borreliosis. The lymphocyte proliferation assay may be of diagnostic value in patients in whom Lyme borreliosis
is strongly clinically suspected and who have nondiagnostic levels of antibodies against B burgdorferi.BACKGROUND AND DESIGN: ... We studied the lymphoproliferative response of peripheral blood mononuclear cells to B burgdorferi antigen from 99 patients (25 with erythema migrans, 16 with acrodermatitis chronica atrophicans, 13 with lymphadenosis benigna
cutis, and 45 with localized scleroderma) and 21 control subjects. ...RESULTS:
The 21 healthy seronegative controls had an SI of 3.3 +/- 2.0 (mean +/- SD). Compared with that of control subjects, the SIs were significantly elevated in patients with erythema migrans (9.8 +/- 9.1), acrodermatitis
chronica atrophicans (11.8 +/- 8.2), and lymphadenosis benigna cutis (7.2 +/- 6.2). ... Elevated titers of antibodies to B
burgdorferi
were present in six (24%) of 25 patients with
erythema
migrans
, five (38%) of 13 patients with
lymphadenosis
benigna
cutis, and 13 (29%) of 45 patients with localized scleroderma. All 16 patients with
acrodermatitis
chronica
atrophicans
had markedly elevated antibody titers.
CONCLUSIONS:
Our findings show that a significant
lymphoproliferative
response to B
burgdorferi
occurs in the majority of patients with
cutaneous
manifestations of Lyme
borreliosis
.
The lymphocyte proliferation assay may be of diagnostic value in patients in whom Lyme
borreliosis
is strongly clinically suspected and who have
nondiagnostic
levels of antibodies against B
burgdorferi
.
Slide24Lyme
-Borreliose: Diagnostik
Indirekter (immunologischer) Nachweis einer
Borrelien
-Infektion
Lymphozytentransformationstest
(LTT)
Breier
et al. 1995.
Lymphocyte
proliferation
test
in
cutaneous
manifestations
of Lyme borreliosis
. Wien Med Wochenschr 145:170
ABSTRACT
The humoral immunoreactivity in Lyme borreliosis is a well
characterized parameter for
establishing
the
diagnosis
of
a
Borrelia
burgdorferi
(
Bb) infection. Since patients with seronegative Lyme
borreliosis
have
been
described
,
lymphocyte
proliferation
tests
may
be used
for detecting patients who only develop a
cellular
immunoreactivity
against Bb
organisms. We performed a the lymphocyte proliferation assays in order to determine the cellular immunoreactivity to these
spirochetes in given
patients with histologically confirmed
diagnosis of erythema migrans (EM), acrodermatitis
chronica
atrophicans
(ACA),
and
for
control
purposes
patients
with
Lyme
disease
non
associated
dermatoses
(NLDH)
and
healthy
volunteers
(G). … .
We
detected
elevated
cellular
immunoreactivity
of
peripheral
mononuclear
cells
in 33% EM-
patients
(3/9)
and
in 23% (3/13) ACA
patients
tested
.
Patients
tested
before
and
after
antibiotic
therapy
showed
a
significant
decrease
of
stimulation
index
after
antimicrobial
treatment
(p < 0.05).
Patients
,
who
are
mostly
seronegative
at
the
onset
of
cutaneous
eruption
exhibit
in a
third
of
patients
an
elevated
cellular
immune
response
to
Bb
.
Therefore
lymphocyte
proliferation
assays
can
be
recommended
as
an additional
test
system
in
case
of
lack
of
serological
response
. The
significant
decrease
of
stimulation
index
after
antimicrobial
therapy
indicates
for
downregulation
of
the
cellular
immune
response
to
these
spirochetes
investigated
,
and
counts
for
the
specificity
of
this
test
system
.
Breier
et al. 1995.
Lymphocyte
proliferation
test
in
cutaneous
manifestations
of
Lyme
borreliosis
.
Wien Med
Wochenschr
145:170.
ABSTRACT
The humoral
immunoreactivity
in
Lyme
borreliosis
is
a well
characterized
parameter
for
establishing
the
diagnosis
of
a
Borrelia
burgdorferi
(
Bb
)
infection
.
Since
patients
with
seronegative
Lyme
borreliosis
have
been
described
,
lymphocyte
proliferation
tests
may
be
used
for
detecting
patients
who
only
develop
a
cellular
immunoreactivity
against
Bb
organisms
.
We
performed
a
the
lymphocyte
proliferation
assays
in order
to
determine
the
cellular
immunoreactivity
to
these
spirochetes
in
given
patients
with
histologically
confirmed
diagnosis
of
erythema
migrans
(EM),
acrodermatitis
chronica
atrophicans
(ACA),
and
for
control
purposes
patients
with
Lyme
disease
non
associated
dermatoses
(NLDH)
and
healthy
volunteers
(G). … .
We
detected
elevated
cellular
immunoreactivity
of
peripheral
mononuclear
cells
in 33% EM-
patients
(3/9)
and
in 23% (3/13) ACA
patients
tested
.
Patients
tested
before
and
after
antibiotic
therapy
showed
a
significant
decrease
of
stimulation
index
after
antimicrobial
treatment
(p < 0.05).
Patients
,
who
are
mostly
seronegative
at
the
onset
of
cutaneous
eruption
exhibit
in a
third
of
patients
an
elevated
cellular
immune
response
to
Bb
.
Therefore
lymphocyte
proliferation
assays
can
be
recommended
as
an additional
test
system
in
case
of
lack
of
serological
response
.
The
significant
decrease
of
stimulation
index
after
antimicrobial
therapy
indicates
for
downregulation
of
the
cellular
immune
response
to
these
spirochetes
investigated
,
and
counts
for
the
specificity
of
this
test
system
.
Slide25Lyme
-Borreliose: Diagnostik
Indirekter (immunologischer) Nachweis einer
Borrelien
-Infektion
Lymphozytentransformationstest
(LTT)
Huppertz
et al. 1996.
Lymphoproliferative
responses to
Borrelia
burgdorferi
in the diagnosis of Lyme
arthritis in children and adolescents.
Eur J Pediatr 155:297.
ABSTRACTTo assess the contribution of the lymphocyte proliferation assay in response to borrelial
antigens to establishing a diagnosis of Lyme arthritis (LA) the response to two strains of Borrelia burgdorferi was tested in peripheral blood lymphocytes of 103 children and adolescents with arthritis, among them 55 with LA and 48 control patients. Patients with LA had a significantly higher response to
borrelial antigens than control patients. However, there were several patients with false positive and false negative test results. Specificity and sensitivity of the test were 78% and 77%. In patients with LA the test may turn positive after antibiotic therapy and remain positive for up to 19 months after the disappearance of arthritis. The test does not aid in prognosis or follow up. In one patient with seronegative LA specific lymphocyte proliferation and polymerase chain reaction for
borrelial fla sequences in urine were positive.
CONCLUSION
Rarely the lymphocyte proliferation assay may aid in finding the correct diagnosis when clinical presentation and anti-
borrelial
serology do not match.
ABSTRACT
To assess the contribution of the lymphocyte proliferation assay in response to
borrelial
antigens to establishing a diagnosis of Lyme arthritis (LA) the response to two strains of
Borrelia
burgdorferi
was tested in peripheral blood lymphocytes of 103 children and adolescents with arthritis, among them 55 with LA and 48 control patients. Patients with LA had a significantly higher response to
borrelial
antigens than control patients. However, there were several patients with false positive and false negative test results. Specificity and sensitivity of the test were 78% and 77%. In patients with LA the test may turn positive after antibiotic therapy and remain positive for up to 19 months after the disappearance of arthritis. The test does not aid in prognosis or follow up. In one patient with
seronegative
LA specific lymphocyte proliferation and polymerase chain reaction for
borrelial
fla
sequences in urine were positive.
CONCLUSION
Rarely the lymphocyte proliferation assay may aid in finding the correct diagnosis when clinical presentation and anti-
borrelial
serology do not match.
ABSTRACT
To assess the contribution of the lymphocyte proliferation assay in response to
borrelial
antigens to establishing a diagnosis of Lyme arthritis (LA) the response to two strains of
Borrelia
burgdorferi
was tested in peripheral blood lymphocytes of 103 children and adolescents with arthritis, among them 55 with LA and 48 control patients. Patients with LA had a significantly higher response to borrelial antigens than control patients. However, there were several patients with false positive and false negative test results. Specificity and sensitivity of the test were 78% and 77%. In patients with LA the test may turn positive after antibiotic therapy and remain positive for up to 19 months after the disappearance of arthritis. The test does not aid in prognosis or follow up.
In one patient with
seronegative
LA specific lymphocyte proliferation and polymerase chain reaction for borrelial fla
sequences in urine were positive.CONCLUSIONRarely the lymphocyte proliferation assay may aid in finding the correct diagnosis when clinical presentation and anti-borrelial serology do not match.
Slide26Lyme
-Borreliose: Diagnostik
Indirekter (immunologischer) Nachweis einer
Borrelien
-Infektion
Lymphozytentransformationstest
(LTT)
Breier
et al. 1996.
Lymphoproliferative
responses to
Borrelia
burgdorferi
in circumscribed
scleroderma.
Br J Dermatol 134:285.
ABSTRACTHumoral immune responses to Borrelia burgdorferi
(Bb) have been reported to occur in certain patients with circumscribed scleroderma (CS) (morphoea). Together with the isolation of spirochaetes
from CS skin biopsies, this finding was taken to suggest Bb as the aetiological agent of CS. ... For this purpose, peripheral blood mononuclear cells from CS patients and, as controls, from patients with various manifestations of LB, and from healthy volunteers without any evidence of Bb infection, were exposed to Bb organisms for 5 days and then assayed for DNA synthesis. Stimulation indices (SI) > 10 were scored positive. By performing lymphocyte proliferation tests we found: (i) that not only patients with various manifestations of LB but also a considerable percentage of
seropositive (five of 13 = 38%) and seronegative (six of 26 = 23%) CS patients exhibit an elevated Bb-induced lymphocyte proliferation; (ii) that the magnitude of the cellular response seen in CS patients is comparable to that encountered in patients with established Bb manifestations; and (iii) that, within a given patient, antibiotic therapy can result in a significant reduction of this response. These results support a causative role of Bb in at least some CS patients. Bb-induced lymphocyte responses were also seen in both
seropositive
and
seronegative
erythema
chronicum
migrans
patients. These findings show that the pattern of Bb-specific immune responses is more complex than previously thought, and underscore the importance of lymphocyte function assays in evaluating the diagnosis of potential Bb infection in
seronegative
patients.
ABSTRACT
Humoral immune responses to Borrelia burgdorferi (Bb) have been reported to occur in certain patients with circumscribed scleroderma (CS) (
morphoea
). Together with the isolation of
spirochaetes
from CS skin biopsies, this finding was taken to suggest Bb as the
aetiological
agent of CS. ... For this purpose, peripheral blood mononuclear cells from CS patients and, as controls, from patients with various manifestations of LB, and from healthy volunteers without any evidence of Bb infection, were exposed to Bb organisms for 5 days and then assayed for DNA synthesis. Stimulation indices (SI) > 10 were scored positive. By performing lymphocyte proliferation tests we found: (
i
) that not only patients with various manifestations of LB but also a considerable percentage of
seropositive
(five of 13 = 38%) and
seronegative
(six of 26 = 23%) CS patients exhibit an elevated Bb-induced lymphocyte proliferation; (ii) that the magnitude of the cellular response seen in CS patients is comparable to that encountered in patients with established Bb manifestations; and (iii) that, within a given patient, antibiotic therapy can result in a significant reduction of this response. These results support a causative role of Bb in at least some CS patients.
Bb-induced lymphocyte responses were also seen in both
seropositive
and
seronegative
erythema chronicum migrans patients. These findings show that the pattern of Bb-specific immune responses is more complex than previously thought, and
underscore the importance of lymphocyte function assays in evaluating the diagnosis of potential Bb infection in
seronegative patients.
Slide27Lyme
-Borreliose: Diagnostik
Indirekter (immunologischer) Nachweis einer
Borrelien
-Infektion
Lymphozytentransformationstest
(LTT)
Vaz
et al. 2001.
Cellular and
humoral
immune responses to
Borrelia
burgdorferi
antigens in patients
with culture-positive early Lyme disease. Infect Immun 69:7437.
ABSTRACTWe determined cellular and humoral immune responses to Borrelia
burgdorferi lysate and to recombinant
flagellin (FlaB), OspC, and OspA in acute- and convalescent-phase samples from 39 culture-positive patients with
erythema migrans and in 20 healthy control subjects. During the acute illness, a median of 4 days after the onset of
erythema
migrans
, 51% of the patients had proliferative cellular responses and 72% had antibody responses to at least one of the
borrelial
antigens tested. During convalescence, at the conclusion of antibiotic therapy, 64% of the patients had proliferative cellular reactivity and 95% had antibody reactivity with at least one of the
spirochetal
antigens tested. In both acute- and convalescent-phase samples, cellular immune responses were found as frequently to
OspA
as to
OspC
and
FlaB
. Although antibody responses were also frequently seen to OspC and FlaB, only a few patients had marginal antibody reactivity with OspA. The percentage of patients with proliferative responses was similar in those with clinical evidence of localized or disseminated infection, whereas humoral
reactivity was found more often in those with disseminated disease. We conclude that cellular and
humoral
responses to B.
burgdorferi
antigens are often found among patients with early Lyme disease. In contrast with the other antigens tested, cellular but not
humoral
reactivity was often found with
OspA
.
ABSTRACT
We determined cellular and
humoral
immune responses to
Borrelia
burgdorferi
lysate and to recombinant flagellin (FlaB), OspC, and OspA in acute- and convalescent-phase samples from 39 culture-positive patients with
erythema
migrans
and in 20 healthy control subjects. During the acute illness, a median of 4 days after the onset of erythema migrans
, 51% of the patients had proliferative cellular responses and 72% had antibody responses to at least one of the borrelial antigens tested. During convalescence, at the conclusion of antibiotic therapy, 64% of the patients had proliferative cellular reactivity and 95% had antibody reactivity with at least one of the spirochetal antigens tested. In both acute- and convalescent-phase samples, cellular immune responses were found as frequently to OspA as to OspC and FlaB. Although antibody responses were also frequently seen to OspC and FlaB, only a few patients had marginal antibody reactivity with OspA. The percentage of patients with proliferative responses was similar in those with clinical evidence of localized or disseminated infection, whereas humoral reactivity was found more often in those with disseminated disease. We conclude that cellular and humoral responses to B. burgdorferi antigens are often found among patients with early Lyme disease. In contrast with the other antigens tested, cellular but not
humoral reactivity was often found with
OspA.
Slide28Lyme
-Borreliose: Diagnostik
Indirekter (immunologischer) Nachweis einer
Borrelien
-Infektion
Lymphozytentransformationstest
(LTT)
Vaz
et al. 2001.
Cellular and
humoral
immune responses to
Borrelia
burgdorferi
antigens in patients
with culture-positive early Lyme disease. Infect Immun 69:7437.
Results
Cellular immune responses to B. burgdorferi did not correlate with humoral
immune reactivity. Moreover, there were patients with weak cellular reactivity and strong humoral responses or vice versa (data not shown). Overall, cellular reactivity with OspA was as frequent and as strong as the cellular responses to FlaB and OspC
.
Slide29Lyme
-Borreliose: Diagnostik
Indirekter (immunologischer) Nachweis einer
Borrelien
-Infektion
Lymphozytentransformationstest
(LTT)
von
Baehr
et al. 2001.
Improving the in vitro antigen specific T cell proliferation assay: the use of
interferon-
a
to elicit antigen specific stimulation and decrease bystander proliferation.
J
Immunol Methods 251:63.
ABSTRACTThe measurement of the proliferative response of primed T cells to an antigenic stimulus (lymphocyte transformation assay: LTT) is commonly used for determining T cell immune responsiveness. However, the ratio between the spontaneous and the antigen-triggered response (stimulation index) is frequently quite low (<3-5) making the interpretation difficult. We modified the assay by the addition of interferon-alpha and the use of fresh autologous serum instead of human AB pool serum. These measures significantly enhanced the stimulation index following stimulation with tetanus
toxoid, Candida albicans
and tick-borne encephalitis (TBE) viral antigen in studies of sensitized patients. There was no concomitant increase in false positive results. Kinetic studies showed a reduced nonspecific background proliferation of non-stimulated cultures particularly between days 4 and 6 of culture. Furthermore, the positive effect of interferon-alpha were confirmed in studies of patients with contact allergy to nickel and gold. We conclude that this modified form of proliferation assay significantly increases the signal to noise ratio which can be attained. This may be of particular value when looking at T cell responses in immunocompromised patients or in diagnostic attempts to detect very low frequencies of antigen-specific T cells.
ABSTRACTThe measurement of the proliferative response of primed T cells to an antigenic stimulus (lymphocyte transformation assay: LTT) is commonly used for determining T cell immune responsiveness. However, the ratio between the spontaneous and the antigen-triggered response (stimulation index) is frequently quite low (<3-5) making the interpretation difficult.
We modified the assay by the addition of interferon-alpha and the use of fresh
autologous
serum instead of human AB pool serum.
These measures significantly enhanced the stimulation index following stimulation with tetanus
toxoid
, Candida
albicans
and tick-borne encephalitis (TBE) viral antigen in studies of sensitized patients. There was no concomitant increase in false positive results.
Kinetic studies showed a reduced nonspecific background proliferation of non-stimulated cultures particularly between days 4 and 6 of culture.
Furthermore, the positive effect of interferon-alpha were confirmed in studies of patients with contact allergy to nickel and gold. We conclude that this modified form of proliferation assay
significantly increases the signal to noise ratio which can be attained.
This may be of particular value when looking at T cell responses in
immunocompromised
patients or in diagnostic attempts to detect very low frequencies of antigen-specific T cells.
Slide30Lyme
-Borreliose: Diagnostik
Indirekter (immunologischer) Nachweis einer
Borrelien
-Infektion
Lymphozytentransformationstest
(LTT)
Valentine-Thon et al. 2008.
A
novel
lymphocyte
transformation
test
(LTT-MELISA)
for
Lyme
borreliosis.
Diagn Microbiol Infect Dis
57:27.ABSTRACT
Diagnosis of active Lyme borreliosis (LB) remains a challenge in clinically ambiguous, serologically indeterminant, and polymerase chain reaction-negative patients. Lymphocyte transformation tests (LTTs) have been applied to detect specific cellular immune reactivity, but their clinical application has been severely hampered by the poorly defined Borrelia antigens and
nonstandardized LTT formats used. In this study, we describe the development and clinical relevance of a novel LTT using a validated format (MELISA) together with well-defined recombinant Borrelia
-specific antigens. From an initial screening of 244 patients with suspected
Borrelia
infection or disease, 4 informative recombinant antigens were selected:
OspC
(
Borrelia
afzelii
), p41-1 (
Borrelia
garinii
), p41-2 (B.
afzelii), and p100 (B. afzelii). Thereafter, 30 seronegative healthy controls were tested in LTT-MELISA(R) to determine specificity, 68 patients were tested in parallel to determine reproducibility, and 54 lymphocyte-reactive symptomatic patients were tested before and after antibiotic therapy to assess clinical relevance. Most (86.2%) of the 36.9% (90/244) LTT-MELISA positive patients were seropositive and showed symptoms of active LB. Specificity was 96.7% and reproducibility 92.6%. After therapy, most patients (90.7%) showed negative or markedly reduced lymphocyte reactivity correlating with clinical improvement. This novel LTT-MELISA assay appears to correlate with active LB and may have diagnostic relevance in confirming LB in clinically and serologically ambiguous cases.
ABSTRACT
Diagnosis of active Lyme
borreliosis
(LB) remains a challenge in clinically ambiguous, serologically
indeterminant
, and polymerase chain reaction-negative patients. Lymphocyte transformation tests (LTTs) have been applied to detect specific cellular immune reactivity, but their clinical application has been severely hampered by the poorly defined
Borrelia
antigens and
nonstandardized
LTT formats used. In this study, we describe the development and clinical relevance of a novel LTT using a validated format (MELISA) together with well-defined recombinant
Borrelia
-specific antigens. From an initial screening of 244 patients with suspected
Borrelia
infection or disease, 4 informative recombinant antigens were selected:
OspC
(
Borrelia
afzelii), p41-1 (Borrelia garinii), p41-2 (B.
afzelii), and p100 (B.
afzelii). Thereafter, 30
seronegative healthy controls were tested in LTT-MELISA(R) to determine specificity, 68 patients were tested in parallel to determine reproducibility, and 54 lymphocyte-reactive symptomatic patients were tested before and after antibiotic therapy to assess clinical relevance. Most (86.2%) of the 36.9% (90/244) LTT-MELISA positive patients were
seropositive and showed symptoms of active LB. Specificity was 96.7% and reproducibility 92.6%. After therapy, most patients (90.7%) showed negative or markedly reduced lymphocyte reactivity correlating with clinical improvement. This novel LTT-MELISA assay appears to correlate with active LB and may have diagnostic relevance in confirming LB in clinically and serologically ambiguous cases.
Slide31Lyme
-Borreliose: Diagnostik
Indirekter (immunologischer) Nachweis einer
Borrelien
-Infektion
Lymphozytentransformationstest
(LTT)
von
Baehr
et al. 2007.
Untersuchungen zur diagnostischen Wertigkeit des
Lymphozytentransforma
-
tionstestes
bei Patienten mit Borreliose.
LaboratoriumsMedizin
31:149.ABSTRACTBorrelienspezifische
Antikörper sind erst mehrere Wochen nach Infektion nachweisbar und allein kein Beweis für eine aktive Borreliose. … Es wurde dazu ein Lymphozytentransformationstest (LTT) mit drei Borrelienlysatantigenen (
Borrelia B. sensu
stricto, B. afzelii und B. garinii) sowie rekombinantem
OspC entwickelt und mit Untersuchungen von seronegativen (n=100) und
seropositiven
Gesunden (n=36) sowie
seropositiven
Patienten mit klinischer Borreliose (n=44) validiert. Die Sensitivität des
Borrelien
-LTT für eine klinische Borreliose vor antibiotischer Behandlung wurde mit 91%, die Spezifität mit 94% ermittelt. Bei 820 Patienten mit klinischem Verdacht auf Borreliose wurde eine Übereinstimmung positiver und negativer serologischer und LTT-Ergebnisse von 77,3% gefunden. Serologisch positiv und im LTT negativ waren 165 Patienten (20,1%), überwiegend mit Borreliose nach antibiotischer Therapie. Serologisch negativ und im LTT positiv waren 21 Patienten (2,6%), davon sieben mit
Erythema
migrans
. Nach antibiotischer Behandlung wird der
Borrelien
-LTT bei Patienten mit Frühmanifestationen der Borreliose (n=90) negativ bis grenzwertig, bei Spätmanifestationen (n=70) rückläufig mit verbleibender Restaktivität. Verlaufsuntersuchungen über ein Jahr von sechs Patienten mit Frühmanifestationen zeigten nur eine Reaktivierung. Bei acht von zehn Patienten mit Spätmanifestationen wurden häufig Reaktivierungen bzw. persistierend positive LTT-Reaktionen beobachtet. Als Indikation für den
Borrelien
-LTT wird deshalb besonders die Verlaufskontrolle bei disseminierten Borrelieninfektionen vorgeschlagen
ABSTRACT
Borrelienspezifische
Antikörper sind erst mehrere Wochen nach Infektion nachweisbar und allein kein Beweis für eine aktive Borreliose. … Es wurde dazu ein
Lymphozytentransformationstest
(LTT) mit drei
Borrelienlysatantigenen
(
Borrelia
B
.
sensu
stricto
,
B.
afzelii
und B. garinii) sowie rekombinantem OspC entwickelt und mit Untersuchungen von seronegativen (n=100) und seropositiven Gesunden (n=36) sowie seropositiven Patienten mit klinischer Borreliose (n=44) validiert.
Die Sensitivität des
Borrelien-LTT für eine klinische Borreliose vor antibiotischer Behandlung wurde mit 91%, die Spezifität mit 94% ermittelt.
Bei 820 Patienten mit klinischem Verdacht auf Borreliose wurde eine Übereinstimmung positiver und negativer serologischer und LTT-Ergebnisse von 77,3% gefunden. Serologisch positiv und im LTT negativ waren 165 Patienten (20,1%), überwiegend mit Borreliose nach antibiotischer Therapie. Serologisch negativ und im LTT positiv waren 21 Patienten (2,6%), davon sieben mit
Erythema migrans. Nach antibiotischer Behandlung wird der Borrelien-LTT bei Patienten mit Frühmanifestationen der Borreliose (n=90) negativ bis grenzwertig, bei Spätmanifestationen (n=70) rückläufig mit verbleibender Restaktivität. Verlaufsuntersuchungen über ein Jahr von sechs Patienten mit Frühmanifestationen zeigten nur eine Reaktivierung. Bei acht von zehn Patienten mit Spätmanifestationen wurden häufig Reaktivierungen bzw. persistierend positive LTT-Reaktionen beobachtet. Als Indikation für den Borrelien-LTT wird deshalb besonders die Verlaufskontrolle bei disseminierten Borrelieninfektionen vorgeschlagen
Slide32Lyme
-Borreliose: Diagnostik
Indirekter (immunologischer) Nachweis einer
Borrelien
-Infektion
Bewertung des
Lymphozytentransformationstests
als diagnostisches Verfahren
durch Fachgremien
S1-Leitlinie „
Neuroborreliose
“ der Deutschen Gesellschaft der Neurologie (gültig bis 2015)
“
Nicht
geeignet
zur
Labordiagnostik
der Lyme-Borreliose sind folgende
Tests: Lymphozyten
-Transformationstest (LTT), … .”„Der LTT misst die Stimulierbarkeit von Lymphozyten durch Borrelienantigene. Insbesondere bestehen Bedenken bezüglich der Spezifität dieses Tests (falsch positive Befunde).“
S1-Leitlinie „
Kutane Manifestationen der
Lyme
-Borreliose
“ der Deutschen Dermatologischen Gesell-
schaft
(gültig bis 2014, in Überarbeitung)
“
Im LTT reagieren nachweislich nicht nur
Borrelien
-spezifische T-Zellen, sondern auch T-Zellen mit anderen Spezifitäten. Dadurch treten auch bei gesunden Probanden häufig falsch positive Ergebnisse auf. Die Methode eignet sich deshalb nicht zur Diagnostik.“
“
Nicht
geeignet
zur
Labordiagnostik
auf.
Die Methode eignet sich deshalb nicht zur Diagnostik.
“
Slide33Lyme
-Borreliose: Diagnostik
Indirekter (immunologischer) Nachweis einer
Borrelien
-Infektion
Bewertung des
Lymphozytentransformationstests
als diagnostisches Verfahren
durch Fachgremien
Diagnostik der
Lyme-Borrreliose
. Stellungnahme der Kommission für Infektionskrankheiten und
Impffragen der Deutschen Akademie für Kinder- und Jugendmedizin (DAKJ).
Monatsschr
Kinderheilkd 2011, 159:368.
“Trotz verfeinerter Testbedingungen und des Einsatzes rekombinanter Antigene gelang es nicht, [für den Lymphozytentransformationstest] die gleiche Spezifität zu erreichen wie bei den serologischen Methoden mit
Enzymimmunotest und Immunoblot. … Entsprechend muss von dessen Verwendung abgeraten werden.“
„Fazit für die Praxis: Die Kommission fordert die Kostenträger auf, die Übernahme der Kosten für nicht indizierte Untersuchungen nicht zu übernehmen. Zu diesen gehören … und der Lymphozytentrans-formationstest.“
“Trotz verfeinerter Testbedingungen und des Einsatzes rekombinanter Antigene gelang es nicht, [für den Lymphozytentransformationstest] die gleiche Spezifität zu erreichen wie bei den serologischen Methoden mit
Enzymimmunotest
und
Immunoblot
. …
Entsprechend muss von dessen Verwendung abgeraten werden
.“
„Fazit für die Praxis: Die Kommission fordert die Kostenträger auf, die
Übernahme der Kosten
für nicht indizierte Untersuchungen
nicht zu übernehmen
. Zu diesen gehören … und der
Lymphozytentrans
-formationstest.“
Slide34Lyme
-Borreliose: Diagnostik
Indirekter (immunologischer) Nachweis einer
Borrelien
-Infektion
Bewertung des
Lymphozytentransformationstests
als diagnostisches Verfahren
durch Fachgremien
Leitlinie „
Diagnostik und Therapie der
Lyme
-Borreliose
“ der Deutschen Borreliose-Gesellschaft (2011)
“
Ein positives Ergebnis des LTT-
Borrelien
ist verdächtig, nicht aber beweisend für eine aktive
Borrelien
-Infektion.“
„Die Indikationen für den LTT-Borrelien sind: Nachweis einer aktiven Borrelien
-Infektion bei seropositiven
Patienten mit vieldeutiger Symptomatik seronegativem oder serologisch als grenzwertig beurteiltem Ergebnis von Patienten mit dringendem klinischen Verdacht auf eine Lyme-Borreliose
Therapiekontrolle ca. 4-6 Wochen nach Beendigung eines antibiotischen Behandlungszyklus
Verlaufskontrolle bei klinischem Verdacht auf ein Rezidiv
Neuinfektion.“
“
Ein positives Ergebnis des LTT-
Borrelien
ist verdächtig, nicht aber beweisend für eine aktive
Borrelien
-Infektion.
“
„Die
Indikationen für den LTT-
Borrelien
sind:
Slide35Lokale Farbreaktion
Bildung eines sog. Spots
Zugabe von enzymkonjugierten zytokinspezifischen Antikörpern
Zugabe eines Chromogens
als Enzymsubstrat
Zytokinproduktion
während der 24-stündigen Stimulation mit
Borrelien
-Antigen
Beschichtung der Membran mit
zytokinspezifischen
Antikörpern
Zugabe der Zellen
unstimuliert
stimuliert
Lyme
-Borreliose: Diagnostik
Indirekter (immunologischer) Nachweis einer
Borrelien
-Infektion
T-
Cellspot
Borrelien
Testverfahren: ELISPOT
Bei positivem Ergebnis aktive
Borrelien
-Infektion wahrscheinlich
Bestimmung der
Borrelien
-spezifischen IFN-
g
produzierenden
TH1-Effektorzellen nach Stimulation (24 h) mit
Borrelien-Lysat
bzw.
OspC
Slide36Lyme
-Borreliose: Diagnostik
Indirekter (immunologischer) Nachweis einer
Borrelien
-Infektion
T-
Cellspot
Borrelien
Slide37Lyme
-Borreliose: Diagnostik
Indirekter (immunologischer) Nachweis einer
Borrelien
-Infektion
T-
Cellspot
Borrelien
Slide38Lyme
-Borreliose: Diagnostik
Indirekter (immunologischer) Nachweis einer
Borrelien
-Infektion
T-
Cellspot
Borrelien
Slide39Lyme
-Borreliose: Diagnostik
Indirekter (immunologischer) Nachweis einer
Borrelien
-Infektion
T-
Cellspot
Borrelien
healthy
controls
Lyme
disease
non-
Lyme
patients
Western
Blot
Slide40Lyme
-Borreliose: Diagnostik
Indirekter (immunologischer) Nachweis einer
Borrelien
-Infektion
3HT-Memory Spot
Borrelien
versus T-
Cellspot
Borrelien
3HT-Memory-Spot
T-
Cellspot
Methode
ProliferationsmessungELISPOTNachweis
ZellteilungIFN-g-ProduktionTestdauer5 bis 6 Tage (Langzeitkultur)
24 Stunden (Kurzzeitkultur)Zelluläre ReaktionGedächtnis-T-Zellen
Effektor-T-ZellenAntigenspezifitätJaJaFrühestmögliche positive Reaktion
2 bis 4 Wochen nach Infektion10 bis 14 Tage nach InfektionUnterscheidung aktive vs. abgelaufene Infektion
Nein
Ja