Update - BCA

V1
Update: State of the Art-Borreliose
Diagnostik
Ärztliche Fortbildung
Augsburg, 27.2.2016
Dr. H.G.Maxeiner , BCA Laboratory, Augsburg, Germany
1
Themen
Grundlagen:
Diagnostik:
- Klinische Präsentation
- Borreliensystematik
- Epidemiologie
- Anzucht
- Molekulare Diagnostik
- Immunogene Proteine der Borrelien
- Pleomorphologie
- Serologie
- Zelluläre Teste Elispot
- Marker: CD57
- Co-infektionen
2
Lyme disease
The etiologic agent, Borrelia burgdorferi, was recovered first in 1982
from the vector tick Ixodes dammini (now I. scapularis) by
Burgdorfer et al.
(Science 216:1317–1319)
B. burgdorferi is a helically shaped bacterium with multiple
endoflagella. The cells, configured with 3 to 10 loose coils, are 10 to
30 um in length and 0.2 to 0.5 um in width. Lack of genes for
essential biochemical functions prompts the need for an intracellular
environment.
B. burgdorferi strain B31 spirochetes imaged
with Differential Interference Contrast microscopy
(DIC), Scale bar 20 µm.
Ixodes ricinus, JC Schou, Biopix.
3
Ausbreitung und Späte Lyme
Erregerverteilung innerhalb von Tagen bis Wochen:
Haut : Erythema migrans, Lymphozytom, Acrodermatitis,
Nervensystem : Bannwarths Syndrom,
Herz : Karditis (4-10%)
Gelenke : Arthritis, Mono oder Oligoartikulär)
Späte Lyme Erkrankung: Kann sich in einigen unbehandelten
Fällen nach Monaten und Jahren entwickeln (definiert:
nach mehr als 6 Monaten) Präsentiert sich als Athritis,
Neuroborreliose oder Akrodermatitis.
“Post treatment lyme syndrome” : In bis zu 10% der
Patienten können die Symptome für Monate bis Jahre
persitieren, obwohl mit Antibiotika behandelt wurde.
4
Neuroborreliose Liquor und Serodiagnostik
• Liquor : pleocytosis , 10–1000 leucos/mm3, AI-index, Q-Alb,
oligoclonal IgG bands
- Mikroskopie : Sehr niedrige Sensitivität und keine Spezifität.
• PCR: Sensitivität im Liquor in der frühen Phase 10% – 30%, später
sehr niedrig. Spezifität nahe 100% . Besonders indiziert bei
normalen AI oder Immundefizienz.
• Kultur: Liquorsensitivität : 10% – 30%,
50–70% in Hautbiopsaten bei EM, Blut: <10%
• Diagnostische Serologische Sensitivität im ELISA-Suchtest in der
Frühphase bis zu 70%,
Spätphase : Nur IgG bis zu 90%.
5
Diagnostic Neuroborreliosis : Antibody Index (AI)
The AI is recommended by the
European Federation of Neurological Societies (EFNS)
Zajkowska et al deployed specific antibodies produced
intrathecally, the Antibody Index (AI) for IgG and IgM in CSF versus
peripheral serum in LNB.
•26 out of 27 patients with LNB had pathologic AI-IgG.
•21/27 LNB patients had at least one positive (>1.5) IgG-AI against
either VlsE, p100, p58, p39, p18, or OspC.
And none of these patients showed positive OspA-IgG-AI.
Furthermore, NB patients showed dysfunction of the blood-CSF
barrier (QAlb). (average QAlb in NB and controls: 13.8 and 5.6).
Immunol Lett. 2015 Jul 22;168(1):58-6
6
Neuroborreliose chemokine CXCL13
• Several authors report the chemokine CxCL13 levels in the CSF as a new
diagnostic marker for LNB:
• Hytönen (2014) reported CXCL13 concentrations in CSF of untreated LNB
patients significantly higher as compared to Controls: non-LNB group viral
CNS infection samples or noninfectious neuroinflammatory conditions
• Schmidt (2011) assessed sensitivity of CXCL13 with 94.1%, (higher than the
AI 85.7%) and specificity of 96.1% (equal to the AI) (CNSlymphoma,bacterial meningitis)
• Senel (2010) confirmed CSF-CXCL13 significantly elevated in NBL(n=28) as
compared with other neurological diseases, systemic borreliosis (9),
Guillain-Barré syndrome (11), Bell's palsy (19), other cranial nerve palsies
(5), cephalgia (20), bacterial CNS infections (16) and viral CNS infections
(18).
• CXCL13 was fast responding to antibiotic therapy, decreasing within 1
week.
7
Themen
Grundlagen:
Diagnostik:
- Klinische Präsentation
- Borreliensystematik
- Epidemiologie
- Anzucht
- Molekulare Diagnostik
- Immunogene Proteine der Borrelien
- Pleomorphologie
- Serologie
- Zelluläre Teste Elispot
- Marker: CD57
- Co-infektionen
8
Borrelia Species
11 (20) Borrelia species within the B. burgdorferi sensu lato complex have been
described worldwide
• North America: 3 species : Borrelia andersonii, B. burgdorferi sensu stricto,
and Borrelia bissettii
• Europe: 5 species : B. burgdorferi sensu stricto, B. garinii, Borrelia afzelii,
Borrelia valaisiana, and Borrelia lusitaniae
• Asia: 7 species: B. garinii, B. afzelii, B. valaisiana, Borrelia japonica, Borrelia
tanukii, Borrelia turdi, and Borrelia sinica
B. miyamotoi
discovered in 1992 and now, since tested more frequently, found in Norway and
The Netherlands and worldwide.
(Bold : described as human pathogens)
9
B. bissettii
• In 3 out of 27 of Borrrelia PCR-positive Californien individuals
an infection with an organism closely related to B. bissettii was
found.
Girard, J clin microbiol 2011 mar;49(3):945-54
• A murine model for B. bissettii revealed an infection dynamics
and humoral response similar to B. burgdorferi.
Leydet et al. 2015
• From Europe, B. bissettii was reported from Slovenia in a
cardiac valve tissue (endocarditis) and aortic valve stenosis
from the Czech Republic.
J Clin Microbiol 46, 3540-3543 , FEMS Microbiol Lett 292, 274-281
10
Borrelia miyamotoi
Borrelia miyamotoi belongs to the relapsing fever group
B. miyamotoi is transmitted by the same vectors as Borrelia
burgdorferi s.l..
In Europe, Asia and North America, B. miyamotoi infection rates in
Ixodes persulcatus, Ixodes scapularis, Ixodes pacificus and Ixodes
ricinus are between 0.5% and 5% . In the Netherlands, in I. ricinus
is 2.4–4.7%. B. miyamotoi is present in wild rodents, indicating
enzootic circulation.
Recently described for northeastern US: 11,515 patients tested, 97
PCR positive , 24% of cases were hospitalized.
Molloy et al., Ann Intern Med.. 2015 Jul 21;163(2):91-8.
11
Themen
Grundlagen:
Diagnostik:
- Klinische Präsentation
- Borreliensystematik
- Epidemiologie
- Anzucht
- Molekulare Diagnostik
- Immunogene Proteine der Borrelien
- Pleomorphologie
- Serologie
- Zelluläre Teste Elispot
- Marker: CD57
- Co-infektionen
12
Epidemiologie Europa
• In Europa idt die geschätzte Inzidenz der Lyme Erkrankung
zwischen 0,6 und 155 pro 100 000 Einwohner.
• In Deutschland werden etwa 60 000–100 000 Fälle pro Jahr
an neuen Fällen geschätzt . Meldepflicht besteht in 9
Ländern, Ersteinführung 2002.
• Die TK schätzt die Zahl der gemeldeten Wanderröte Fälle auf
800 000 in 2009.
13
Lyme Epidemiology for the US
For the period 1992-1998: total 88,967 cases of Lyme disease
In 1992: 9,896 ; in 1998: 16,802
MMWR CDC Surveill Summ. 2000 Apr 28;49(3):1-11
Between 1992 and 2011: a total of 439,738 cases of Lyme
disease were reported by the CDC
Orloski et al., 2000; Centers for Disease Control and Prevention, 2012
For 2014 the CDC released: Total 33,461 (confirmed 25,359;
probable 8,102) highest: New England total 11,292
MMWR , September 18, 2015 / 64(36);1019-1033
Estimation by Kuehn: 300,000 US cases of Lyme annually
JAMA. 2013;310:1110
14
Incidences in Bavaria
Disease
N
%
EM
5860
98,8
Neuroborreliosis
104
1,7
Arthritis
155
2,5
Sum
6107
100
Übersicht über die Anzahl der Fälle nach Erkrankungsform und dem
jeweiligen Anteil an den Gesamtmeldungen (Meldezeitraum 1. April 2013
bis 31. März 2014). In: Epidemiologisches Bulletin 23. Februar 2 015 / Nr. 8
15
Seroprevalence of B.burgdorferi s.l. in Germany
Estimated seroprevalence of Borrelia burgdorferi sensu lato IgG among the male
and female population, Germany, 2008–2011.
Wilking H, Fingerle V, Klier C, Thamm M, Stark K. Antibodies against Borrelia burgdorferi sensu lato among adults, Germany, 2008–2011. Emerg Infect Dis. 2015 Jan
16
Themen
Grundlagen:
Diagnostik:
- Klinische Präsentation
- Borreliensystematik
- Epidemiologie
- Anzucht
- Molekulare Diagnostik
- Immunogene Proteine der Borrelien
- Pleomorphologie
- Serologie
- Zelluläre Teste Elispot
- Marker: CD57
- Co-infektionen
17
Cultivation of Borreliae from clinical samples
Site
Erythema migrans
US
>50%
Culture yield (%)
US max
Europe
86%
>40%
EU max
88%
Acrodermatitis chronica
atrophicans
Borrelia Lymphocytoma
-
-
>22%
60%
-
-
24%
-
CSF
-
-
10%
17%
>40%
-
1%
9%
Synovial fluid
Blood
Aguero-Rosenfeld et al, 2005, CLINICAL MICROBIOLOGY REVIEWS
18
Erregerkultivierung
• Blut:
Plasma ist effizienter als Serum als Vollblut
• Die Ausbeute korreliert positiv mit dem Volumen des
eingesetzten Materials
19
Themen
Grundlagen:
Diagnostik:
- Klinische Präsentation
- Borreliensystematik
- Epidemiologie
- Anzucht
- Molekulare Diagnostik
- Immunogene Proteine der Borrelien
- Pleomorphologie
- Serologie
- Zelluläre Teste Elispot
- Marker: CD57
- Co-infektionen
20
PCR: Sensitivities for detection of B. burgdorferi
Clinical specimen
region
EM
ACA
Blood,plasma,serum
US
Europe
Europe
US
Europe
CSF
US
Europe
Synovial fluid
US
Europe
Median % sensitivity range
69
64
73
76
14
18
10
38
73
23
78
36-88
59-67
36-88
54-100
0-100
0-59
4-100
12-100
25-93
12-100
42-100
83
66
76-100
42-85
First PCR
in1989
Aguero-Rosenfeld et
al, 2005, CLINICAL
MICROBIOLOGY
REVIEWS
21
Urine
• Hyde described in 1989 the detection of Borrelia antigen in titres up to
1:128 in human urine and in infected mice.
• Priem (1997) achived by PCR in
- lyme arthritis
sensitivities of 85% in synovial fluid (SF),
79% with urine samples, and 91% with paired SF-urine samples
- and in neuroborreliosis
79% CSF , 45% with urine samples, and 87% with
paired CSF-urine specimens from neuroborreliosis patients
• Douglas et al (2011) report an experimental assay for urine antigen
detection enhancing sensitivity by nanotechnology.(Biomaterials 2011 Feb;32(4))
22
Themen
Grundlagen:
Diagnostik:
- Klinische Präsentation
- Borreliensystematik
- Epidemiologie
- Anzucht
- Molekulare Diagnostik
- Immunogene Proteine der
Borrelien
- Pleomorphologie
- Serologie
- Zelluläre Teste Elispot
- Marker: CD57
23
Immunogenic proteins expressed by Borrelia I
• P 41 kDa flagellar protein flagellin or FlaB: Strong IgG and IgM responses to
this protein are developed within a few days after infection with B.
burgdorferi sensu lato , but highly cross-reactive with antigens in other
bacteria and mammalian tissues (neural tissues, synovium, and myocardium).
• P37 kDa flagellar outer sheath protein or FlaA, immunodominant in early
disease.
• OspC (21 to 25 kDa) immunodominant antigen, early after infection,
expressed during tick feeding, plasmid-encoded. Conserved epitopes within
the OspC led to a synthetic peptide containing the conserved C-terminal 10
amino acids of OspC , the pepC10.
At least 69 OspC groups described in the B burgdorferi sensu lato species.
OspC is used in assays to distinguish B.afzelii, B.burgdorferi and B.garinii in the
early stage by IgM antibodies
Function: bind host plasminogen and thereby support dissemination of the
24
Borrelia (Onder et al., 2012)
Immunogenic proteins expressed by Borrelia II
• 39-kDa protein BmpA, and with similar seize BmpB, BmpC, BmpD
Early phase marker, low sensitivity of 60-70%, but highly specific >95%
• DbpA, or Osp17, (17 kDa), Decorin binding protein A, DbpA (and a DbpB
peptide) detect both IgM and IgG, between 80 and 88% in
early and late Lyme disease patients. DbpA and B also contribue to arthritis and
persistence of B. burgdorferi as shown in a murine model.
• OspA (31 kDa) and OspB (34 kDa), low expression during early stage.
Antibody response was used in a recombinant OspA vaccine, which was
commercially available in the United States for use in humans until March 2002
25
Immunogenic proteins expressed by Borrelia III
• VlsE, Vmp-like sequence expressed protein, (34 to 35 kDa), linear plasmid
encoded. One invariable region (IR6) encodes a conserved 25-mer
oligopeptide , C6 peptide, within the variable portion of VlsE, is highly
immunogenic. Assays may use the VlsE to differentiate B.garinii, B.afzelii,
B.burgdorferi species by IgG serum antibodies.
Further proteins :
P35/BBK32 is a fibronectin binding protein, and highly specific
immunodominant
26
Autoimmun Diagnostika GmbH
Pleomorphic forms of Borrelia spp.
Ebingerstraße 4
D-72479 Straßberg
Tel.: +49 (0) 7434/9364-0
[email protected]
Blebs / Loops
Biofilms
Barbour & Hayes 1986
Kersten et al 1995
Kraiczy et al 2001
Round Bodies
Round Bodies
Broroson et al 2009
Speroplast
Muric et al 1996
CYSTs, L forms, CWD
Alban et al 2000
Al-Robaly et al 2010
Brorson & Brorson 1997, 1998a, 1998b, 1999, 2006
Gruntar et al 2001
Hulinski et al. 1994
Kersten et al 1995
MacDonald 1988
MacDonald 2006, 2013
Miklossy et al 2008
Mursic et al 1996
Murgia & Cinco 2004
Murgia, Piazzetta & Cibnco 2002
Oliverira et al 2010
Sapi et al 2011
Schaller & Neubert 1994
Stricker & Johnson 2011
Aberer et al 1996
Barbour and Hayes 1996
Burgdorferi et al 1989
Eisendle et al 2008
Kurtti et al 1987
MacDonald 2013
Miklossy et al 2008
Sapi & MacDonald 2008
Sapi et al 2012
Srivastava Sy, de Silva AM 2009
Copyright by Dr. Leona Gilbert
Themen
Grundlagen:
Diagnostik:
- Klinische Präsentation
- Borreliensystematik
- Epidemiologie
- Anzucht
- Molekulare Diagnostik
- Immunogene Proteine der Borrelien
- Pleomorphologie
- Serologie
- Zelluläre Teste Elispot
- Marker: CD57
- Co-infektionen
28
Serologie
Historisch wurden zuerst der indirekte immunofluorescens-antikörpertest (IFA)
entwickelt, mit dem Nachteil einer subjektiven Bewertung.
Deweiteren wurde ein Antigentest mit Zellmembranbindung entwickelt, mit der
Möglichkeit einer automatisierten Auswertung (FIAX).
Diese Systeme wurden durch das EIA System ersetzt, z.B. dem “enzyme-linked
immunosorbent assay” , dem ELISA
-- Als Antigene werden ganze Organsimen eingesetzt , z.B. von B. burgdorferi zur
Suche von
IgG, IgM Antikörpern
-- Oder auch einzelne, recombinante synthetische Peptide aus der VlsE Sequenz
IR6 oder C6 peptide
Western Blots (WB) oder immunochromographische Verfahren oder dot blot
Systeme
29
Sensitivität der serologischen Methoden
Sensitivität:
• Am allgemeinen weniger als 60% (40-60%) in der akutenPhase
bei Patienten mit EM innerhalb der ersten Woche.
• Anstieg der Sensitivität bei unbehandelten Patienten. Höher bei
Patienten mit multiplen EMs und mit Symptomen.
Organbefall (ausserhalb der Haut), Karditis, Neuroborreliose oder
ein spätes Stadien (z.B. Arthritis) führt zum Anstieg der
serologischen Sensitivität.
30
Improving early serodiagnosis by recombinant proteins
Signorino et al performed epitope mapping of linear epitopes in
oligopeptide permease A2 (OppA2) expressed during early
infection. Two of the peptides were identified as new candidates
with sufficient specificity and sensitivity.
Clinical and Vaccine Immunology,p.704–711,2014, Vol 21,No 5
To detected seroreactivity in EM,early carditis or neuroborreliosis
IgM immunoblotting of the recombinat proteins BBA65, BBA70
and BBA73 was performed with similar sensitivity as to the OspC.
For IgG the proteins BBA69 and BBA73 reacted in the early
phase.
Several samples were scored negative according the CDC
algorithm.
Weiner et al .Clin Vaccine Immunol. 2015 Sep 16
31
IgM Antikörpernachweis während einer Infektion
IgM Antikörper:
Während früher Lyme-Borreliosis, sind IgM Antikörper
gerichted gegen:
- OspC
- p41 kDa flagellar antigens, FlaB
- FlaA (37 kDa)
- VlsE in 77%-89%
Variable Quantitäten von IgM
Gegen BmpA (39 kDa)
32
IgG antibody detection during infection
Während der fühen Phase der Lyme-Borreliosis sind IgG Antikörper
gerichted gegen:
- OspC
- Flagellin (41 kDa).
- BmpA (39 kDa) in 35%- 85% während EM, und in 33% bis 64% in
convaleszenten Seren.
- VlsE in 65%-88%
33
CDC criteria for Positive of IgM serology
United States (IgM CDC citeria)
based on a study of EM patients using B burgdorferi sensu
stricto strain 297 (for IgG isolat G39/40). According to
these criteria, a positive IgM blot is defined by the
presence of
At least two out of three particular immunoreactive bands
OspC (23-25kDa)
41 kDa
39 kDa
34
US Criteria for positive IgG serology
United States (IgG CDC citeria)
The IgG criteria are based B. burgdorferi sensu stricto isolate G39/40 and
sera from patients with various manifestations of LB.
The criteria require the presence of at least 5 of 10 particular bands
83-93, 66, 58, 45, 41, 39, 30, 28, 23-25(OspC), or 18 kDa
However: IGeneX, US based specialized Lyme disease Laboratory, uses in
house criteria in addition to the CDC criteria:
IgG or IgM are positive if 2 or more bands out of 5 are present:
23-25(OspC), 31, 34, 41, 83-89 kDa
But likely to change: 2 out of 6 bands: 23, 31,34, 39, 41 and 93kDa;
indeterminate if only bands 31 and 41kDa or 31 and 93kDa are present;
IgM WB is considered negative if only bands 41 and 93kDa are present
35
Europe: Criteria for positive serology I
Hauser (1999) proposed to differentiated B. afzelii from B.
garinii by immunoreactivity in the WB :
WB IgG, at least two bands among
p83/100, p58, p43, p39, p30, OspC, p21, p17, and p14 for
B.afzelii and at least one band among p83/100, p39, p30,
OspC, p21, and p17b for B. garinii.
WB IgM, at least one band among p39, OspC, and p17 or a
strong p41 band for B. afzelii and at least one band among
p39 and OspC or a strong p41 band for B. garinii.
36
Europe: Criteria II
Schulte-Spechtel showed 2003 adding VlsE
increased sensitivity about 20% (60%-89%)
Interpretation of WB results depend on proteins displayed and
quantity. Example:
Bands: VlsE, p18,p19,p20,p21, OpC(p25) , p39, p58, p83,
Lipid Bb, Lipid Ba
If IgG VlsE strong positive, this is sufficient for fulfilling positivity
If VlsE weekly is positive at least one more band has to react.
If VlsE is negative at least 2 other bands must be reactive
37
Antigens in commercially available kits IgG
Antigen
Prevalence IgG
Specificity IgG
VlsE
67%
96 % / 98% / 96%
VlsE Ba
66%
99%
VlsE Bb
89%
99%
VlsE Bg
68%
95%
Lipid Ba
25%
100%
Lipid Bb
25%
100%
p83
54%
95%
p39
61%
99%
OspC
49%
96%
p58 (BB_A34)
21%
98%
p21 (BB_K53)
9%
99%
p20 (BB_Q03)
7%
100%
p19 (BB_N38)
9%
99%
p18 (BB_P38)
22%
99%
Data provided by the manufacturer and literature
38
2 tier
increased
sensitivity
between 38%
for EM acute
phase to 97%
for arthritis.
39
Laboratory comparison
Fallon et al (2014) compared 4 US laboratories on serum samples from 37 patients with posttreatment Lyme
syndrome (40 medically healthy controls). (Clinical Infectious Diseases® 2014;59(12):1705–10)
Combining IgG and IgM results will result in higher sensitivity (Lab B).
Criteria
?/+ ELISA
C6 ELISA
WB IgM (CDC)
WB IgM (laboratory)
WB IgG (CDC)
WB IgG (laboratory)
2-tier: ?/+ ELISA & WB IgG
University Reference Lab Commercial Laboratory Specialty Laboratory A Specialty Laboratory B
CDC
CDC
23 (62.2)
25 (67.6)
...
8 (21.6)
...
21 (56.8)
...
18 (48.6)
...
6 (16.2)
...
16 (43.2)
...
15 (40.5)
In house
In house
25 (67.6)
25 (67.6)
25 (67.6)
23 (62.2)
1 (2.7)
16 (43.2)
1 (2.7)
23 (62.2)
16 (43.2)
18 (48.6)
14 (37.8)
26 (70.3)
14 (37.8)
16 (43.2)
2-tier: C6 ELISA & WB IgG
.
.
15 (40.5)
17 (45.9)
2-tier: ?/+ ELISA & C6 ELISA
...
...
22 (59.5)
40
18 (48.6)
Serology as treatment control
• Marques (2004) demonstrated C6 antibodies at baseline
and at month 6 months ( 3 month following treatment) C6
antibody cannot be used to assess treatment outcome or
the presence of an active infection in patients with
persistent lyme disease.
• Glatz (2008) found no correlation between Immuno Blot
results and treatment-related parameters in patients
treated for Erythema Migrans.
• However: VlsE quantified may emerge as a marker
41
US - test assays performance on European sera
• Branda et al (2013) using US assays for European sera,
sensitivity and specificity was 52% and 100% resp. as
compared to 81% of European laboratory test results.
• The sensitivity of a US C6 ELISA was 88% and comparable
to the conventional 2-tiered testing using European tests
(100% specific).
42
Serology C6 peptide
• 131 sera from Russian patients with erythema
migrans (EM) investigated by C6 peptide based
multiplex for B. garinii or B. afzelii :
• IgM 32.8 % positive
• IgG 72.5% positive
• No increase of sensitivity if additional testing for
OspC- and VlsE-IgM or IgG
Pomelova et al, PLoS One. 2015 Jul 6;10(7)
43
Themen
Grundlagen:
Diagnostik:
- Klinische Präsentation
- Borreliensystematik
- Epidemiologie
- Anzucht
- Molekulare Diagnostik
- Immunogene Proteine der Borrelien
- Pleomorphologie
- Serologie
- Zelluläre Teste Elispot
- Marker: CD57
- Co-infektionen
44
IFN-gamma
The cytokine interferon – gamma is a signal protein in the innate
and adaptive immune system
Function: High levels of IFN-g are expressed by TH1 cells to
- activate macrophages, promoting cytotoxic activities of cells, and
inducing apoptosis of epithelial cells in the skin and mucosa.
- role in the development of a TH1 response and the B-cell isotype
switching to IgG2a
-IFN-g regulates MHC class I and II protein expression and antigen
presentation
-IFN-g inhibits cell growth and apoptosis yet controls the extension
of the immune response by inducing cell death of CD4 T cells
45
IL-2
Produced by CD4 and CD8 T-cells, also by activated
dentritic cells (DC) and Natural Killer Cells (NK) and NK T
(NKT) cells.
Function: IL-2 is essential for the development of T-reg cells.
IL-2 is a B-cell growth factor, stimulates antibody synthesis,
and promotes proliferation and differentiation of NK cells
to increase their cytolytic functions.
Therapeutic use: Recombinant human IL-2 is used in
immunotherapy for cancer and AIDS. Anti–IL-2Ra inhibits
the immune response in patients with autoimmune
diseases and prevents rejection of transplanted organs.
46
Disease activity by cytokine production
Levels of disease activity and viral replication correlate with distinct
functional signatures of T-cell responses assayed by e.g. IFN / IL-2
Giuseppe Pantaleo and Alexandre Harari
47 NATURE
REVIEWS | IMMUNOLOGY, vol 6 417-23, 2006
Autoimmun Diagnostika GmbH
Ebingerstraße 4
D-72479 Straßberg
Tel.: +49 (0) 7434/9364-0
[email protected]
Cellular test in Mycobacteria Tuberculosis diagnostics
Q fever and Interferon-gamma assay
• Schoffelen (2013) tested 1525 individuals from an endemic area with
a risk for chronic Q fever. IFN-γ production was measured after in
vitro stimulation of whole blood with C. burnetii antigens
• In all assay C. burnetii–specific IFN-γ production was higher in
seropositive or Skin Test (ST) positives than in sero-or ST-negatives
Values: IFN-γ 449 ± 82 pg/mL in the positives (n = 219) ;
21 ± 3 pg/mL in negatives (n = 908)
• Sensitivity of 87.0% and specificity of 90.2% were similar to the
combination of serology and Skin Test (83.0% and 95.6%, resp.)
49
IL-2 / Interferon- gamma cellular test
IL2-value
INF-g value
Possible therapeutic implications
low
high
Treatment with specific antibiotic
protocols to consider
Clinical symptom may be considered
before treatment decision (start or
stop)
high
low
Clinical symptoms may be considered
and monitoring of immune answer
50
Themen
Grundlagen:
Diagnostik:
- Klinische Präsentation
- Borreliensystematik
- Epidemiologie
- Anzucht
- Molekulare Diagnostik
- Immunogene Proteine der Borrelien
- Pleomorphologie
- Serologie
- Zelluläre Teste Elispot
- Marker: CD57
- Co-infektionen
51
CD 57 + NK Zellen
Stricker (2001) berichtet über 73 chronische, seropositive Lyme (LD)
Patienten
(53 Bewegungsapparaterkrankungen, 20 neurologische, Krankheitsdauer
3 Monate bis 15 Jahre)
Kontrolle: 10 Patienten mit akuter Lyme Erkrankung (unter einem Monat
alte Infektion ) und 22 AIDS Patienten
Ergebnisse: 31 chronische LD Patienten, die vor antibiotischer Therapie
getested wurden, hatten signifikant erniedrigte CD57 Zellzahlen (Mittel
30 +/-16 Cells/ul; normal: 60-360 Zellen/ul).
Zusätzlich hatten 19 von 37 Patienten (51%), die erst nach der Initiierung
einer antibiotisch Therapie getested wurden eine erniedrigte CD57
Zellzahl (Mittel 66 +/-39 cells/ul)
Alle 5 Patienten, die nach der Beendigung einer antibiotischen Therapie
getested wurden, hatten normale CD57 Zellzahlen(Mittel, 173+/-98
cells/ul)
52
Disease
Pathogen
H.Granolucytic
Aanaplasmosis
Co – infections, Tick borne
Transmission
Reservoir
Anaplasma
phagocytophila
Ticks Ixodes ricinus (Europe) Ixodes scapularis
(USA)
White- footed
mouse
H.Granulocytic
Ehrlichiosis
Ehrlichia
chaffeensis
Ticks Ixodes ricinus (Europe) Ixodes scapularis
(USA)
Red deer, human
Bartonellosis
Bartonella
henselae,
Bartonella
quintana, B.
bacilliformis
Bite or scratch wounds of dog or cat ; cat fleas;
lice (B. quintana) , tick bite .Others: dust mites,
flea bites, flea feces (oral infection), contact
with cats or dogs (paws, saliva, lice , flies,
gadflies), blood trans., mother-child.
domestic and wild
animals
Rickettsiosis
Mediterranian
spotted fever
Rickettsia
helvetica,
Rickettsia conorii
Ticks, mites, fleas, lice
Ticks
Tularemia
Francisella
tularensis
Mosquitos, gadflies, fleas, lice, mites , oral,
inhalation
vertebrates
Q fever
Coxiella burnetii
Oral or inhalation
Cattle, milk, human
Babesiosis
Babesia microti,
Ixodes ricinus (Europe), Ixodes scapularis (USA)
Babesia divergens , blood transfusions , perinatal
Cattle (other verte53
brates)
Associated infections, non-tick transmitted
Disease
Pathogen
Transmission
Reservoir
Mycoplasma infections
primary atypical pneumoniae
Mycoplasma
pneumoniae
Respiratory, small and large
seize droplets, humans
human
Chlamydophila pneumoniae infection
Community aquired pneumoniae
Chlamydophila
pneumoniae
Droplet infection, by person to human
person
Chlamydia trachomatis infection
Chlamydia
Lymphogranuloma venerum, respiratory, trachomatis
also ophtalmia neonatorum, pelvic
inflammation, urethritis, epididymitis
STD
human
Yersinosis
Yersinia
Mesenteric lymphadenitis,
enterocolitica
gastroenteritis , diarrhea, liver abscesses Yersinia
pseudotuberculosis
Fecal oral
vertebrates
Campylobacter jejuni
Infection
gastroenteritis
Fecal-oral
verteGame and domestic animals, brates
particularly poultry, animal
54
products, contaminated water
Campylobacter
jejuni
Zusammenfassung:
• Die Anzahl der pathogenen Borrelia Spezies und die weltweite Verteilung
nimmt zu. Und stellt eine besondere Herausforderung an
Antikörpernachweise da . Der auf dem recombinantem C6 Protein
basierende serologische Nachweis ist derzeit in der Erprobungsphase.
• Die Kriterien für die Bewertung der Western-Blot Banden ist derzeit in
Überarbeitung, insbesondere gilt dies für die USA-CDC Criteria.
• Das nach der Behandlung beschriebene „ post treatment lyme syndrom“
ist nach wie vor eine diagnostische Herausforderung , neue Formen der
Borrelien (round bodies, biofilm) bieten neue Erklärungsansätze.
• Die Quantifizierung der zellulären Immunantwort bietet ein weiteres
diagnostisches Testsystem das zusätzlich zur Therapiekontrolle
eingesetzt werden kann.
• Weitere Immunparameter wie die phänotypisierung
immunkompetenter Zellen, Cytokinspiegelbestimmungen oder
metabolische Biosignaturen sind Wege zu neuen diagnostischen und
Therapiekontrollmöglichkeiten.
55
a dedicated diagnostic may help
56
Vielen
Dank
57
Contact:
Dr. med. Horst-G. Maxeiner
FA für Mikrobiologie und Infektionsepidemiologie
Consultant for clinical microbiology and infection control
BCA-Laboratory
Morellstraße 33
86159 Augsburg
Tel. +49 (0) 821 - 45 5 471 23
Fax +49 (0) 821 - 45 5 471 70
www.infectolab.de
e-mail: [email protected]
58
Additional slides if needed
59
Laboratory comparison II
Table 2. Number and Percentage of False-Positive Serologic Test Results and Discordant Pairs for 40 Medically Healthy Controls (University Reference Laboratory Versus Commercial and
Lyme Specialty Laboratories)
(Fallon et al, Clinical Infectious Diseases® 2014;59(12):1705–10)
University Reference
Commercial
Laboratory
Laboratory
No. Positivea
No. Positivea
P
(%)
Value
(%)
?/+ ELISA
5 (12.5)
C6 ELISA
WB IgM (CDC)
3 (7.5)
..
5 (12.5)
...
WB IgG (CDC)
1 (2.5)
WB IgG (laboratory)
0
+WB IgM or IgG (laboratory)
0
...
0
.
2-tier: ?/+ ELISA & C6 ELISA
..
.
...
5 (12.5)
..
...
...
2-tier: C6 ELISA & WB IgG
+ WB IgM or IgG (CDC)
.
0
WB IgM (laboratory)
2-tier: ?/+ ELISA & WB IgG
.
..
...
0
Disc
Pairs
Specialty Laboratory A
No. Positivea
P
Value
(%)
.683
6
1
.. .
. ..
0
.074
5
1
...
...
1
1.00
1
...
(2.5)
Disc
Pairs
Specialty
Laboratory B
No. Positivea
P
Value
(%)
.125
4
3 (7.5)
.. .
.. .
0
(2.5)
.125
4
(2.5)
.125b
0
...
. ..
Disc Pairs
.683
6
...
...
8 (20.0)
.505
9
4
15 (37.5)
.024
16b
1.00
1
3 (7.5)
.480
2
0
1.00b
1
11 (27.5)
.004
10b
0
0
. ..
0
1 (2.5)
1.000
1
. ..
...
0
...
...
0
...
...
...
...
0
...
...
0
...
...
.074
5
1
(2.5)
.133
4
10 (25.0)
.182
9
1
(2.5)
.133
4
23 (57.5)
<.001
22
Abbreviations: ?/+, indeterminate/positive; CDC, Centers for Disease Control and Prevention; Disc pairs, discordant pairs; ELISA,
enzyme-linked immunosorbent assay; IgG, immunoglobulin G; IgM, immunoglobulin M; WB, Western blot. a Criteria for a positive test are given in Table
1.
60
b Results using in-house criteria at Specialty Laboratories A and B were compared with results using CDC criteria at the university-based reference laboratory
Antigens in commercially avilable kits IgM
Antigen
Sensitivity IgM
Specificity IgM
VlsE
5%
p39
16%
99% / 95% /
93%
99%
OspC
77%-88%
99%
61
Topics:
• Clinical presentation
Neuroborreliosis
• Systematics, species
• Epidemiology
Transmission
• Diagnostic methods:
Cultivation
Molecular
Immunogen proteins expressed by Borrelia
Pleomorphology
Serology
Cellular test
Immunologic marker
• Tick borne co-infections
Elispot
CD57
62
Clinic: early infection
localized erythema migrans (EM), with or without headache, myalgia, arthralgias,
or fever.
Erythrema migrans skin
lesion. Steere A.C. 2001.
63
Dissemination
May be followed within days or weeks:
- Skin : disseminated infection affecting skin with multiple EMs,
lymphocytoma, acrodermatitis chronica atrophicans(ACA)
- Nervous system: facial palsy, meningitis, and
meningoradiculoneuritis (Bannwarth’s syndrome)
- Heart : Lyme carditis is seen in 4% to 10% with
atrioventricular conduction defects, myopericarditis)
- Joints : Lyme arthritis, monoarticular or oligoarticular,
typically involves the knee, more frequent in North American
patients
64
Late stage clinical presentation
• Late Lyme Borreliosis (LB) may develop among some
untreated patients months to a few years after ticktransmitted infection (defined > 6 month)
• The major manifestations of late LB include arthritis,
late neuroborreliosis (peripheral neuropathy or
encephalomyelitis), and Akrodermatitis chronica
atrophicans
• In up to 10% of patients, arthritis may persist for months
or years despite treatment with antimicrobials
65
Neuroborreliose (LNB) Signs and Symptoms
• Neurological symptoms usually within 1–12 weeks (2-6 weeks) after
tick bite
• Peak July to December
• But only 40–50% of the patients remember a tick bite
• Stage I :20–30% report an erythema migrans (EM )
• Stage II : 95% are classified as early LNB
signs and symptoms lasting for less 6 months
• Stage III : 5% have late LNB with duration between 6 months and
several years
• early LNB is often self-limiting; But late LNB has a chronic course
66
Neuroborreliose serology
• Borreliose-specific antibodies in serum and CSF
• two-step approach: where sera that are positive in the ELISA screening
assay are subjected to immunoblot (IB) for confirmation
• Diagnostic serologic sensitivity of
• ELISA screening assays in early LNB is 70–90%, and
• for late LNB (only IgG, as IgM is not diagnostic for
late disease) it is >90–100%
67
Neuroborreliose (LNB) diagnostic by EFNS
1) neurological symptoms
2) cerebrospinal fluid (CSF) pleocytosis
3) specific antibodies produced intrathecally , Antibody Index (AI)
Facultative:
PCR and CSF culture may be corroborative if symptom duration is <6 weeks,
when Bb antibodies may be absent. PCR is otherwise not recommended.
Not recommend:
microscope-based assays, chemokine CXCL13, antigen detection, immune
complexes, lymphocyte transformation test, cyst formation, lymphocyte markers.
According European Federation of Neurological Societies (EFNS) guidelines 2010
68
Neuroborreliose chemokines
• Narayan (2005) concluded form PBMC stimulation experiments that
persistent production of CXCL13 and IgG are features of LNB.
• The chemokine CXCL13 triggers homing and motility of B cells in
lymphoid tissue. Rupprecht et al showed that CXCL13 levels are
increased in acute LNB in the B cell-rich CSF and that CXCL13 is
released by monocytes upon recognition of Borrelia outer surface
proteins by Toll-like receptor 2.
• Hytönen (2014) reported CXCL13 concentrations in CSF of
untreated LNB patients significantly higher (median, 6,480 pg/ml)
than non-LNB group (<7.8 pg/ml), viral CNS infection samples (<7.8
pg/ml), or samples from patients with noninfectious
neuroinflammatory conditions (<7.8 pg/ml).
69
Neuroborreliose chemokine CXCL13
• Schmidt (2011) assessed sensitivity of CXCL13 with 94.1%, this is higher than
the AI (85.7%) and specificity of 96.1% (equal to the AI) (CNSlymphoma,bacterial meningitis)
• Tjernberg (2011) sensitivity of 99% and a specificity of 96% were achieved for
CSF-Serum CXCL13 ratio. CSF-C6 antibodies performed with a sensitivity of 99%
and a specificity of 88.0%. A combination of CSF-Serum CXCL13 ratio and CSFC6 antibodies, evaluated in parallel, revealed a sensitivity of 99% and specificity
of 98%.
• Senel (2010) confirmed CSF-CXCL13 significantly elevated in NBL(n=28)
compared with other neurological diseases (n), systemic borreliosis (9),
Guillain-Barré syndrome (11), Bell's palsy (19), other cranial nerve palsies (5),
cephalgia (20), bacterial CNS infections (16) and viral CNS infections (18).
Sensitivity was 96.4% and specificity 96.9%. CSF CXCL13 was also fast
responding to antibiotic therapy, decreasing within 1 week.
70
Lyme disease
Lack of genes that encode enzymes required for synthesis of amino
acids, fatty acids, enzyme cofactors, and nucleotides. Also lacks genes
coding for tricarboxylic acid cycle enzymes or for compounds involved
in electron transport.
Cell envelop
-No lipopolysaccharides
-pseudo gram negative
-flagella intercellular
om
*
°
p
im
* flagella
° periplamic space
p protoplasmic cylinder
om outer membrane
im inner membrane
Radolf et al 2010
71
Comparison of diagnostic tests for 47 adult patients with
erythema migrans.
72
© 2001 by the Infectious Diseases Society of America
John Nowakowski et al. Clin Infect Dis. 2001;33:2023-2027
Clinical characteristics of 47 adult patients with erythema migrans.
© 2001 by the Infectious Diseases Society of America
John Nowakowski et al. Clin Infect Dis. 2001;33:2023-2027
73
Epidemiology
• US: 1982 to 2005 more than 200,000 LB cases in the United States
have been reported to the CDC, with about 17,000 cases reported
yearly between 1998 and 2001.
• In Europe, the incidence of Lyme borreliosis is estimated to range
from 0.6 to 155/100,000 .
• In Germany, 60,000–100,000 incident cases per year are estimated .
Mandatory reporting of new LB cases was established in 2002, now
including 9 federal states.
TK: 798.000 Neuerkrankungen in 2009, gemeldete „Wanderröten“
74
Incidences in Bavaria
Disease
N
%
EM
5860
98,8
Neuroborreliosis
104
1,7
Arthritis
155
2,5
Sum
6107
100
Übersicht über die Anzahl der Fälle nach Erkrankungsform und dem
jeweiligen Anteil an den Gesamtmeldungen (Meldezeitraum 1. April 2013
bis 31. März 2014). In: Epidemiologisches Bulletin 23. Februar 2 015 / Nr. 8
75
Seroprevalence of B.burgdorferi s.l. in Germany
Estimated seroprevalence of Borrelia burgdorferi sensu lato IgG among the male
and female population, Germany, 2008–2011.
Wilking H, Fingerle V, Klier C, Thamm M, Stark K. Antibodies against Borrelia burgdorferi sensu lato among adults, Germany, 2008–2011. Emerg Infect Dis. 2015 Jan
76
life cycle
Larvae
feeds on 1st HOST
Eggs into
Borrelia-free larvae
Larvae drops and
moults to nymph
Infected with Borrelia
Ixodes spp. ticks have a three-stage life
cycle — larva, nymph and adult — with
one blood meal per stage.
Nymph
Nymph feeds on 2nd HOST
Infected with Borrelia
Female drops and
lays eggs
Infection is acquired by feeding on an
infected reservoir animal, and the
bacterium is retained during the
subsequent stages after each blood meal
and moult.
Expanding Vectors such as mosquitoes,
blood-sucking flies, etc.
Adult
feeds on 3rd HOST
Nymph drops and
moults to adult
Dead-end hosts
Provided by Dr. Leona Gilbert
77
78
Antigen presentation by DCs
Antigen presentation by DCs to naive T cells and other factors (innate immune response substances, vitamins, cytokines in the
environment) induces the T cells to produce ILs and differentiate into TH1, TH2, TH9, TH17, TH22, or follicular TH (TFH) cells.
These T-cell subsets can promote different types of inflammatory responses on the basis of their respective cytokine profiles,
responses to chemokines, and interactions with other cells.
AKDIS ET AL, J Allergy Clin Immunol 2011;127:701-21
79
Gene expression
• Depending on the cycle:
In ticks OspA but not OspC is expressed when residing in the midguts of
unfed ticks. However, during a blood meal by the tick, OspA is
swithched to OspC.
Certain B. burgdorferi sensu lato genes either are expressed only in a
mammalian host or have significantly upregulated expression in that
environment; such gene products include VlsE (71, 230), DbpA (71,
129), BBK32 (96), Erp (202), and Mlp (376) proteins.
80
CD3-/CD57+ T-Lymphocytes
1. Subpopulation of the NK cells
2. Reduction indicates chronic activity of Lyme disease (symptoms > 1 year)
3. Reduction in untreated and inadequately treated Lyme disease
4. After the therapy end of chronic Lyme disease: normalization as an expression of therapeutical success
CD3-/CD57+ T-Lymphocytes
Reference range (mean/range)
Lyme patient:
46 /ul / 8 – 160 /ul
Healthy:
164 /ul / 60 – 354 /ul
81
Two tier testing
Current practise ins lyme serology is the 2 tier testing,
deploying first a screening test (e.g.ELISA method) and in
case of a positiv or borderline result a confirmatory test
such as Western Blot.
2 tier increased sensitivity between 38% for EM acute phase
to 97% for arthritis.
82
Kit based systems
Various manufacturers offer test systems for example :
• Borrelia burgdorferi IDEIA (Oxoid) serum assay is based on purified native flagella
antigen from a cultured strain of Borrelia afzelii (strain DK1)
• The Liaison Borrelia IgM Quant and the Borrelia IgG assays(Diasorin) are based on
recombinant OspC and VlsE antigens in the IgM assay and on VlsE antigens in the
IgG assay and is on a automated instrument (Liaison). Antigens are coated on
magnetic beads and readout by chemiluminescence.
• Euroimmun offers ELISA screening systems and Western Blot with a
semiautomatic, quantifying read out.
• And many more manufacturers share the market
83
Antigens in commercially available kits IgG
Antigen
Prevalence IgG
Specificity IgG
VlsE
67%
96 % / 98% / 96%
VlsE Ba
66%
99%
VlsE Bb
89%
99%
VlsE Bg
68%
95%
Lipid Ba
25%
100%
Lipid Bb
25%
100%
p83
54%
95%
p39
61%
99%
OspC
49%
96%
p58 (BB_A34)
21%
98%
p21 (BB_K53)
9%
99%
p20 (BB_Q03)
7%
100%
p19 (BB_N38)
9%
99%
p18 (BB_P38)
22%
99%
Data provided by the manufacturer and literature
84
Cellular test in Mycobacteria Tuberculosis diagnostics
85
Serology and T cell assay
Giuseppe Pantaleo and Alexandre Harari
NATURE REVIEWS | IMMUNOLOGY, vol 6
417-23, 2006
86
IFN-gamma
Cells:
-innate immune system: NK cells, NKT cells, macrophages, myelomonocytic cells, DCs,
epithelial cells
-adaptive immune systems: TH1 cells, cytotoxic T lymphocytes (CTL), B cells
Receptor: A single IFN-g molecule interacts with 2 ligand-binding IFNGR1 (or IFNGR a)
chains and 2 signal-transducing IFNGR2 (or IFNGR b) chains as members of the class II
cytokine receptor family.
Function: High levels of IFN-g are expressed by TH1 cells to
- activate macrophages, promoting cytotoxic activities of cells, and inducing apoptosis of
epithelial cells in the skin and mucosa.
- role in the development of a TH1 response and the B-cell isotype switching to IgG2a
-IFN-g regulates MHC class I and II protein expression and antigen presentation
-IFN-g also inhibits cell growth and apoptosis yet controls the extension of the immune
87
response by inducing activation-induced cell death of CD4 T cells
IL-2
History: IL-2, discovered about 30 years ago in supernatants of activated T cells
Cells: mainly produced by CD4 and CD8 T-cells, to a lesser extent by activated
dentritic cells (DC) and Natural Killer Cells (NK) and NK T (NKT) cells.
Receptor: 3 subunits: the ligand-specific a chain IL-2Ra (CD25), the b-chain IL2Rb (CD122) (also part of the IL-15R complex), and the common gc . All 3
subunits are required for the high-affinity IL-2R. IL-2Ra is rapidly induced and
participates in formation of a high-affinity quaternary complex, which activates
multiple signal transduction pathways.
Function: IL-2 is essential for the development of T-reg cells. IL-2 is as a B-cell
growth factor, stimulates antibody synthesis, and promotes proliferation and
differentiation of NK cells to increase their cytolytic functions.
Therapy use: Recombinant human IL-2 is used in immunotherapy for cancer and
AIDS. Anti–IL-2Ra inhibits the immune response in patients with autoimmune
88
diseases and prevents rejection of transplanted organs.
Example Borrelia antigens in a commercially available Borrelia Elispot
LTT
• Borrelia burgdorferi Fully Antigen: Borrelia burgdorferi B31-reference
strain (Borrelia burgdorferi sensu stricto)
• Borrelia burgorferi Peptide-Mix: OspA from Borrelia b. s.s, Borrelia afzelii,
Borrelia garinii , OspC native , DbpA recombinant
• Borrelia burgdorferi LFA-1 (Lymphocyte Function Antigen 1):
Borrelia burgdorferi sensu stricto (shared epitope with human cross
reacting antigen)
LFA-1 is Autoimmune diseases associated: collagenosis, Rheumatoid
Arthritis, vasculitis (ANA, CCP-antibodies, ANCA)
Explanation: Native = cultured antigens/ Recombinant: genetic technology produced
89
Disease activity by cytokine production
Levels of disease
activity and viral
replication
correlate with
distinct
functional
signatures
of T-cell
responses
assayed by e.g.
IFN / IL-2
Giuseppe Pantaleo and Alexandre
Harari NATURE REVIEWS |
IMMUNOLOGY, vol 6 417-23, 2006
90
Lyme Epidemiology for the US
For the period 1992-1998:
total 88,967 cases of Lyme disease. (In 1992 : 9,896 ; in 1998 : 16,802)
MMWR CDC Surveill Summ. 2000 Apr 28;49(3):1-11
Between 1992 and 2011, a total of 439,738 cases of Lyme disease were reported by the CDC
Orloski et al., 2000; Centers for Disease Control and Prevention, 2012
In 2014 the CDC released:
Total 33,461 (confirmed 25,359 ; probable 8,102)
highest: New England total 11,292
MMWR , September 18, 2015 / 64(36);1019-1033
Estimation by Kuehn : 300,000 US cases of Lyme disease annually
JAMA. 2013;310:1110
91
B. bissettii
• In 3 out of 27 of Borrrelia PCR-positive Californien individuals
an infection with an organism closely related to B. bissettii was
found.
Girard, J clin microbiol 2011 mar;49(3):945-54
• A murine model for B. bissettii revealed an infection dynamics
and humoral response similar to B. burgdorferi.
Leydet et al. 2015
• From Europe, B. bissettii was reported from Slovenia in a
cardiac valve tissue (endocarditis) and aortic valve stenosis
from the Czech Republic.
J Clin Microbiol 46, 3540-3543 , FEMS Microbiol Lett 292, 274-281
92
Borrelia miyamotoi
Borrelia miyamotoi belongs to the relapsing fever group of the
Borrelia.
B. miyamotoi is transmitted by the same vectors as Borrelia
burgdorferi s.l..
In Europe, Asia and North America, B. miyamotoi infection rates in
Ixodes persulcatus, Ixodes scapularis, Ixodes pacificus and Ixodes
ricinus are between 0.5% and 5% . In the Netherlands, the
infection rate of I. ricinus is 2.4–4.7%. B. miyamotoi iis present in
wild rodents, indicating enzootic circulation.
Recently described for northeastern US: 11,515 patients tested, 97
PCR positive , 24% of cases were hospitalized.
Molloy et al., Ann Intern Med.. 2015 Jul 21;163(2):91-8.
93
Borrelia miyamotoi in the northeastern US
• 11,515 patients tested, 97 PCR positive
• 57 cases : high fever, chills, marked headache,
myalgia, arthralgia. 24% hospitalized. Elevated
liver enzymes, neutropenia, thrombocytopenia.
Molloy et al., Ann Intern Med.. 2015 Jul 21;163(2):91-8.
94
Serology C6 peptide
• 131 sera from Russian patients with erythema migrans (EM)
investigated by C6 peptide based multiplex for B. garinii or B. afzelii :
• IgM 32.8 % positive
• IgG 72.5% positive
• No increase of sensitivity if additional testing for OspC- and VlsE-IgM or
IgG
Pomelova et al, PLoS One. 2015 Jul 6;10(7)
95
OppA2 Linear Epitopes for early serodiagnosis
Signorino et al performed epitope mapping of linear
epitopes in oligopeptide permease A2 (OppA2)
expressed during early infection. Two of the peptides
were identified as new candidates with sufficient
specificity and sensitivity.
Clinical and Vaccine Immunology,p.704–711,2014, Vol 21,No 5
96
Adherence to guidelines
• A dutch study found by interviewing physicians that 82% of testing
were not supported by guidelines and requested by patients with
atypical symptoms.
• 10 % of the requests were from patients with atypical skin lesions, of
which 20 % were positive (Dutch seroprevalence is 5%)
Eur J Clin Microbiol Infect Dis (2014) 33:1803–1808
97
B. Bissettii
• Girard et al analysed the sequence of rrf-rrl and p66 loci in 11%
(3/27) of PCR-positive individuals and found an infection with an
organism closely related to B. bissettii
J clin microbiol 2011 mar;49(3):945-54
• A murine model by Leydet et al. for B. bissettii revealed an infection
dynamics and humoral response similar to B. burgdorferi.
• From Europe, B. bissettii was reported from Slovenia and in a cardiac
valve tissue (endocarditis) and aortic valve stenosis in the Czech
Republic.
J Clin Microbiol 46, 3540-3543 , FEMS Microbiol Lett 292, 274-281
98
Detection
Moniuszko et al investigated skin biopsy and blood samples from 93
patients with EM
DNA was detected in 48% of the skin biopsy and in 2% of blood
samples (if positive 70% tick bite less than 2 weeks prior to sampling).
Seroprevalence was for IgM 35% and for IgG antibodies 30%.
Postepy Dermatol Alergol.. 2015 Feb;32(1):11-4
99
Serology: Recombinat proteins
Recombinat proteins:
EM, early carditis or neuroborreliosis :
IgM immunoblotting of BBA65, BBA70, and BBA73 reacted
comparable to the OspC antigen
IgG the proteins BBA69 and BBA73 reacted in the early phase.
Several samples were scored negative according the CDC
algorithm.
Weiner et al .Clin Vaccine Immunol. 2015 Sep 16.
100
Diagnostic histopathology
Highly divers patterns of EM
• Frequent:
- sparse to mild perivascular and interstitial mixed infiltrate of variable
amount of lymphocytes, eosinophils, neutrophils, and plasma cells
- epidermal changes such as spongiosis and interface change
- Periadnexal infiltrates
• Rare:
- dense inflammatory infiltrate or perineural lymphocytic infiltrates
- pigment incontinence
Miraflor et al. J Cutan Pathol.. 2015 Sep 8.
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Diagnostic Neuroborreliosis
A recent paper of Zajkowska et al antibody synthesis index (ASI)for
IgG and IgM in CSF versus peripheral serum to 27 patients with
clinically defined Bannwarth syndrome.
• 26/27 patients with NB had pathologic ASI-IgG against B.
burgdorferii.
• 21/27 NB patients had at least one positive (>1.5) IgG-ASI against
either VlsE, p100, p58, p39, p18, or OspC.
And none of these patients showed positive OspA-IgG-ASI.
Furthermore, NB patients showed dysfunction of the blood-CSF
barrier (average QAlb in the NB and control groups: 13.8 and 5.6)
Immunol Lett. 2015 Jul 22;168(1):58-6
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