Eur J Clin Microbiol Infect Dis (1998) 17:207-209 9 Springer-Verlag 1998
Note
Serological Evidence of Human Granulocytic Ehrlichiosis
in Switzerland
N. Pusterla, R. Weber, C. Wolfensberger, G. Schfir, R. Zbinden, W. Fierz,
J. E. Madigan, J. S. Dumler, H. Lutz
Abstract T o investigate whether h u m a n granulocytic
ehrlichiosis ( H G E ) is p r e v a l e n t in Switzerland, 1515 h u m a n s e r u m samples f r o m individuals with different risks for tick e x p o s u r e were tested for antibodies to Ehrlichia phagocytophila, a surrogate m a r k e r of the agent of H G E . T h e distribution of titres showed m a r k e d differences b e t w e e n sera of individuals with no or low risk for tick e x p o s u r e and those with a high risk. T h e results of serological testing p r o v i d e d evidence of H G E in Switzerland as well as evidence of two types of coinfections: those with the agent of H G E and BorreIia burgdorferi, and those with the agent of H G E and the central E u r o p e a n t i c k b o r n e encephalitis virus.
K e y words H u m a n granulocytic ehrlichiosis 9 Indirect i m m u n o f l u o r e s c e n t a n t i b o d y test 9 P r e v a l e n c e 9 Switzerland
N. Pusterla ([]), C. Wolfensberger, H. Lutz
Department of Veterinary Internal Medicine, University of Zurich, Winterthurerstrasse 260, CH-8057 Zurich, Switzerland R. Weber
Division of Infectious Diseases and Hospital Epidemiology, Department of Internal Medicine, University Hospital of Zurich, CH-8091 Zurich, Switzerland
G. Sch~ir, R. Zbinden
Department of Medical Microbiology, University of Zurich, CH-8028 Zurich, Switzerland
W. Fierz
Institute of Clinical Microbiology and Immunology, CH-9000 St. Gallen, Switzerland
J. E. Madigan
School of Veterinary Medicine, University of California, Davis, California 95616, USA
J. S. Dumler
Department of Pathology, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21287, USA
Introduction
H u m a n granulocytic ehrlichiosis ( H G E ) is an acute, fe- brile disease with nonspecific s y m p t o m s , often accom- panied by leukopenia, t h r o m b o c y t o p e n i a , anaemia, and a mild elevation of the t r a n s a m i n a s e s [1]. Ticks of the genus Ixodes, which transmit BorreIia burgdorferi and the central E u r o p e a n tickborne encephalitis virus, are also the likely vector of the agent of H G E . I n f o r m a t i o n concerning the agent responsible for H G E and its dis- tribution outside the U S A is sparse. Epidemiological studies in Sweden [2], N o r w a y [3], the U K [4], and n o r t h e r n Switzerland [5] indicate that H G E is e n d e m i c in those countries. Recently, investigators in Slovenia p r o v i d e d serological and m o l e c u l a r evidence of H G E in their country [6]. B e c a u s e of the m a r k e d serological cross-reactivity b e t w e e n the m e m b e r s of the Ehrlichia phagocytophila g e n o g r o u p , Ehrlichia equi and Ehrli- chia phagocytophila, antigen can be used in serological tests for H G E [7].
In the p r e s e n t study, sera f r o m 1515 individuals f r o m five risk groups were investigated to d e t e r m i n e the pre- valence, if any, of H G E in Switzerland.
Materials and Methods
Sera from 1515 persons from five groups representing different risk categories for tick exposure were collected between January 1993 and December 1996 and tested for antibodies to Ehrlichia phagocytophila by indirect immunofluorescence. All persons re- sided in the eastern region of Switzerland.
Group 1 consisted of 373 newborn babies from which umbilical cord blood was collected, and group 2 was composed of 530 ran- dom blood donors. Group 3 consisted of 258 recreational hunters who were assumed to have a high risk of tick exposure due to their weekly hunting activities. Group 4 consisted of 149 patients who had been diagnosed previously with Lyme borreliosis by their own physicians on the basis of clinical evaluation and anti- body to Borrelia burgdorferi screened by enzyme immunoassay (EIA) and confirmed by immunoblotting. The 205 patients in
208
group 5 had been diagnosed previously with acute central Euro- pean fickborne encephalitis on the basis of clinical evaluation and a positive EIA test for antibodies to the central European tick- borne encephalitis virus.
All serum samples were examined for antibodies to Ehrlichia phagocytophila (Swiss strain) by indirect immunofluorescence as described previously [8]. Values _>80 were considered positive, because nonspecific reactivity is likely to occur in less diluted se- rum. Values from the five groups were compared using the chi- square test. P values _<0.05 were considered statistically signifi- cant.
Table 1 lists the reference sera and conjugated antisera used to determine possible cross-reactivity with Ehrlichia phagocytophila
antigen via indirect immunofluorescence. Sera from four human patients with HGE acquired in the USA (provided by J.S. Dump er, the Johns Hopkins University School of Medicine, Baltimore, USA), and sera from three horses experimentally infected with HGE (provided by J.E. Madigan, University of California, Davis, USA) were drawn during the acute phase of infection and again approximately 30 days later.
Results and Discussion
O u r a p p r o a c h was to e x a m i n e g r o u p s o f individuals with different risk factors f o r tick e x p o s u r e . G r o u p 1 c o m p r i s e d n e w b o r n s , c h o s e n b e c a u s e tick e x p o s u r e can
be safely ruled o u t in these subjects; 99.5% of t h e sera f r o m this g r o u p h a d a negative a n t i b o d y titre (Table 2). T w o samples h a d titres o f 80, w h i c h m a y h a v e reflected m a t e r n a l antibodies. U n f o r t u n a t e l y , m a t e r n a l s e r u m samples w e r e n o t available to c o n f i r m o u r suspicion. I n c o m p a r i s o n , 0.85% o f babies b o r n to m o t h e r s living in Switzerland h a d positive titres for B o r r e l i a b u r g d o r f e r i
[9]. G r o u p 2, consisting of r a n d o m l y c h o s e n b l o o d do- nors, was p r e s u m e d to r e p r e s e n t individuals with a rela- tively low, or average, risk of tick e x p o s u r e ; h o w e v e r , b e c a u s e the d o n o r s r e m a i n e d a n o n y m o u s , the true ex- p o s u r e r a t e was u n k n o w n . O f the samples f r o m this g r o u p , 98.9% h a d a titre o f < 8 0 , a n d only six (1.1%) h a d a titre o f 80. G r o u p 3 c o m p r i s e d hunters, w h o w e r e c h o s e n b e c a u s e they, b y virtue of their r e c r e a t i o n a l ac- tivity, can be e x p e c t e d to h a v e a h i g h e r risk o f tick bites c o m p a r e d with t h e a v e r a g e p o p u l a t i o n . This expecta- tion was b o r n e o u t b y a statistically significant h i g h e r i n c i d e n c e o f positive titres ( 9 % ) in this g r o u p as c o m - p a r e d with g r o u p s 1 a n d 2 (p < 0.001).
A s e x p e c t e d , the highest s e r o p r e v a l e n c e o c c u r r e d in in- dividuals s e r o p o s i t i v e f o r B o r r e l i a b u r g d o r f e r i (12.7%) or the central E u r o p e a n t i c k b o r n e encephalitis virus (19.5%). C o m p a r e d with g r o u p s 1 a n d 2, these differ-
Table 1 Reference sera and conjugated antisera used to determine specificity
Reference serum Species Conjugated antiserum
Coxiella burnetti a bovine FITC conjugated
Ehrlichia canis a canine FITC conjugated
Ehrlichia risticii a equine FITC conjugated
Ehrlichia chaffeensis b human FITC conjugated
Rickettsia rickettsii b human FITC conjugated
Rickettsia typhi b human FITC conjugated
Brucella abortus a bovine FITC conjugated
A n a p l a s m a marginale a bovine FITC conjugated
Babesia bovis a bovine FITC conjugated
Babesia divergens a bovine FITC conjugated
rabbit anti-bovine IgG ~ rabbit anti-dog IgG d goat anti-horse IgG e goat anti-human IgG f goat anti-human IgG f goat anti-human IgG f rabbit anti-bovine IgG ~ rabbit anti-bovine IgG ~ rabbit anti-bovine IgG ~ rabbit anti-bovine IgG r
a Reference sera were obtained from the U.S. National Veterinary Services Laboratories (Ames,
Iowa)
b Reference sera obtained from J. S. Dumler, the Johns Hopkins University School of Medicine (Baltimore, Maryland)
c Rabbit anti-bovine/FITC, Nordic Immunological Laboratories b.v., The Netherlands
aFluorescein_conjugated affinipure rabbit anti-dog IgG, Jackson ImmunoResearch Laboratories, Inc., USA
e Goat anti-horse IgG affin. FITC-conjugated, Chemical Credential, USA
f Fluorescein-conjugated affinipure goat anti-human IgG, Jackson ImmunoResearch Laboratories, Inc., USA
Table 2 Distribution of titres to Ehrlichia phagocytophila in five groups of individuals with different risk categories for tick exposure
Group No. (%) (no. of samples)
Titer < 80 Titer = 80 Titer = 160 Newborns (373) 371 (99.5) 2 (0.5) 0 Blood donors (530) 524 (98.9) 6 (1.1) 0 Hunters (258) 235 (91.0) 19 (7.4) 4 (1.6) Patients with Lyme 130 (87.3) 13 (8.7) 6 (4.0)
borreliosis (149)
Patients with CETE (205) 165 (80.5) 35 (17.1) 5 (2.4) Total (1515) 1425 (94.0) 75 (5.0) 15 (1.0) CETE, central European tickborne encephalitis
ences were statistically significant (p < 0.001). O t h e r in- vestigators f r o m N o r w a y [3] and Switzerland [5] h a v e studied patients with L y m e disease for antibodies to the Ehrlichia equi antigen, and found a c o m p a r a b l e sero- p r e v a l e n c e of 10.2% and 17.1%, respectively. U n l i k e Borrelia burgdorferi, the virus causing central E u r o - p e a n tickborne encephalitis has a m a r k e d l y h e t e r o g e - neous g e o g r a p h i c distribution in Switzerland and oc- curs p r e d o m i n a n t l y in so-called natural loci [10]. This distribution m a y explain the low discrepancy b e t w e e n the n u m b e r of positive s e r u m samples in groups 4 and 5.
R e f e r e n c e sera used for d e t e r m i n a t i o n of cross-reactivi- ty had a n t i b o d y titres to Ehrlichia phagocytophila of 10 or 20; the exception was Ehrlichia chaffeensis seroposi- tive r e f e r e n c e sera, which had a titre to Ehrlichia pha- gocytophila of 40. All acute and postinfection sera with d o c u m e n t e d acute H G E infection showed seroconver- sion: a two- to four-fold increase in titres was o b s e r v e d in sera f r o m humans, and a four- to seven-fold increase in titres was found in sera f r o m equines.
O f the four h u m a n patients with H G E f r o m w h o m paired sera were collected, two had titres of 40 thirty days after the acute phase. According to the criteria de- scribed, these results would have b e e n classified as negative. T h e m i n i m a l increase in titre in these two pa- tients might b e explained by the relatively rapid recov- ery of these individuals following antibiotic t r e a t m e n t and the fact that a d e c r e a s e d bacterial load m a y h a v e f a v o u r e d a low seroresponse. A n o t h e r possible expla- nation is that antigenic structural diversity could exist a m o n g otherwise indistinguishable granulocytic ehrli- chial isolates. Nevertheless, we a s s u m e d that the specif- icity and sensitivity of o u r anti-Ehrlichia phagocytophi- la a n t i b o d y test was acceptable, and thus, the Ehrlichia phagocytophila antigen was suitable for detection of se- r u m antibodies to the agent of H G E acquired in Eu- rope.
W e realise that findings f r o m serological examinations to detect an infection that has not yet b e e n identified in Switzerland by clinical observations, by direct detection of the organism, or by m o l e c u l a r techniques, must be i n t e r p r e t e d with caution. H o w e v e r , several observa- tions indicate that the occurrence of H G E in Switzer- land is possible and, in fact, very likely. First, the p r o b - able vector for the agent of H G E , Ixodes ricinus, has a wide distribution in Switzerland and, d e p e n d i n g on the
209 region, m a y transmit Borrelia burgdorferi and/or the central E u r o p e a n t i c k b o r n e encephalitis virus. Second, we recently r e p o r t e d a new subspecies of the Ehrlichia phagocytophila g e n o g r o u p in dogs in Switzerland [11]; of the 16S r R N A gene this subspecies had a 100% ho- m o l o g y with that of the agent of H G E [12].
Acknowledgements This study was supported by the Kommis-
sion zur F~Srderung des akademischen Nachwuchses and the Swiss Federal Office of Public Health. The authors wish to thank Dr. med. Walter Bossard, Dr. reed. David Nadal, and Dr. med. vet. Felix Grimm for supplying serum samples.
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