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Assessing the risk of human granulocytic anaplasmosis and lyme borreliosis after a tick bite in Bavaria, Germany
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Assessing the risk of human granulocytic anaplasmosis and lyme borreliosis after a tick bite in Bavaria, Germany Beatrix von Wissmann a,b , Wolfgang Hautmann a , Andreas Sing a , Cecilia Hizo-Teufel a , Volker Fingerle a,∗ a National Reference Centre for Borrelia, Consultant Laboratory for Ehrlichia (until 2013), Bavarian Health and Food Safety Authority, Veterinärstr. 2, 85764 Oberschleißheim, Germany b Applied Epidemiology, Robert Koch Institute, DGZ-Ring 1, 13086 Berlin, Germany
a r t i c l e
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Keywords: Anaplasma phagocytophilum Ixodes ricinus Borrelia burgdorferi Risk assessment Tick bite
a b s t r a c t To date, only isolated incidences of human granulocytic anaplasmosis (HGA) have been reported in Europe. However, entomological studies in Bavaria, Germany showed a prevalence of Anaplasma phagocytophilum of between 2 and 9.5% in the tick vector Ixodes ricinus. In this study we assessed the risk of pathogenic A. phagocytophilum infection after a tick bite in Bavaria. The seroprevalence of anti-Borrelia burgdorferi sensu lato (s.l.) antibodies was investigated as an indicator of past exposure, seroconversion as actual exposure of participants to ticks. Patients with a tick bite in the preceding four weeks were recruited by participating doctors. Questionnaires on demographics, tick exposure and clinical signs were completed by patients and doctors, respectively. Two blood samples, taken at an interval of two weeks, were tested for antibodies against A. phagocytophilum and B. burgdorferi s.l. One of 107 recruited patients showed serological evidence of an acute infection of A. phagocytophilum but had no clinical signs. Four out of six patients with serological evidence of an acute B. burgdorferi s.l. infection, presented with erythema migrans. A seroprevalence of 7.5% for A. phagocytophilum and 13.1% for B. burgdorferi s.l. was detected. The comparatively high seroprevalence of B. burdorferi s.l. and A. phagocytophilum antibodies indicate frequent past exposure of participants to ticks. The finding of one acute infection of A. phagocytophilum in the absence of clinical signs, supports entomological evidence that the strains of A. phagocytophilum predominantly present in the Bavarian tick population may cause transient infections but are of low pathogenicity in humans. © 2015 Published by Elsevier GmbH.
1. Introduction Human granulocytic anaplasmosis (HGA) was first described in the United States in the 1990s (Bakken et al., 1994; Chen et al., 1994; Dumler et al., 2005). It is caused by Anaplasma phagocytophilum, an obligate intracellular bacterium. Initially, the causative pathogen was grouped in the genus Ehrlichia and named E. phagocytophila or Human Granulocytic Ehrlichiosis (HGE) agent. The pathogen was later reclassified as the species A. phagocytophilum (Dumler et al., 2001), together with the veterinary pathogens previously known as E. equi and Cytoecetes phagocytophila (Dumler et al., 2005). Despite being classified as a single species, phylogenetic analysis has identified different genetic variants of A. phagocytophilum associated
∗ Corresponding author. Tel.: +49 9131 68085870. E-mail address: [email protected] (V. Fingerle).
with predilection for different host species (Scharf et al., 2011; Huhn et al., 2014). Only certain variants have been isolated from human HGA cases and appear to be associated with pathogenicity in immunocompetent humans. The most frequent manifestations of HGA, reported in 70–95% of clinical cases, are pyrexia, malaise, myalgia and headaches, with a minority of cases presenting with arthalgia, involvement of the gastrointestinal or respiratory tract, the liver or the central nervous system. On rare occasions the infection is fatal. Laboratory findings include thrombocytopenia, leucopenia, elevated C-reactive protein, and elevated liver enzymes (Aguero-Rosenfeld et al., 1996; Bakken et al., 1996; Walker and Dumler, 1996). HGA is endemic in the Midwest and the East Coast of the US. Reported cases rose continuously and have now possibly reached a plateau at around 2500 cases a year reported in 2011, 2012 and 2013 (CDC, 2012, 2013, 2014, 2015; Thomas et al., 2009).
http://dx.doi.org/10.1016/j.ijmm.2015.08.026 1438-4221/© 2015 Published by Elsevier GmbH.
Please cite this article in press as: von Wissmann, B., et al., Assessing the risk of human granulocytic anaplasmosis and lyme borreliosis after a tick bite in Bavaria, Germany. Int. J. Med. Microbiol. (2015), http://dx.doi.org/10.1016/j.ijmm.2015.08.026
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The geographical distribution of A. phagocytophilum in the US corresponds to the focal distribution of B. burgdorferi, with which it shares the tick vector species Ixodes (I.) scapularis and I. pacificus. Despite comparatively low absolute case numbers of HGA across the US, the incidence of up to 58/100,000 population reported in some endemic areas in the US is of similar magnitude to the reported incidence of Lyme disease in these areas (Bakken et al., 1996; Ijdo et al., 2000, http://www.cdc.gov/lyme/stats/ chartstables/casesbyyear.html). In Europe, A. phagocytophilum is transmitted to humans by I. ricinus. The first serological evidence of a human infection with A. phagocytophilum was reported in the mid-1990s in Switzerland (Brouqui et al., 1995). The first confirmed clinical HGA case was described in Slovenia in 1997, with a moderately severe but self-limiting illness including fever, headache, nausea, vomiting, malaise, intense myalgia, and arthralgia (Petrovec et al., 1997). Despite an A. phagocytophilum prevalence of 0.25–24.4% in the tick vector across Europe (Cinco et al., 1997; Alberdi et al., 1998), only sporadic clinical cases of HGA have been reported in Europe (Blanco and Oteo, 2002). In Germany no autochthonous human clinical infections with A. phagocytophilum have been reported to date. However, a range of studies showed a seroprevalence between 1.0% and 4.4% for blood donors and control populations without a specific history of tick bites (Fingerle et al., 1997; Hunfeld and Brade, 1999; BätzingFeigenbaum et al., 2000; Randolph, 2002; Kowalski et al., 2006). A seroprevalence ranging from 14.0 to 19.5% was reported for forestry workers (Fingerle et al., 1997; Bätzing-Feigenbaum et al., 2000). In individuals with acute Lyme disease or serological evidence of past exposure to B. burgdorferi sensu lato (s.l.), an A. phagocytophilum seroprevalence of 4.5–13.1% was detected (Fingerle et al., 1997; Hunfeld and Brade, 1999; Kowalski et al., 2006). In comparison, the seroprevalence for B. burgdorferi s.l. in Germany ranges from 13.4% to 34.6% in people with high occupational risk of exposure to ticks (Oehme et al., 2002; Reimer et al., 2002; Rieger et al., 2002) and from 1.8 to 12.0% in people with leisure time exposure or no known exposure to ticks (Nübling et al., 2002; Rieger et al., 2002; Wilking et al., 2015). Since A. phagocytophilum is transmitted by the same vector (I. ricinus) as B. burgdorferi s.l. in Europe, transmission to humans is presumed to be governed by similar risk factors. A. phagocytophilum prevalence of 1.6–9.5% was detected in I. ricinus in Germany, with a higher prevalence in adult ticks than in nymphs (Fingerle et al., 1999; Silaghi et al., 2008; Schorn et al., 2011a,b). A prevalence of B. burgdorferi s.l. of up to 36.2% (178/492) was detected in ticks in Bavaria (Fingerle et al., 1999). In contrast to B. burgdorferi s.l., which is endemic across Bavaria, the presence of A. phagocytophilum in vector ticks appears to be focal, with significant differences in prevalence in the tick vector reported between localities (Fingerle et al., 1999) and types of habitats (Silaghi et al., 2008). Higher infection rates were observed in ticks from a variety of inner city parks than in ticks from woodland habitats (Silaghi et al., 2008) suggesting separate sylvatic and urban A. phagocytophilum transmission cycles. Nevertheless, the influence of the host community structure on A. phagocytophilum transmission cycles still remains unclear. Partial sequencing of the 16SrRNA gene identified 6 different A. phagocytophilum variants of I. ricinus from a range of Bavarian inner city parks. Two out of 45 infections showed 100% sequence similarity of the 16SrRNA target with the original human infective HGA agent. However, the variant of A. phagocytophilum infection most frequently detected in ticks in Bavaria (Ehrlichia sp. Frankonia 2) (Silaghi et al., 2008; Schorn et al., 2011a) has, to date, only been isolated from a single clinical HGA case from Slovenia (Scharf et al., 2011) but has been found more frequently in dogs and cats (Silaghi et al., 2008; Scharf et al., 2011). Based on the available data this variant is suspected to be of low pathogenicity in humans. No clinical infections of A. phagocytophilum in humans have been
recorded in Bavaria to date. Potential reasons include episodes of HGA not being recorded due to lack of awareness or misdiagnosis. An alternative reason could be that little or no transmission of A. phagocytophilum to humans takes place in Bavaria. A closed transmission cycle between ticks and vertebrate hosts such as dogs or rodents could result in maintenance of the observed prevalence of A. phagocytophilum in ticks with few or no spill-over infections in humans. It is also conceivable that transmission of A. phagocytophilum to humans does take place on a regular basis, but that the variant of A. phagocytophilum present in the tick vector in Bavaria is specific to other host species, causing only transient infections without relevant clinical signs in humans. No studies assessing the risk of acute A. phagocytophilum infections in humans have previously been carried out in Bavaria. The primary aim of this study was to investigate whether there is a risk of infection with A. phagocytophilum after tick bite in Bavaria. The risk of B. burgdorferi s.l. infection after a tick bite was investigated in parallel as an indicator of the level of past exposure to tick-borne pathogens experienced by participants. As a secondary aim, the activity of the participants at the time of tick exposure as well as regular leisure or occupational activities were investigated as potential risk factors for contracting a tick-borne infection.
2. Methods 2.1. Study design and sample size A cross-sectional study was designed. The target population were residents of Bavaria over 18 years of age, experiencing a tick bite in Bavaria in the preceding 4 weeks. As determined according to Cannon and Roe (1982), a sample size of 100 participants was required for proof of presence of acute A. phagocytophilum infections after tick bite in Bavaria. This sample size would permit the detection of at least one acute infection of A. phagocytophilum, with a probability of over 85%, assuming an acute infection in 2% of people after a tick bite (Cannon and Roe, 1982). All general practice (GP) and internal medicine surgeries, who were registered for the syndromic or virological influenza sentinel surveillance in Bavaria in June 2010 (n = 134), were asked to participate in the study. Participating surgeries recruited patients over 18 years of age with recent tick exposure. Recent tick exposure was defined as patients who presented to their doctor with a tick bite or reported a history of a tick bite in the 4 weeks preceding the time point of the first sample.
2.2. Biological samples After written consent was obtained, venous blood samples were collected from participating patients on two occasions, with an interval of two weeks between sampling time points. Submission of a third blood sample was requested if interpretations of the serological results from the first two time points were ambiguous.
2.3. Survey For each of the sampling time points, doctors were asked to fill in a short structured questionnaire recording any clinical signs, the history of chronic diseases and any prescription of antibiotics. After giving consent to participate in the study, patients were asked to fill in a structured questionnaire with questions on demographics including place of residence, geographical location and activity at the time of tick exposure (if remembered) as well as regular leisure activities and potential occupational risk factors for tick exposure.
Please cite this article in press as: von Wissmann, B., et al., Assessing the risk of human granulocytic anaplasmosis and lyme borreliosis after a tick bite in Bavaria, Germany. Int. J. Med. Microbiol. (2015), http://dx.doi.org/10.1016/j.ijmm.2015.08.026
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2.4. Laboratory analysis All laboratory analysis was carried out at the German National Reference Centre for Borrelia and Consiliar Laboratory for Ehrlichia. For the detection of antibodies against A. phagocytophilum, an immunofluorescence antibody (IFA) test kit for IgG and IgM was used according to the manufacturer’s recommendations (Focus Diagnostics, Cypress, California). Semi-quantitative endpoint titres were obtained by serial dilution of reactive samples. IgG titres of ≥1:64 and IgM titres of ≥1:20 were regarded as positive. Quantitative endpoint titres were performed for IgG and IgM positive samples. For the detection of antibodies against B. burgdorferi the Enzygnost Lyme link, an enzyme-linked immunosorbent assay (ELISA) (Siemens Healthcare Diagnostics GmbH, Eschborn, Germany) was used. The test was automatically processed on a BEP® III (Siemens Health Diagnostics GmbH, Eschborn, Germany) and interpreted as recommended by the manufacturer. Positive or borderline results were confirmed by a line blot (Borrelia Europe plus TpN17 LINE, Virotech, Rüsselsheim, Germany). The test was performed and interpreted according to manufacturers’ recommendations. 2.5. PCR DNA extraction was performed using the High Pure PCR Template Preparation Kit (Roche Diagnostics) according to the manufacturers’ instructions. Extraction controls were included in each run. A nested PCR targeting the 16S rRNA gene was done as described previously (Massung et al., 1998) from the first sample submitted by each participant. DNA from a human patient isolate of HGA served as positive control (Goodman et al., 1996). Amplified DNA was separated by agarose gel electrophoresis, stained with ethidium bromide, and visualized under UV illumination. 2.6. Case definitions Laboratory confirmed acute infection with A. phagocyophilum: A laboratory confirmed acute infection with A. phagocytophilum was defined as one of the following findings:
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between samples, was defined as serological evidence of past exposure to B. burgdorferi s.l.
2.7. Statistical analysis For a descriptive analysis of participants continuous variables were summarised using median and range. Categorical variables were presented using absolute numbers and the proportions in percent. The univariable association between case status and exposure to individual risk factors was tested using the Chi-squared test (2 ) or the Fisher’s exact test (FE) where appropriate. The effect was quantified by estimation of the relative risk (RR) and the respective 95% confidence interval (CI). In the case of small numbers exact Poisson regression was used for estimation. Statistical significance was assumed when p < 0.05 and the 95% confidence interval of the relative risk excluded 1. When interpretation of the p-value and the 95% confidence interval were contradictory, the more conservative interpretation (not significant) was reported. Data analysis was carried out in STATA 10.1.
3. Results 3.1. Descriptive analysis A total of 107 participants were included in the study which was performed between July 2010 and August 2011. The number of participants recruited by each of the 24 participating surgeries ranged between one and 22. Fifty of the participants were male and 54 were female, there was no information on gender for three participants. The median age of participants was 59 years (range: 18–89 years). There was no information on age for five participants. The participants were resident in six of the seven Bavarian regions, with eight to 32 participants per region. There were no participants from the region of Schwaben.
3.2. Laboratory and clinical findings 1. PCR positive for A. phagocytophilum from blood confirmed by sequencing of the amplicon. 2. IgM seroconversion (seroconversion = change from negative to positive) OR a four-fold (two titre steps) or higher increase in positive IgM titre between samples tested in parallel. 3. IgG seroconversion OR a four-fold or higher increase in positive IgG titre between samples when tested in parallel. Laboratory confirmed past exposure to A. phagocytophilum: Positive anti-A. phagocytophilum IgG and/or IgM in both consecutive samples tested in parallel, with the titre remaining stable between samples, was defined as serological evidence of past exposure to A. phagocytophilum. Laboratory confirmed acute infection with B. burgdorferi s.l. A laboratory confirmed acute infection with B. burgdorferi s.l. was defined as one of the following combinations of anti- B. burgdorferi s.l. IgM and anti- B. burgdorferi s.l. IgG results of serology: 1. IgM seroconversion OR a two-fold or higher increase in positive IgM values between samples when tested in parallel. 2. IgG seroconversion OR a two-fold or higher increase in IgG units between samples when tested in parallel. Laboratory confirmed past exposure to B. burgdorferi s.l. Positive anti-B. burdorferi s.l. IgG and/or IgM in both consecutive samples tested in parallel, with the values remaining stable
Serological evidence of an acute infection with A. phagocytophilum was found for one out of 107 participants (0.9%). The IgM titre of this participant declined significantly between samples when tested in parallel from 1:160 in the first sample (on the day of tick removal by the doctor) to 1:80 in the second sample and 1:20 in the third sample, with two to three weeks between sampling time points. IgG seroconversion took place from the second to the third sampling time point. None of the blood-samples tested positive for A. phagocytophilum by PCR. The participant presented without clinical signs throughout the study and no antibiotics were prescribed. Serological evidence of an acute infection with B. burgdorferi s.l. was detected for six out of 107 participants (5.6%); two presented without clinical signs, two presented with an erythema migrans, one reported an erythema migrans and malaise and another suffered from an erythema migrans, myalgia and malaise. Antibiotics were prescribed to three of the four clinical cases and one of the two asymptomatic cases with acute B. burgdorferi s.l. infection. None of the blood samples tested were positive for A. phagocytophilum or B. burgdorferi s.l. by PCR. Serological evidence of past exposure to A. phagocytophilum was found for eight participants (7.5%) and to B. burgdorferi s.l. for 14 participants (13.1%). One of these participants showed serological evidence of past exposure to both A. phagocytophilum and B. burgdorferi s.l.
Please cite this article in press as: von Wissmann, B., et al., Assessing the risk of human granulocytic anaplasmosis and lyme borreliosis after a tick bite in Bavaria, Germany. Int. J. Med. Microbiol. (2015), http://dx.doi.org/10.1016/j.ijmm.2015.08.026
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Table 1 Proportion of participants with serological evidence of acute infection with A. phagocytophilum or B. burgdorferi s.l. or with serological evidence of past exposure to A. phagocytophilum or B. burgdorferi s.l, by age group, Bavaria 2010–2011. Age group*
Acute infection A. phagocytophilum
Past exposure A. phagocytophilum
Acute infection B. burgdorferi**
Past exposure B. burgdorferi
18–40 >40–65 >65
0.0% (0/16) 2.0% (1/51) 0.0% (0/35)
6.3% (1/16) 5.9% (3/51) 11.4% (4/35)
0.0% (0/16) 7.8% (4/51) 2.9% (1/35)
6.3% (1/16) 15.7% (8/51) 14.3% (5/35)
* **
n = 102, missing = 5. 1 acute B. burgdorferi s.l. infection amongst participant with missing age.
Table 2 Proportion of participants with serological evidence of acute infection with A. phagocytophilum or B. burgdorferi s.l. by region of tick exposure and proportion of participants with serological evidence of past exposure to A. phagocytophilum or B. burgdorferi s.l, by region of residence, Bavaria 2010–2011 (n = 107). Region: resi-dence/exposure
Mittelfranken Niederbayern Oberbayern Oberfranken Oberpfalz Unterfranken
Acute infections by region of tick exposure
Past infections by region of residence
A. phagocytophilum
B. burgdorferis.l.
A. phagocytophilum
B. burgdorferis.l.
0.0% (0/13) 0.0% (0/14) 2.9% (1/34) 0.0% (0/28) 0.0% (0/11) 0.0% (0/7)
0.0% (0/13) 14.3% (2/14) 0.0% (0/34) 10.7% (3/28) 9.1% (1/11) 0.0% (0/7)
15.4% (2/13) 0.0% (0/15) 6.3% (2/32) 10.7% (3/28) 0.0% (0/11) 12.5% (1/8)
7.7% (1/13) 6.7% (1/15) 15.6% (5/32) 17.9% (5/28) 9.1% (1/11) 12.5% (1/8)
3.3. Analytical statistics 3.3.1. Gender There were no statistically significant differences between female and male participants in the risk of acute infections (IRR = 0.93; 95%CI: 0.02–∞; p = 0.99) or past exposure to A. phagocytophilum (RR = 0.55; 95%CI: 0.14–2.2; p = 0.47). There was a lower risk of acute B. burgdorferi s.l. infections (RR = 0.19; 95%CI: 0.02–1.5; p = 0.10) and of past exposure to B. burgdorferi s.l. (RR = 0.37; 95%CI: 0.1–1.1; p = 0.08) for female than for male participants, however the difference was not statistically significant. 3.3.2. Age distribution Table 1 summarises the distribution of participants with acute infections and past exposure across age groups. The highest proportion of acute infections with both A. phagocytophilum (2.0%) and B. burgdorferi s.l. (7.8%) were detected in the age group between 40–65 years. In the age group over 65 the proportion of participants with evidence of past exposure to A. phagocytophilum was higher (11.4%) than in the two younger age groups (age group 18–40: 6.3%, age group >40–65: 5.9%). There was a higher proportion of participants with evidence of past exposure to B. burgdorferi s.l. in the two age groups over 40 (age group >40–65: 15.7%, age group >65: 14.3%) than in the age group between 18 and 40 (6.3%). However none of the differences between age groups were statistically significant. 3.3.3. Regional distribution Table 2 summarises the distribution of participants with acute infections by the region in which tick exposure took place, and the distribution of participants with past exposure by region of residence. There was no significant association between the region of Bavaria in which tick exposure took place and the proportion of participants with an acute A. phagocytophilum (p = 0.80) or an acute B. burgdorferi s.l. infection (p = 0.11). There was no significant association between the region of residence and the proportion of participants with serological evidence of past exposure to A. phagocytophilum (p = 0.34) or B. burgdorferi s.l. (p = 0.86) (Table 2). 3.3.4. Association of acute infections with activity or type of location at time of tick exposure Table 3 summarises the activities or type of location where participants contracted the most recent tick bite, which resulted in their study participation, and the risk of acute A. phagocytophilum
Table 3 Proportion of participants with acute A. phagocytphilum or B. burgdorferi s.l. infection by the type of exposure during which the most recent tick bite occured. Exposure*
Proportion of exposed participants with acute infection
Gardening or being in the garden Activity in forest Outdoor sports (e.g. walking, jogging) Occupational exposure Other Not specified *
A. phagocytophilum
B. burgdorferi s.l.
0% (0/50) 2.9% (1/34) 0% (0/29) 0% (0/4) 0% (0/11) 0% (0/6)
6.0% (3/50) 2.9% (1/34) 13.8% (4/29) 0% (0/4) 0% (0/11) 0% (0/6)
Multiple categories could be named.
or acute B. burgdorferi s.l. infection in each exposure category. There was no significant association between any of the exposures and the risk of an acute infection with A. phagocytophilum. There was an increased risk of an acute infection with B. burgdorferi s.l. for participants who did outdoor sports; this association approached statistical significance (RR = 4.9%; 95%CI: 0.95–25.3%, p = 0.06). 3.3.5. Association of past pathogen exposure with regular activities Table 4 summarises occupational or regular leisure activities of participants, during which tick exposure may occur, and the risk Table 4 Proportion of participants with past exposure to A. phagocytphilum or B. burgdorferi s.l. by regular outdoor leisure activity or occupation. Exposure*
Proportion of exposed participants with past exposure to A. phagocytophilum
Proportion of exposed participants with past exposure to B. burgdorferi s.l.
Occupational exposure (all) Hunting Other Leisure activities (all) Jogging Cycling Walking Hunting Gardening Other
13.3% (2/15) 100% (2/2) 0% (0/13) 8.9% (8/90) 6.7% (1/15) 2.9% (1/35) 5.6% (3/54) 50.0% (2/4) 6.8% (4/59) 5.3% (1/19)
20% (3/15) 50% (1/2) 15.4% (2/13) 13.3% (12/90) 13.3% (2/15) 8.6% (3/35) 16.7% (9/54) 50.0% (2/4) 5.1% (3/59) 10.5% (2/19)
*
Multiple categories could be named.
Please cite this article in press as: von Wissmann, B., et al., Assessing the risk of human granulocytic anaplasmosis and lyme borreliosis after a tick bite in Bavaria, Germany. Int. J. Med. Microbiol. (2015), http://dx.doi.org/10.1016/j.ijmm.2015.08.026
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of past exposure to A. phagocytophilum or B. burgdorferi s.l. in each exposure category. Being a hunter (leisure and occupational) was significantly associated with past exposure to A. phagocytophilum (RR = 8.2; 95%CI: 2.3–28.5, p < 0.05). Gardening was significantly associated with a reduced risk of past exposure to B. burgdorferi s.l. (RR = 0.2; 95%CI: 0.06–0.66, p < 0.01). The risk of past exposure to A. phagocytophilum associated with gardening was also reduced, however this was not statistically significant (RR = 0.7, 95%CI: 0.2–2.7; p = 0.72) There were no other statistically significant associations between regular activities and serological evidence of past exposure to either A. phagocytophilum or B. burgdorferi s.l.
4. Discussion One acute infection of A. phagocytophilum was detected by clear IgG-seroconversion during the course of the study, providing evidence that transmission of this pathogen to humans does take place in Bavaria. However, here it is of interest to note that most probably the measured immunological reaction against A. phagocytophilum was not induced by the detected tick bite but most probably resulted from a preceding – undetected – tick bite. Usually a measurable antibody response against A. phagocytophilum has still not developed in very early disease but is highly sensitive from two to four weeks after disease onset (Childs et al., 1999; Thomas et al., 2009). Therefore, in our case-tick bite serology would have been negative at least in the first serum sample that was taken directly after removal of the tick. Notably, in a long-term follow up study on 96 patients with erythema migrans, 47% of them reported an additional tick bite, a finding that further support our assumption of an overlooked preceding tick bite (Nowakowski et al., 2003). That HGA transmission occurs in Bavaria is further supported by the detection of prevalence titres to A. phagocytophilum in eight participants, indicative of past exposure. Previous entomological studies indicated that the variant of A. phagocytophilum predominantly present in the tick vector in Bavaria belongs to a cluster that is not usually associated with symptomatic HGA (Silaghi et al., 2008; Schorn et al., 2011a). Congruent with this is that no clinical signs were associated with the laboratory confirmed acute A. phagocytophilum infection detected in the current study. However, it is important to note that detection of a single asymptomatic A. phagocytophilum infection is insufficient evidence to entirely rule out the occurrence of clinically manifest HGA in Bavaria, but points to an at least very rare occurrence. Therefore, HGA should be included in the differential diagnosis for febrile illnesses after a tick bite. However, the observation does support the hypothesis that the cluster of A. phagocytophilum predominantly present in the tick vector in Bavaria is associated with low or no pathogenicity when transmitted to humans. This would also explain the absence of reports of clinical HGA cases in Bavaria, despite a prevalence of up to 9.5% in the tick vector (Schorn et al., 2011b). The proportion of participants with acute A. phagocytophilum infections after tick bite was lower than the proportion with acute B. burgdorferi s.l. infections. This difference was not statistically significant, but a lower risk of exposure to A. phagocytophilum was also reflected in a lower proportion of participants with past exposure to A. phagocytophilum than with past exposure to B. burgdorferi s.l. Even though only 15/107 participants had occupational tick exposure, the seroprevalence for A. phagocytophilum and B. burgdorferi detected in the current study was higher than the seroprevalence previously reported for populations without occupational exposure to ticks or a history of tick borne infection. This indicates that leisure time exposure considerably contributes to the risk of exposure to tick borne pathogens. No significant differences in the proportion of participants with acute infections or past exposure to either A. phagocytophilum or
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B. burgdorferi s.l. by region could be detected. However, the assessment of regional differences in the risk of infection was restricted to six of the seven Bavarian regions due to a lack of participants from the 7th region, and the sample size per region was small. Stratified sampling by region would be required to allow more detailed investigations of geographical variations in A. phagocytophilum or B. burgdorferi s.l. seroprevalence. The study was designed to provide proof of presence or absence of acute A. phagocytophilum infection after tick bites in Bavaria. Due to the sample size, the study had insufficient power to detect small differences between risk factors. Nonetheless, investigation of the type of activity during which tick exposure took place showed that outdoor sports were associated with an increased risk of acute infections of B. burgdorferi s.l. and hunting was associated with past exposure to A. phagocytophilum and B. burgdorferi s.l. As only a single acute infection of A. phagocytophilum was detected, no significant association with any particular activity could be shown. However the participant in question acquired the documented tick bite whilst being in woodlands. On the other hand, this infection was most probably acquired from a different tick bite a few days previously, a finding that illustrates the difficulties in interpreting results from tick-bite studies. Activities in the garden apparently posed a comparatively low risk of acquisition of A. phagocytophilum or B. burgdorferi s.l. infections, as reflected in a lower seroprevalence associated with this category. This may be due to differences in the prevalence of infection in the tick vector depending on the type of habitat. An alternative explanation is a lower frequency of contact with ticks, and thus a lower cumulative risk of exposure to tick borne pathogens associated with gardening as compared to hunting. However, a large number of participants (50/107) in the current study remember contracting their most recent tick bite in their gardens and half of the acute B. burgdorferi s.l infections could be attributed to ticks contracted whilst gardening. Only 1/5 of the participants with past exposure to B. burgdorferi s.l., but half of the participants with past exposure to A. phagocytophilum listed gardening as a regular leisure activity. This is likely to be a reflection of a large part of the population in Bavaria spending time in the garden, whilst only a much smaller fraction of the population are involved in hunting. Despite a comparatively low risk of exposure to tick borne pathogens associated with gardening, the large number of people involved in this activity is therefore likely to result in a high absolute burden of tick borne infections. Public health measures to increase awareness for tick borne infections should therefore also be targeted at the large part of the population who are involved in comparatively low risk outdoor activities such as gardening, cycling or walking. 5. Conflict of interest statement The authors declare no conflict of interest. Acknowledgements The authors are grateful to Wiltrud Strehle and Silvia Stockmeier for excellent technical assistance, Katharina Alpers und Manuel Dehnert for valuable discussions and help with the study design and the Bavarian Ministry for Health and Care and the Robert-KochInstitut, Berlin, for funding the study. Volker Fingerle and Andreas Sing are ESGBOR members. References Aguero-Rosenfeld, M.E., Horowitz, H.W., Wormser, G.P., McKenna, D.F., Nowakowski, J., Munoz, J., Dumler, J.S., 1996. Human granulocytic ehrlichiosis: a case series from a medical center in New York State. Ann. Intern. Med. 125, 904–908.
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Assessing the risk of human granulocytic anaplasmosis and lyme borreliosis after a tick bite in Bavaria, Germany Beatrix von Wissmann a,b , Wolfgang Hautmann a , Andreas Sing a , Cecilia Hizo-Teufel a , Volker Fingerle a,∗ a National Reference Centre for Borrelia, Consultant Laboratory for Ehrlichia (until 2013), Bavarian Health and Food Safety Authority, Veterinärstr. 2, 85764 Oberschleißheim, Germany b Applied Epidemiology, Robert Koch Institute, DGZ-Ring 1, 13086 Berlin, Germany
a r t i c l e
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Keywords: Anaplasma phagocytophilum Ixodes ricinus Borrelia burgdorferi Risk assessment Tick bite
a b s t r a c t To date, only isolated incidences of human granulocytic anaplasmosis (HGA) have been reported in Europe. However, entomological studies in Bavaria, Germany showed a prevalence of Anaplasma phagocytophilum of between 2 and 9.5% in the tick vector Ixodes ricinus. In this study we assessed the risk of pathogenic A. phagocytophilum infection after a tick bite in Bavaria. The seroprevalence of anti-Borrelia burgdorferi sensu lato (s.l.) antibodies was investigated as an indicator of past exposure, seroconversion as actual exposure of participants to ticks. Patients with a tick bite in the preceding four weeks were recruited by participating doctors. Questionnaires on demographics, tick exposure and clinical signs were completed by patients and doctors, respectively. Two blood samples, taken at an interval of two weeks, were tested for antibodies against A. phagocytophilum and B. burgdorferi s.l. One of 107 recruited patients showed serological evidence of an acute infection of A. phagocytophilum but had no clinical signs. Four out of six patients with serological evidence of an acute B. burgdorferi s.l. infection, presented with erythema migrans. A seroprevalence of 7.5% for A. phagocytophilum and 13.1% for B. burgdorferi s.l. was detected. The comparatively high seroprevalence of B. burdorferi s.l. and A. phagocytophilum antibodies indicate frequent past exposure of participants to ticks. The finding of one acute infection of A. phagocytophilum in the absence of clinical signs, supports entomological evidence that the strains of A. phagocytophilum predominantly present in the Bavarian tick population may cause transient infections but are of low pathogenicity in humans. © 2015 Published by Elsevier GmbH.
1. Introduction Human granulocytic anaplasmosis (HGA) was first described in the United States in the 1990s (Bakken et al., 1994; Chen et al., 1994; Dumler et al., 2005). It is caused by Anaplasma phagocytophilum, an obligate intracellular bacterium. Initially, the causative pathogen was grouped in the genus Ehrlichia and named E. phagocytophila or Human Granulocytic Ehrlichiosis (HGE) agent. The pathogen was later reclassified as the species A. phagocytophilum (Dumler et al., 2001), together with the veterinary pathogens previously known as E. equi and Cytoecetes phagocytophila (Dumler et al., 2005). Despite being classified as a single species, phylogenetic analysis has identified different genetic variants of A. phagocytophilum associated
∗ Corresponding author. Tel.: +49 9131 68085870. E-mail address: [email protected] (V. Fingerle).
with predilection for different host species (Scharf et al., 2011; Huhn et al., 2014). Only certain variants have been isolated from human HGA cases and appear to be associated with pathogenicity in immunocompetent humans. The most frequent manifestations of HGA, reported in 70–95% of clinical cases, are pyrexia, malaise, myalgia and headaches, with a minority of cases presenting with arthalgia, involvement of the gastrointestinal or respiratory tract, the liver or the central nervous system. On rare occasions the infection is fatal. Laboratory findings include thrombocytopenia, leucopenia, elevated C-reactive protein, and elevated liver enzymes (Aguero-Rosenfeld et al., 1996; Bakken et al., 1996; Walker and Dumler, 1996). HGA is endemic in the Midwest and the East Coast of the US. Reported cases rose continuously and have now possibly reached a plateau at around 2500 cases a year reported in 2011, 2012 and 2013 (CDC, 2012, 2013, 2014, 2015; Thomas et al., 2009).
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The geographical distribution of A. phagocytophilum in the US corresponds to the focal distribution of B. burgdorferi, with which it shares the tick vector species Ixodes (I.) scapularis and I. pacificus. Despite comparatively low absolute case numbers of HGA across the US, the incidence of up to 58/100,000 population reported in some endemic areas in the US is of similar magnitude to the reported incidence of Lyme disease in these areas (Bakken et al., 1996; Ijdo et al., 2000, http://www.cdc.gov/lyme/stats/ chartstables/casesbyyear.html). In Europe, A. phagocytophilum is transmitted to humans by I. ricinus. The first serological evidence of a human infection with A. phagocytophilum was reported in the mid-1990s in Switzerland (Brouqui et al., 1995). The first confirmed clinical HGA case was described in Slovenia in 1997, with a moderately severe but self-limiting illness including fever, headache, nausea, vomiting, malaise, intense myalgia, and arthralgia (Petrovec et al., 1997). Despite an A. phagocytophilum prevalence of 0.25–24.4% in the tick vector across Europe (Cinco et al., 1997; Alberdi et al., 1998), only sporadic clinical cases of HGA have been reported in Europe (Blanco and Oteo, 2002). In Germany no autochthonous human clinical infections with A. phagocytophilum have been reported to date. However, a range of studies showed a seroprevalence between 1.0% and 4.4% for blood donors and control populations without a specific history of tick bites (Fingerle et al., 1997; Hunfeld and Brade, 1999; BätzingFeigenbaum et al., 2000; Randolph, 2002; Kowalski et al., 2006). A seroprevalence ranging from 14.0 to 19.5% was reported for forestry workers (Fingerle et al., 1997; Bätzing-Feigenbaum et al., 2000). In individuals with acute Lyme disease or serological evidence of past exposure to B. burgdorferi sensu lato (s.l.), an A. phagocytophilum seroprevalence of 4.5–13.1% was detected (Fingerle et al., 1997; Hunfeld and Brade, 1999; Kowalski et al., 2006). In comparison, the seroprevalence for B. burgdorferi s.l. in Germany ranges from 13.4% to 34.6% in people with high occupational risk of exposure to ticks (Oehme et al., 2002; Reimer et al., 2002; Rieger et al., 2002) and from 1.8 to 12.0% in people with leisure time exposure or no known exposure to ticks (Nübling et al., 2002; Rieger et al., 2002; Wilking et al., 2015). Since A. phagocytophilum is transmitted by the same vector (I. ricinus) as B. burgdorferi s.l. in Europe, transmission to humans is presumed to be governed by similar risk factors. A. phagocytophilum prevalence of 1.6–9.5% was detected in I. ricinus in Germany, with a higher prevalence in adult ticks than in nymphs (Fingerle et al., 1999; Silaghi et al., 2008; Schorn et al., 2011a,b). A prevalence of B. burgdorferi s.l. of up to 36.2% (178/492) was detected in ticks in Bavaria (Fingerle et al., 1999). In contrast to B. burgdorferi s.l., which is endemic across Bavaria, the presence of A. phagocytophilum in vector ticks appears to be focal, with significant differences in prevalence in the tick vector reported between localities (Fingerle et al., 1999) and types of habitats (Silaghi et al., 2008). Higher infection rates were observed in ticks from a variety of inner city parks than in ticks from woodland habitats (Silaghi et al., 2008) suggesting separate sylvatic and urban A. phagocytophilum transmission cycles. Nevertheless, the influence of the host community structure on A. phagocytophilum transmission cycles still remains unclear. Partial sequencing of the 16SrRNA gene identified 6 different A. phagocytophilum variants of I. ricinus from a range of Bavarian inner city parks. Two out of 45 infections showed 100% sequence similarity of the 16SrRNA target with the original human infective HGA agent. However, the variant of A. phagocytophilum infection most frequently detected in ticks in Bavaria (Ehrlichia sp. Frankonia 2) (Silaghi et al., 2008; Schorn et al., 2011a) has, to date, only been isolated from a single clinical HGA case from Slovenia (Scharf et al., 2011) but has been found more frequently in dogs and cats (Silaghi et al., 2008; Scharf et al., 2011). Based on the available data this variant is suspected to be of low pathogenicity in humans. No clinical infections of A. phagocytophilum in humans have been
recorded in Bavaria to date. Potential reasons include episodes of HGA not being recorded due to lack of awareness or misdiagnosis. An alternative reason could be that little or no transmission of A. phagocytophilum to humans takes place in Bavaria. A closed transmission cycle between ticks and vertebrate hosts such as dogs or rodents could result in maintenance of the observed prevalence of A. phagocytophilum in ticks with few or no spill-over infections in humans. It is also conceivable that transmission of A. phagocytophilum to humans does take place on a regular basis, but that the variant of A. phagocytophilum present in the tick vector in Bavaria is specific to other host species, causing only transient infections without relevant clinical signs in humans. No studies assessing the risk of acute A. phagocytophilum infections in humans have previously been carried out in Bavaria. The primary aim of this study was to investigate whether there is a risk of infection with A. phagocytophilum after tick bite in Bavaria. The risk of B. burgdorferi s.l. infection after a tick bite was investigated in parallel as an indicator of the level of past exposure to tick-borne pathogens experienced by participants. As a secondary aim, the activity of the participants at the time of tick exposure as well as regular leisure or occupational activities were investigated as potential risk factors for contracting a tick-borne infection.
2. Methods 2.1. Study design and sample size A cross-sectional study was designed. The target population were residents of Bavaria over 18 years of age, experiencing a tick bite in Bavaria in the preceding 4 weeks. As determined according to Cannon and Roe (1982), a sample size of 100 participants was required for proof of presence of acute A. phagocytophilum infections after tick bite in Bavaria. This sample size would permit the detection of at least one acute infection of A. phagocytophilum, with a probability of over 85%, assuming an acute infection in 2% of people after a tick bite (Cannon and Roe, 1982). All general practice (GP) and internal medicine surgeries, who were registered for the syndromic or virological influenza sentinel surveillance in Bavaria in June 2010 (n = 134), were asked to participate in the study. Participating surgeries recruited patients over 18 years of age with recent tick exposure. Recent tick exposure was defined as patients who presented to their doctor with a tick bite or reported a history of a tick bite in the 4 weeks preceding the time point of the first sample.
2.2. Biological samples After written consent was obtained, venous blood samples were collected from participating patients on two occasions, with an interval of two weeks between sampling time points. Submission of a third blood sample was requested if interpretations of the serological results from the first two time points were ambiguous.
2.3. Survey For each of the sampling time points, doctors were asked to fill in a short structured questionnaire recording any clinical signs, the history of chronic diseases and any prescription of antibiotics. After giving consent to participate in the study, patients were asked to fill in a structured questionnaire with questions on demographics including place of residence, geographical location and activity at the time of tick exposure (if remembered) as well as regular leisure activities and potential occupational risk factors for tick exposure.
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2.4. Laboratory analysis All laboratory analysis was carried out at the German National Reference Centre for Borrelia and Consiliar Laboratory for Ehrlichia. For the detection of antibodies against A. phagocytophilum, an immunofluorescence antibody (IFA) test kit for IgG and IgM was used according to the manufacturer’s recommendations (Focus Diagnostics, Cypress, California). Semi-quantitative endpoint titres were obtained by serial dilution of reactive samples. IgG titres of ≥1:64 and IgM titres of ≥1:20 were regarded as positive. Quantitative endpoint titres were performed for IgG and IgM positive samples. For the detection of antibodies against B. burgdorferi the Enzygnost Lyme link, an enzyme-linked immunosorbent assay (ELISA) (Siemens Healthcare Diagnostics GmbH, Eschborn, Germany) was used. The test was automatically processed on a BEP® III (Siemens Health Diagnostics GmbH, Eschborn, Germany) and interpreted as recommended by the manufacturer. Positive or borderline results were confirmed by a line blot (Borrelia Europe plus TpN17 LINE, Virotech, Rüsselsheim, Germany). The test was performed and interpreted according to manufacturers’ recommendations. 2.5. PCR DNA extraction was performed using the High Pure PCR Template Preparation Kit (Roche Diagnostics) according to the manufacturers’ instructions. Extraction controls were included in each run. A nested PCR targeting the 16S rRNA gene was done as described previously (Massung et al., 1998) from the first sample submitted by each participant. DNA from a human patient isolate of HGA served as positive control (Goodman et al., 1996). Amplified DNA was separated by agarose gel electrophoresis, stained with ethidium bromide, and visualized under UV illumination. 2.6. Case definitions Laboratory confirmed acute infection with A. phagocyophilum: A laboratory confirmed acute infection with A. phagocytophilum was defined as one of the following findings:
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between samples, was defined as serological evidence of past exposure to B. burgdorferi s.l.
2.7. Statistical analysis For a descriptive analysis of participants continuous variables were summarised using median and range. Categorical variables were presented using absolute numbers and the proportions in percent. The univariable association between case status and exposure to individual risk factors was tested using the Chi-squared test (2 ) or the Fisher’s exact test (FE) where appropriate. The effect was quantified by estimation of the relative risk (RR) and the respective 95% confidence interval (CI). In the case of small numbers exact Poisson regression was used for estimation. Statistical significance was assumed when p < 0.05 and the 95% confidence interval of the relative risk excluded 1. When interpretation of the p-value and the 95% confidence interval were contradictory, the more conservative interpretation (not significant) was reported. Data analysis was carried out in STATA 10.1.
3. Results 3.1. Descriptive analysis A total of 107 participants were included in the study which was performed between July 2010 and August 2011. The number of participants recruited by each of the 24 participating surgeries ranged between one and 22. Fifty of the participants were male and 54 were female, there was no information on gender for three participants. The median age of participants was 59 years (range: 18–89 years). There was no information on age for five participants. The participants were resident in six of the seven Bavarian regions, with eight to 32 participants per region. There were no participants from the region of Schwaben.
3.2. Laboratory and clinical findings 1. PCR positive for A. phagocytophilum from blood confirmed by sequencing of the amplicon. 2. IgM seroconversion (seroconversion = change from negative to positive) OR a four-fold (two titre steps) or higher increase in positive IgM titre between samples tested in parallel. 3. IgG seroconversion OR a four-fold or higher increase in positive IgG titre between samples when tested in parallel. Laboratory confirmed past exposure to A. phagocytophilum: Positive anti-A. phagocytophilum IgG and/or IgM in both consecutive samples tested in parallel, with the titre remaining stable between samples, was defined as serological evidence of past exposure to A. phagocytophilum. Laboratory confirmed acute infection with B. burgdorferi s.l. A laboratory confirmed acute infection with B. burgdorferi s.l. was defined as one of the following combinations of anti- B. burgdorferi s.l. IgM and anti- B. burgdorferi s.l. IgG results of serology: 1. IgM seroconversion OR a two-fold or higher increase in positive IgM values between samples when tested in parallel. 2. IgG seroconversion OR a two-fold or higher increase in IgG units between samples when tested in parallel. Laboratory confirmed past exposure to B. burgdorferi s.l. Positive anti-B. burdorferi s.l. IgG and/or IgM in both consecutive samples tested in parallel, with the values remaining stable
Serological evidence of an acute infection with A. phagocytophilum was found for one out of 107 participants (0.9%). The IgM titre of this participant declined significantly between samples when tested in parallel from 1:160 in the first sample (on the day of tick removal by the doctor) to 1:80 in the second sample and 1:20 in the third sample, with two to three weeks between sampling time points. IgG seroconversion took place from the second to the third sampling time point. None of the blood-samples tested positive for A. phagocytophilum by PCR. The participant presented without clinical signs throughout the study and no antibiotics were prescribed. Serological evidence of an acute infection with B. burgdorferi s.l. was detected for six out of 107 participants (5.6%); two presented without clinical signs, two presented with an erythema migrans, one reported an erythema migrans and malaise and another suffered from an erythema migrans, myalgia and malaise. Antibiotics were prescribed to three of the four clinical cases and one of the two asymptomatic cases with acute B. burgdorferi s.l. infection. None of the blood samples tested were positive for A. phagocytophilum or B. burgdorferi s.l. by PCR. Serological evidence of past exposure to A. phagocytophilum was found for eight participants (7.5%) and to B. burgdorferi s.l. for 14 participants (13.1%). One of these participants showed serological evidence of past exposure to both A. phagocytophilum and B. burgdorferi s.l.
Please cite this article in press as: von Wissmann, B., et al., Assessing the risk of human granulocytic anaplasmosis and lyme borreliosis after a tick bite in Bavaria, Germany. Int. J. Med. Microbiol. (2015), http://dx.doi.org/10.1016/j.ijmm.2015.08.026
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Table 1 Proportion of participants with serological evidence of acute infection with A. phagocytophilum or B. burgdorferi s.l. or with serological evidence of past exposure to A. phagocytophilum or B. burgdorferi s.l, by age group, Bavaria 2010–2011. Age group*
Acute infection A. phagocytophilum
Past exposure A. phagocytophilum
Acute infection B. burgdorferi**
Past exposure B. burgdorferi
18–40 >40–65 >65
0.0% (0/16) 2.0% (1/51) 0.0% (0/35)
6.3% (1/16) 5.9% (3/51) 11.4% (4/35)
0.0% (0/16) 7.8% (4/51) 2.9% (1/35)
6.3% (1/16) 15.7% (8/51) 14.3% (5/35)
* **
n = 102, missing = 5. 1 acute B. burgdorferi s.l. infection amongst participant with missing age.
Table 2 Proportion of participants with serological evidence of acute infection with A. phagocytophilum or B. burgdorferi s.l. by region of tick exposure and proportion of participants with serological evidence of past exposure to A. phagocytophilum or B. burgdorferi s.l, by region of residence, Bavaria 2010–2011 (n = 107). Region: resi-dence/exposure
Mittelfranken Niederbayern Oberbayern Oberfranken Oberpfalz Unterfranken
Acute infections by region of tick exposure
Past infections by region of residence
A. phagocytophilum
B. burgdorferis.l.
A. phagocytophilum
B. burgdorferis.l.
0.0% (0/13) 0.0% (0/14) 2.9% (1/34) 0.0% (0/28) 0.0% (0/11) 0.0% (0/7)
0.0% (0/13) 14.3% (2/14) 0.0% (0/34) 10.7% (3/28) 9.1% (1/11) 0.0% (0/7)
15.4% (2/13) 0.0% (0/15) 6.3% (2/32) 10.7% (3/28) 0.0% (0/11) 12.5% (1/8)
7.7% (1/13) 6.7% (1/15) 15.6% (5/32) 17.9% (5/28) 9.1% (1/11) 12.5% (1/8)
3.3. Analytical statistics 3.3.1. Gender There were no statistically significant differences between female and male participants in the risk of acute infections (IRR = 0.93; 95%CI: 0.02–∞; p = 0.99) or past exposure to A. phagocytophilum (RR = 0.55; 95%CI: 0.14–2.2; p = 0.47). There was a lower risk of acute B. burgdorferi s.l. infections (RR = 0.19; 95%CI: 0.02–1.5; p = 0.10) and of past exposure to B. burgdorferi s.l. (RR = 0.37; 95%CI: 0.1–1.1; p = 0.08) for female than for male participants, however the difference was not statistically significant. 3.3.2. Age distribution Table 1 summarises the distribution of participants with acute infections and past exposure across age groups. The highest proportion of acute infections with both A. phagocytophilum (2.0%) and B. burgdorferi s.l. (7.8%) were detected in the age group between 40–65 years. In the age group over 65 the proportion of participants with evidence of past exposure to A. phagocytophilum was higher (11.4%) than in the two younger age groups (age group 18–40: 6.3%, age group >40–65: 5.9%). There was a higher proportion of participants with evidence of past exposure to B. burgdorferi s.l. in the two age groups over 40 (age group >40–65: 15.7%, age group >65: 14.3%) than in the age group between 18 and 40 (6.3%). However none of the differences between age groups were statistically significant. 3.3.3. Regional distribution Table 2 summarises the distribution of participants with acute infections by the region in which tick exposure took place, and the distribution of participants with past exposure by region of residence. There was no significant association between the region of Bavaria in which tick exposure took place and the proportion of participants with an acute A. phagocytophilum (p = 0.80) or an acute B. burgdorferi s.l. infection (p = 0.11). There was no significant association between the region of residence and the proportion of participants with serological evidence of past exposure to A. phagocytophilum (p = 0.34) or B. burgdorferi s.l. (p = 0.86) (Table 2). 3.3.4. Association of acute infections with activity or type of location at time of tick exposure Table 3 summarises the activities or type of location where participants contracted the most recent tick bite, which resulted in their study participation, and the risk of acute A. phagocytophilum
Table 3 Proportion of participants with acute A. phagocytphilum or B. burgdorferi s.l. infection by the type of exposure during which the most recent tick bite occured. Exposure*
Proportion of exposed participants with acute infection
Gardening or being in the garden Activity in forest Outdoor sports (e.g. walking, jogging) Occupational exposure Other Not specified *
A. phagocytophilum
B. burgdorferi s.l.
0% (0/50) 2.9% (1/34) 0% (0/29) 0% (0/4) 0% (0/11) 0% (0/6)
6.0% (3/50) 2.9% (1/34) 13.8% (4/29) 0% (0/4) 0% (0/11) 0% (0/6)
Multiple categories could be named.
or acute B. burgdorferi s.l. infection in each exposure category. There was no significant association between any of the exposures and the risk of an acute infection with A. phagocytophilum. There was an increased risk of an acute infection with B. burgdorferi s.l. for participants who did outdoor sports; this association approached statistical significance (RR = 4.9%; 95%CI: 0.95–25.3%, p = 0.06). 3.3.5. Association of past pathogen exposure with regular activities Table 4 summarises occupational or regular leisure activities of participants, during which tick exposure may occur, and the risk Table 4 Proportion of participants with past exposure to A. phagocytphilum or B. burgdorferi s.l. by regular outdoor leisure activity or occupation. Exposure*
Proportion of exposed participants with past exposure to A. phagocytophilum
Proportion of exposed participants with past exposure to B. burgdorferi s.l.
Occupational exposure (all) Hunting Other Leisure activities (all) Jogging Cycling Walking Hunting Gardening Other
13.3% (2/15) 100% (2/2) 0% (0/13) 8.9% (8/90) 6.7% (1/15) 2.9% (1/35) 5.6% (3/54) 50.0% (2/4) 6.8% (4/59) 5.3% (1/19)
20% (3/15) 50% (1/2) 15.4% (2/13) 13.3% (12/90) 13.3% (2/15) 8.6% (3/35) 16.7% (9/54) 50.0% (2/4) 5.1% (3/59) 10.5% (2/19)
*
Multiple categories could be named.
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of past exposure to A. phagocytophilum or B. burgdorferi s.l. in each exposure category. Being a hunter (leisure and occupational) was significantly associated with past exposure to A. phagocytophilum (RR = 8.2; 95%CI: 2.3–28.5, p < 0.05). Gardening was significantly associated with a reduced risk of past exposure to B. burgdorferi s.l. (RR = 0.2; 95%CI: 0.06–0.66, p < 0.01). The risk of past exposure to A. phagocytophilum associated with gardening was also reduced, however this was not statistically significant (RR = 0.7, 95%CI: 0.2–2.7; p = 0.72) There were no other statistically significant associations between regular activities and serological evidence of past exposure to either A. phagocytophilum or B. burgdorferi s.l.
4. Discussion One acute infection of A. phagocytophilum was detected by clear IgG-seroconversion during the course of the study, providing evidence that transmission of this pathogen to humans does take place in Bavaria. However, here it is of interest to note that most probably the measured immunological reaction against A. phagocytophilum was not induced by the detected tick bite but most probably resulted from a preceding – undetected – tick bite. Usually a measurable antibody response against A. phagocytophilum has still not developed in very early disease but is highly sensitive from two to four weeks after disease onset (Childs et al., 1999; Thomas et al., 2009). Therefore, in our case-tick bite serology would have been negative at least in the first serum sample that was taken directly after removal of the tick. Notably, in a long-term follow up study on 96 patients with erythema migrans, 47% of them reported an additional tick bite, a finding that further support our assumption of an overlooked preceding tick bite (Nowakowski et al., 2003). That HGA transmission occurs in Bavaria is further supported by the detection of prevalence titres to A. phagocytophilum in eight participants, indicative of past exposure. Previous entomological studies indicated that the variant of A. phagocytophilum predominantly present in the tick vector in Bavaria belongs to a cluster that is not usually associated with symptomatic HGA (Silaghi et al., 2008; Schorn et al., 2011a). Congruent with this is that no clinical signs were associated with the laboratory confirmed acute A. phagocytophilum infection detected in the current study. However, it is important to note that detection of a single asymptomatic A. phagocytophilum infection is insufficient evidence to entirely rule out the occurrence of clinically manifest HGA in Bavaria, but points to an at least very rare occurrence. Therefore, HGA should be included in the differential diagnosis for febrile illnesses after a tick bite. However, the observation does support the hypothesis that the cluster of A. phagocytophilum predominantly present in the tick vector in Bavaria is associated with low or no pathogenicity when transmitted to humans. This would also explain the absence of reports of clinical HGA cases in Bavaria, despite a prevalence of up to 9.5% in the tick vector (Schorn et al., 2011b). The proportion of participants with acute A. phagocytophilum infections after tick bite was lower than the proportion with acute B. burgdorferi s.l. infections. This difference was not statistically significant, but a lower risk of exposure to A. phagocytophilum was also reflected in a lower proportion of participants with past exposure to A. phagocytophilum than with past exposure to B. burgdorferi s.l. Even though only 15/107 participants had occupational tick exposure, the seroprevalence for A. phagocytophilum and B. burgdorferi detected in the current study was higher than the seroprevalence previously reported for populations without occupational exposure to ticks or a history of tick borne infection. This indicates that leisure time exposure considerably contributes to the risk of exposure to tick borne pathogens. No significant differences in the proportion of participants with acute infections or past exposure to either A. phagocytophilum or
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B. burgdorferi s.l. by region could be detected. However, the assessment of regional differences in the risk of infection was restricted to six of the seven Bavarian regions due to a lack of participants from the 7th region, and the sample size per region was small. Stratified sampling by region would be required to allow more detailed investigations of geographical variations in A. phagocytophilum or B. burgdorferi s.l. seroprevalence. The study was designed to provide proof of presence or absence of acute A. phagocytophilum infection after tick bites in Bavaria. Due to the sample size, the study had insufficient power to detect small differences between risk factors. Nonetheless, investigation of the type of activity during which tick exposure took place showed that outdoor sports were associated with an increased risk of acute infections of B. burgdorferi s.l. and hunting was associated with past exposure to A. phagocytophilum and B. burgdorferi s.l. As only a single acute infection of A. phagocytophilum was detected, no significant association with any particular activity could be shown. However the participant in question acquired the documented tick bite whilst being in woodlands. On the other hand, this infection was most probably acquired from a different tick bite a few days previously, a finding that illustrates the difficulties in interpreting results from tick-bite studies. Activities in the garden apparently posed a comparatively low risk of acquisition of A. phagocytophilum or B. burgdorferi s.l. infections, as reflected in a lower seroprevalence associated with this category. This may be due to differences in the prevalence of infection in the tick vector depending on the type of habitat. An alternative explanation is a lower frequency of contact with ticks, and thus a lower cumulative risk of exposure to tick borne pathogens associated with gardening as compared to hunting. However, a large number of participants (50/107) in the current study remember contracting their most recent tick bite in their gardens and half of the acute B. burgdorferi s.l infections could be attributed to ticks contracted whilst gardening. Only 1/5 of the participants with past exposure to B. burgdorferi s.l., but half of the participants with past exposure to A. phagocytophilum listed gardening as a regular leisure activity. This is likely to be a reflection of a large part of the population in Bavaria spending time in the garden, whilst only a much smaller fraction of the population are involved in hunting. Despite a comparatively low risk of exposure to tick borne pathogens associated with gardening, the large number of people involved in this activity is therefore likely to result in a high absolute burden of tick borne infections. Public health measures to increase awareness for tick borne infections should therefore also be targeted at the large part of the population who are involved in comparatively low risk outdoor activities such as gardening, cycling or walking. 5. Conflict of interest statement The authors declare no conflict of interest. Acknowledgements The authors are grateful to Wiltrud Strehle and Silvia Stockmeier for excellent technical assistance, Katharina Alpers und Manuel Dehnert for valuable discussions and help with the study design and the Bavarian Ministry for Health and Care and the Robert-KochInstitut, Berlin, for funding the study. Volker Fingerle and Andreas Sing are ESGBOR members. References Aguero-Rosenfeld, M.E., Horowitz, H.W., Wormser, G.P., McKenna, D.F., Nowakowski, J., Munoz, J., Dumler, J.S., 1996. Human granulocytic ehrlichiosis: a case series from a medical center in New York State. Ann. Intern. Med. 125, 904–908.
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Please cite this article in press as: von Wissmann, B., et al., Assessing the risk of human granulocytic anaplasmosis and lyme borreliosis after a tick bite in Bavaria, Germany. Int. J. Med. Microbiol. (2015), http://dx.doi.org/10.1016/j.ijmm.2015.08.026