- Email: [email protected]
Candidatus Neoehrlichia mikurensis and Borrelia burgdorferi sensu lato detected in the blood of Norwegian patients with erythema migrans
Ticks and Tick-borne Diseases xxx (xxxx) xxx–xxx
Contents lists available at ScienceDirect
Ticks and Tick-borne Diseases journal homepage: www.elsevier.com/locate/ttbdis
Original article
Candidatus Neoehrlichia mikurensis and Borrelia burgdorferi sensu lato detected in the blood of Norwegian patients with erythema migrans ⁎
H. Quarstena, , A. Grankvistb, L. Høyvollc, I.B. Myred, T. Skarpaasa, V. Kjellandd,e, C. Wennerasb, S. Noraasa a
Sørlandet Hospital Health Enterprise, Department of Medical Microbiology, Egsveien 100, PO-Box 416, NO-4604, Kristiansand, Norway University of Gothenburg, Department of Infectious Diseases, Guldhedsgatan 10, 413 46 Göteborg, Sweden c Sørlandet Hospital Health Enterprise, Medical Department, Egsveien 100, PO-Box 416, NO-4604 Kristiansand, Norway d University of Agder, Faculty of Engineering and Science, Department of Natural Sciences, Gimlemoen 25, PO-Box 422, NO-4604 Kristiansand, Norway e Sørlandet Hospital Health Enterprise, Research Unit, Egsveien 100, PO-Box 416, NO-4604 Kristiansand, Norway b
A R T I C L E I N F O
A B S T R A C T
Keywords: Tick-borne disease Candidatus Neoehrlichia mikurensis Borrelia burgdorferi sensu lato Lyme borreliosis Real-time PCR
The most common tick-borne human disease in Norway is Lyme borreliosis. Ticks in Norway also harbour less known disease-causing agents such as Candidatus Neoehrlichia mikurensis, Borrelia miyamotoi and Rickettsia helvetica. However, human infections caused by these pathogens have never been described in Norway. The main aims of the study were to evaluate the contribution of several tick-borne bacterial agents, other than Borrelia burgdorferi sensu lato, to zoonotic diseases in Norway and to determine their clinical pictures. Blood samples from 70 symptomatic tick-bitten adults from the Agder counties in southern Norway were screened for seven tick-borne pathogens by using a commercial multiplex PCR-based method and by singleplex real-time PCR protocols. Most patients (65/70) presented with a rash clinically diagnosed as erythema migrans (EM). The most frequently detected pathogen DNA was from Ca. N. mikurensis and was found in the blood of 10% (7/70) of the patients. The Ca. N. mikurensis-infected patients presented with an EM-like rash as the only symptom. B. burgdorferi s.l. DNA was present in the blood of 4% (3/70) of the study participants. None had detectable Anaplasma phagocytophilum, B. miyamotoi, Rickettsia typhus group or spotted fever group, Francisella tularensis, Coxiella burnetii or Bartonella spp. DNA in the blood. The commercially available multiplex PCR bacteria flow chip system failed to identify half of the infected patients detected by corresponding real-time PCR protocols. The recovery of Ca. N. mikurensis DNA was higher in the pellet/plasma fraction of blood than from whole blood. To conclude, Ca. N. mikurensis appeared to be the etiological agent in patients with EM in a surprisingly large fraction of tick-bitten persons in the southern part of Norway.
1. Introduction In Norway, the only frequently diagnosed human tick-borne disease is Lyme borreliosis (LB) with 300–400 cases of disseminated disease reported annually in a population of 5 million. Other diseases transmitted by ticks are rarely diagnosed; only 10–20 cases of tick-borne encephalitis (TBE) are reported yearly, and occasional cases of human granulocytic anaplasmosis and babesiosis have been described (Kristiansen et al., 2001; Morch et al., 2015). The incidence of human tick-borne disease in Norway is restricted by the distribution of Ixodes ricinus, the only significant tick vector of
human pathogens in the region. I. ricinus in Norway have been shown to harbour Borrelia burgdorferi sensu lato, Borrelia miyamotoi, Anaplasma phagocytophilum, Babesia spp., Candidatus Neoehrlichia mikurensis, Rickettsia helvetica and TBE-virus (Jenkins and Kristiansen, 2013; Kjelland et al., 2015; Oines et al., 2012; Quarsten et al., 2015). Ca. N. mikurensis, B. miyamotoi and R. helvetica are novel pathogens that have been associated with human disease in various European countries (Nilsson et al., 1999; Platonov et al., 2011; Welinder-Olsson et al., 2010), but not in Norway. It is unknown whether this is due to the existence of non-pathogenic strains, strains of low virulence giving rise to mild disease or due to low awareness in the healthcare sector leading
Abbreviations: LB, Lyme borreliosis; EM, erythema migrans; PCR-FCS, multiplex PCR bacteria flow chip system; SFG, spotted fever group; TG, typhus group; TBE, tick-borne encephalitis; Spp, species ⁎ Corresponding author. E-mail address: [email protected] (H. Quarsten). http://dx.doi.org/10.1016/j.ttbdis.2017.05.004 Received 26 January 2017; Received in revised form 9 May 2017; Accepted 10 May 2017 1877-959X/ © 2017 Elsevier GmbH. All rights reserved.
Please cite this article as: Quarsten, H., Ticks and Tick-borne Diseases (2017), http://dx.doi.org/10.1016/j.ttbdis.2017.05.004
Ticks and Tick-borne Diseases xxx (xxxx) xxx–xxx
H. Quarsten et al.
(< 10 weeks before blood sampling) tick bite. Out of 81 patients who were recruited to the study, 70 patients fulfilled the study criteria and were included in the study. Eleven patients did not meet the inclusion criteria; six did not have a recent tick bite, two patients were tick-bitten but did not contract any symptoms, and three patients either lacked written consent, critical information (about tick bite, symptoms and treatment) or the correct type of blood samples. Patients with characteristic EM diagnosed by a GP who did not recall tick bites (9/70) were accepted as study participants. The included patients answered questions regarding recent history of tick bite, EM, other symptoms and antibiotic treatment, but no patient histories were taken. 2.3. Ethical statements The study was approved by the Norwegian Regional Committee for Medical and Health Research Ethics, the South-Eastern region (2014/ 178), and all participants provided written informed consent to take part in the study. 2.4. DNA sample preparation Blood samples (2–8 ml EDTA blood and serum) were collected from the study participants by their GP and sent to the Department of Medical Microbiology (Kristiansand, Norway) by postal service. DNA was isolated from two different fractions of EDTA blood on the day of arrival in the laboratory: (1) Whole blood fraction; 200 μl blood was collected after inverting of the tube 5–6 times and used for extraction of total DNA by QIAamp DNA Mini Kit (Qiagen, Venlo, The Netherlands) as described by the manufacturer's instructions. (2) Pellet/plasma fraction; the remaining blood was centrifuged at 100 × g for 15 min to remove erythrocytes and all plasma/buffy coat was collected and centrifuged at 10,000 × g for 60 min in order to pellet bacteria together with leukocytes and platelets (Cerar et al., 2008). All the pelleted material was resuspended in 200 μl plasma and either processed immediately or stored at −70 °C until processing. The pellet/plasmasample was incubated in ATL lysis buffer (QIAamp DNA Mini kit) supplemented with proteinase K (1 mg/ml final concentration) for 1 h at 56 °C. Thereafter, the sample was processed as described by the manufacturer's instructions (QIAamp DNA Mini kit). All DNA extracts were stored at −20 °C for later analyses.
Fig. 1. The present distribution of I. ricinus in Norway depicted by a multi-source analysis (Jore et al., 2011). The distribution of ticks is mainly in coastal regions up to an altitude of 583 m above sea level and 66° north. The colour intensity represents the density of ticks from no ticks in the light yellow areas to high density in the darkest areas. The study patients were recruited from the coastal area in the south. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of the article.)
to under-diagnosis of certain tick-borne diseases. The focus of the study was (a) to evaluate to what extent tick-borne bacterial agents other than B. burgdorferi s.l. cause infectious disease in Norway, (b) to describe the clinical picture of non-B. burgdorferi s.l. tick-borne infections, (c) to evaluate a novel commercially available multiplex PCR bacteria flow chip system (PCR-FCS) for the detection of tick-borne bacteria by comparing performance with conventional established real-time PCR protocols, and (d) to evaluate which fraction of blood to use for molecular detection of tick-borne pathogens.
2.5. Tick-borne bacteria flow chip system A tick-borne bacteria flow chip system (PCR-FCS) from Master Diagnostica (Granada, Spain) was used for detection of DNA from tickborne bacteria by multiplex PCR, followed by automated reverse dot blot hybridisation with specific probes directed against Borrelia spp., A. phagocytophilum, Rickettsia typhus group (TG) and spotted fever group (SFG), Francisella tularensis, Bartonella spp., Coxiella burnetii and Ehrlichia (Ca. N. mikurensis, E. chaffeensis and E. ewingii). The samples were analysed according to the manufacturer's instructions. All samples were analysed in one replicate.
2. Materials and methods 2.1. Patient recruitment strategy Information regarding the study was disseminated to the public in the Agder counties (Fig. 1) through the internet, local newspapers, radio and posters set up at general practitioner (GP) offices in order to recruit patients with flu-like illness and/or EM after recent (within 10 weeks before inclusion in study) tick bite. The health workers at the general practices received information regarding the study by internet, email and ordinary postal service.
2.6. Real-time PCR All samples were tested by singleplex real-time PCR protocols for detection of B. burgdorferi s.l. (two independent assays targeting the ospA and 16S rRNA genes), B. miyamotoi (16S rRNA), A. phagocytophilum (groESL), Rickettsia TG and SFG (gltA), F. tularensis (fopA), Bartonella spp. (gltA), C. burnetii (icd) and Ca. N. mikurensis (groEL). Sequences, concentrations and thermocycling conditions of the TaqMan-based realtime PCR directed against a 169-bp fragment of the Ca. N. mikurensis groEL gene have been reported previously (Grankvist et al., 2015b). The sequences and final concentrations of all other primers and probes (except for B. miyamotoi) including the PCR thermocycling conditions used for the real-time PCR protocols have been published elsewhere
2.2. Patients From June 2014 through December 2015, patients were included in the study by their GPs. The inclusion criteria for participation in the study were (1) age 18 years or older, (2) ongoing symptoms after recent 2
Ticks and Tick-borne Diseases xxx (xxxx) xxx–xxx
H. Quarsten et al.
(Quarsten et al., 2015). The primers and probe for the B. miyamotoispecific real-time PCR (Tsao et al., 2004) were used at 0.5 μM and 0.2 μM, respectively, under the same thermocycling conditions employed in the B. burgdorferi s.l. protocols. All protocols were performed using 5 μl of DNA in a 15 μl reaction mixture consisting of 5 mM MgCl2, 0.5 U uracil DNA-glycosylase (Eurogentec S.A. Seraing, Belgium), and LightCycler FastStart DNA master mix (Roche) with primers and probe(s). All samples were analysed in one replicate by each real-time PCR protocol, except for the Ca. N. mikurensis protocol where samples were analysed in duplicate. All positive samples, except for one, were confirmed positive in a re-run. Samples positive for B. burgdorferi s.l. were confirmed and genotyped by direct sequencing of the rrs (16S)–rrlA (23S) intergenic spacer as previously described (Kjelland et al., 2010).
Table 1 Tick-borne bacteria detected in patient blood by two molecular methods. No. of patients with bacterial DNA in blood (%) Ca. N. mikurensis B. burgdorferi s.l. DNA DNA
Bacterial DNA (total)
Real-time singleplex PCR n = 70 PCR-FCS n = 69 n = 1 NAa
7 (10)
3 (4)
10 (14)
4 (6)
1 (1)
5 (7)
Both methods
4 (6)
1 (1)
5 (7)
Abbreviations: PCR-FCS multiplex PCR bacteria flow chip system (Master Diagnostica). a NA: not analysed; the sample was negative in all real-time protocols.
2.7. B. burgdorferi s.l. serology
blood. B. burgdorferi s.l. DNA was identified in 4% (3/70) of the patient samples by B. burgdorferi s.l.-specific real-time PCR protocols (ospA and/or 16S rRNA); 2/3 of the samples were successfully sequenced as B. garinii and B. afzelii, respectively. The third isolate could not be typed, possibly due to low amount of bacterial DNA in the sample. No A. phagocytophilum, Rickettsia spp., F. tularensis, C. burnetii or Bartonella spp. DNA were detected in any of the patient samples.
All patient serum samples collected at the start of the study, as well as follow-up serum samples from patients positive for bacterial DNA in the blood were analysed for antibodies against B. burgdorferi s.l. by using the Siemens Enzygnost Borreliosis/IgM and Lymelink VlsE/IgG ELISA kits (Marburg, Germany) with the manufacturer's cut-off levels and interpretation criteria. For detection of IgM and IgG in serum, the 98% specific diagnostic cut-offs were OD = 0.29 and 0.17, respectively.
3.3. Comparison of molecular tests and choice of blood fraction 2.8. Nucleotide sequence accession numbers Patient samples were additionally analysed by the multiplex PCRFCS kit (Table 1). Only 6% (4/69) Ca. N. mikurensis- and 1% (1/69) Borrelia spp.-infected samples were identified, i.e. the kit missed 3/7 Ca. N. mikurensis- and 2/3 B. burgdorferi s.l.-infected samples that were detected by the singleplex real-time PCR protocols. No samples were identified as infected by the PCR-FCS method only. Most of the samples with discrepant results had high cycle threshold values in real-time PCR, indicating that the real-time protocols have higher sensitivity than the PCR-FCS. DNA was isolated from two different fractions of patient blood: whole blood and a pellet/plasma fraction where extracellular and leukocyte-associated bacteria were expected to be concentrated by centrifugation. Among the Ca. N. mikurensis-positive patients, 6/7 had detectable Ca. N. mikurensis DNA in the pellet/plasma fraction, but only 3/7 were positive in the whole blood fraction (Table 2). Regarding B. burgdorferi s.l. DNA, one isolate was only recovered from the pellet/ plasma fraction, one isolate was only recovered from the whole blood fraction, and one isolate was recovered from both fractions (Table 2).
The sequences obtained in this study have been deposited in GenBank with accession numbers KY_782011 (B. afzelii) and KY_782012 (B. garinii). 2.9. Statistics Fisher's exact test was employed at a significance level of P < 0.05 (GraphPad Prism 6.0 software, San Diego, CA) in order to compare the frequency of symptoms other than EM in the Ca. N. mikurensis DNApositive (1/7) and pathogen DNA-negative patient groups (29/60). 3. Results 3.1. Patients The study comprised 70 patients presenting with the symptoms EM and/or flu-like illness after tick bite. The study cohort had a female to male ratio of 1.6:1 and a median age of 55 years. There were no restrictions regarding underlying diseases and/or current medication. Most study patients (61/70) reported a recent tick bite, less than ten weeks before the time of blood sampling. Nine patients were included with characteristic EM, diagnosed by a GP, without recalling a tick bite. Ten of the patients reported they had had antibiotics treatment within the previous three months. All except for five study participants developed EM. The ones without a rash experienced flu-like symptoms such as muscle pain, headache, fatigue, fever, and chills. The patients who had EM reported varying degrees of flu-like illness.
3.4. Patient symptoms Of the 70 patients included, 65 patients had EM alone or together Table 2 Detection of tick-borne bacterial DNA in blood sample fractions. PCR results
3.2. Detection of bacterial pathogen DNA in patient blood DNA from the whole blood and pellet/plasma fractions from each patient's blood sample was analysed by the real-time PCR methods for detection of the seven tick-borne pathogens. Patients from whom bacterial DNA was detected in either one or both of the blood fractions were considered infected. Bacterial DNA was demonstrated in 14% (10/ 70) of the patient samples in total (Table 1). Two out of the seven tested tick-borne bacterial pathogens were identified in blood of the symptomatic tick-bitten patients. Ca. N. mikurensis DNA was detected in 10% (7/70) of the patients and was the pathogen DNA most often detected in 3
Bacteria
Patient (no.)
Whole blood
Plasma/pellet
Ca. N. mikurensis
1 2 3 4 5 6 7 Positive fractions
Neg Neg Neg Pos Pos Pos Neg 3/7
Pos Pos Pos Neg Pos Pos Pos 6/7
B. burgdorferi s.l.
8 9 10 Positive fractions
Pos Neg Pos 2/3
Pos Pos Neg 2/3
Ticks and Tick-borne Diseases xxx (xxxx) xxx–xxx
H. Quarsten et al.
problems) during treatment but recovered completely after switching to doxycycline. Furthermore, B. burgdorferi s.l. DNA was not detected in the follow-up sample. Since there are no serological Ca. N. mikurensis assays available, it was not possible to screen Ca. N. mikurensis-positive patient samples for specific antibodies. However, no change in serological B. burgdorferi s.l. status was observed in the follow-up serum samples of Ca. N. mikurensis-patients, hence there was no serologic evidence of co-infections with B. burgdorferi s.l.
Table 3 Symptoms reported by the study participants. Number of patients Pathogen DNA detected in blood
Erythema migrans
Other symptoms
Ca. N. mikurensis B.burgdorferi s.l.
7/7 3/3
None
55/60
Fatigue 1/7 Myalgia 2/3 Unspecified ear problems 1/3 Flu-like symptoms 29/60a
Total
65/70
a
3.5. Patient follow-up The patients identified as infected (in total 10/70) by PCR, all presented with an EM rash at the time of inclusion and blood sampling. They were treated with antibiotics (penicillin or doxycycline) according to Norwegian guidelines for treatment of LB. New blood samples drawn after six weeks or later following the first detection of bacterial DNA in 9/10 patients were all negative for bacterial DNA. One patient did not provide a follow-up sample.
Muscle pain, headache, fatigue, fever, chills, pain.
with various degrees of flu-like illness and five patients had flu-like symptoms only after recent tick bite (Table 3). All of the Ca. N. mikurensis-positive patients presented with EM. Whereas 1/7 of the patients in the Ca. N. mikurensis DNA-positive group reported symptoms (fatigue) other than EM, flu-like symptoms were experienced by 29/60 in the pathogen DNA-negative patient group (Not significant; Fisher's exact test). None of the Ca. N. mikurensis patients presented with flu-like symptoms, in contrast, two out of three B. burgdorferi s.l. PCR-positive patients experienced muscle pain and one of them also reported unspecified ear problems.
4. Discussion In this study, Ca. N. mikurensis (10%) and B. burgdorferi s.l. (4%) DNA was detected in the blood of recently tick-bitten, symptomatic adult persons from the Agder counties in southern Norway. Among the 70 patients examined, none had detectable A. phagocytophilum, B. miyamotoi, Rickettsia TG or SFG, F. tularensis, C. burnetii or Bartonella spp. DNA in the blood. We provide the first record of human Ca. N. mikurensis infections in Norway. Ca. N. mikurensis is widespread among ticks in Europe (Wenneras, 2015). The pathogen has previously been detected in Norwegian ticks with a prevalence of 2–12% (Jenkins and Kristiansen, 2013). A high prevalence of Ca. N. mikurensis in ticks collected in southern Norway (17% (13/76); own unpublished data), may explain the relatively high infection rate documented in the study patients with 10% of them being infected by Ca. N. mikurensis. In comparison, 1.4% (7/291) of tick-bitten patients with EM in the Netherlands had Ca. N. mikurensis DNA in their blood, and it was estimated that the prevalence of Ca. N. mikurensis in ticks obtained from tick-bitten individuals was 5% (Jahfari et al., 2016). Establishment of a serological test for monitoring Ca. N. mikurensis antibodies,
3.4.1. B. burgdorferi s.l. serology All acute serum samples (except for one, due to lack of material) were analysed for B. burgdorferi s.l.-specific IgM and IgG antibodies. The B. burgdorferi s.l. IgG seroprevalence in this cohort was 39%: IgG alone was detected in close to 28% of the samples, whereas in nearly 12% of the samples both IgM and IgG were detected. Presence of IgM alone (12%) was considered to be unspecific. Two out of the three patients with B. burgdorferi s.l. DNA in blood at the time of inclusion had detectable B. burgdorferi s.l.-specific IgM or IgM/IgG in serum (Table 4). Their antibody level remained unchanged after antibiotic treatment. The third B. burgdorferi s.l. DNA-positive patient seroconverted for B. burgdorferi s.l. IgM and IgG during treatment with penicillin. This particular patient complained of a lack of improvement of symptoms (muscle pain and unspecified ear
Table 4 Borrelia antibody titers in acute and follow-up serum samples of patients with bacterial DNA in blood. Borrelia burgdorferi s.l. serum antibody titer (% cut off) Findings
Case no.
Sample type
IgM−/IgG−
Ca. N. mikurensis
1
Acute Follow-up Acute Follow-up Acute Follow-up Acute Follow-up Acute Follow-up Acute Follow-up Acute Follow-up
–/– nt/nt –/– –/– nt/nt –/–
Acute Follow-up Acute Follow-up Acute Follow-up
–/–
2 3 4 5 6 7 B. burgdorferi s.l.
8 9 10
IgM+/IgG−
IgM−/IgG+
IgM+/IgG+
Interpretation No change in antibody status
–/314 109/383 –/1503 –/1530 286/203 262/163 229/225 143/222 Seroconversion 657/837 –/284 –/201 222/– 166/–
Abbreviations: nt: not tested.
4
No change in antibody status
Ticks and Tick-borne Diseases xxx (xxxx) xxx–xxx
H. Quarsten et al.
an increased B. burgdorferi s.l. DNA yield compared to its recovery from whole blood. However, our data do not support this notion, probably due to small sample size of B. burgdorferi s.l.-positive findings. Consequently, we recommend isolating DNA from both the whole blood and the plasma/pellet fraction in order to increase the chance of detection of tick-borne pathogens. It should be emphasised that serology, not PCR analysis of blood samples, is the preferred method for diagnosing LB. However, a novel pathogenic Borrelia species, B. mayonii, causes LB with an unusually high bacteraemia and PCR analysis of blood may have a potential role for diagnosing infections caused by this pathogen (Pritt et al., 2016).
which is lacking, will in the future provide more information on the seroprevalence and risk of transmission of Ca. N. mikurensis to human beings after tick bites. The first reports of Ca. N. mikurensis as the cause of human disease were published in 2010 and concerned immunocompromised patients (Fehr et al., 2010; von Loewenich et al., 2010; Welinder-Olsson et al., 2010). Among this category of patients, symptoms such as remitting fever and pain in joints and muscles may be rather severe (Grankvist et al., 2014). Moreover, a high incidence of thromboembolic complications is seen among immunocompromised Ca. N. mikurensis-infected patients (Wenneras, 2015). However, Ca. N. mikurensis infections have also been demonstrated in immunocompetent, healthy subjects with varying clinical presentations ranging from no symptoms at all (WelcFaleciak et al., 2014) or only EM in Europe (Grankvist et al., 2015b) to febrile disease in China (Li et al., 2012). This seemingly lower virulence among European versus Asian strains of Ca. N. mikurensis may be due to the documented genotypic differences between Asian and European isolates (Grankvist et al., 2015a; Wenneras, 2015). Although no personal histories were taken, we assume that the majority, if not all patients in this study, were immunocompetent, including the Ca. N. mikurensis-infected patients. Thus, their lack of fever and other flu-like symptoms are in accordance with previous case histories of neoehrlichiosis among non-immunocompromised patients in Europe. Possible co-infection of B. burgdorferi s.l. and Ca. N. mikurensis in a patient has previously been reported in Sweden (Grankvist et al., 2015b). Whether any of the seven Ca. N. mikurensis-infected patients in the present study were co-infected with B. burgdorferi s.l. cannot be determined with certainty. It is estimated that up to 20% of skin biopsies collected from EM-like rashes are negative for B. burgdorferi s.l. DNA (Sjowall et al., 2011), which raises the question whether other microbes might give rise to similar rashes, Ca. N. mikurensis, for instance. It has been reported that prolonged Ca. N. mikurensis DNA carriage may occur in immunocompetent persons: two Ca. N. mikurensispositive patients with EM who were treated with penicillin, which does not eradicate Ca. N. mikurensis, due to suspected LB had detectable Ca. N. mikurensis DNA in follow-up blood samples collected 1–3 months later (Grankvist et al., 2015b). However, Ca. N. mikurensis DNA was not detected in the 6-week follow-up blood samples in the present study, indicating that all patients had either been successfully treated with antibiotics (the two who received doxycycline) or recovered spontaneously (the five who were treated with penicillin). Patient samples were analysed both by real-time PCR protocols and the commercial multiplex PCR-FCS method. The PCR-FCS has previously been demonstrated to have slightly lower sensitivity for the detection of pathogens in ticks compared with singleplex PCRs (Quarsten et al., 2015). In the present study, the PCR-FCS-method identified only half of the patients with tick-borne infections compared with the real-time PCR protocols. The most probable reason for the marked difference in performance is that the patient group in this study had low quantities of bacteria in the blood, which is to be expected in immunocompetent (Grankvist et al., 2015b) compared to immunocompromised individuals (Grankvist et al., 2014, 2015b). Thus, a small difference in sensitivity will have a major influence on the number of infected immunocompetent patients that may be identified. In patients with high-level bacteraemia, the discrepancy in the performance of the methods may not be as prominent. The tissue tropism of Ca. N. mikurensis is not known, though it is likely that infection of endothelial cells is involved (Kawahara et al., 2004). The pellet/plasma fraction was clearly superior for detection of Ca. N. mikurensis in blood in this study. Possible explanations are that Ca. N. mikurensis has invaded or been phagocytosed by white blood cells and/or that free bacteria derived from disrupted endothelial cells are more easily recovered from this fraction. Along this line, the B. burgdorferi s.l. spirochetes being extracellular organisms should be enriched by centrifugation of the pellet/plasma fraction, resulting in
5. Conclusions This study provides the first record of Ca. N. mikurensis infections in recently tick-bitten individuals in Norway. The Ca. N. mikurensisinfected patients all presented with an EM-like skin rash. Ca. N. mikurensis is optimally detected in the pellet/plasma fraction where the pathogen is likely to be concentrated. Real-time PCR methods identified twice as many Ca. N. mikurensis- and B. burgdorferi s.l.infected individuals as a commercial tick-borne bacteria flow chip kit (PCR-FCS). Whether a causal relationship exists between Ca. N. mikurensis and EM remains to be established. Acknowledgment This work was partly supported by The EU-Interreg ÖKS project ScandTick Innovation. References Cerar, T., Ogrinc, K., Cimperman, J., Lotric-Furlan, S., Strle, F., Ruzic-Sabljic, E., 2008. Validation of cultivation and PCR methods for diagnosis of Lyme neuroborreliosis. J. Clin. Microbiol. 46, 3375–3379. Fehr, J.S., Bloemberg, G.V., Ritter, C., Hombach, M., Luscher, T.F., Weber, R., Keller, P.M., 2010. Septicemia caused by tick-borne bacterial pathogen Candidatus Neoehrlichia mikurensis. Emerg. Infect. Dis. 16, 1127–1129. Grankvist, A., Andersson, P.O., Mattsson, M., Sender, M., Vaht, K., Hoper, L., Sakiniene, E., Trysberg, E., Stenson, M., Fehr, J., Pekova, S., Bogdan, C., Bloemberg, G., Wenneras, C., 2014. Infections with the tick-borne bacterium “Candidatus Neoehrlichia mikurensis” mimic noninfectious conditions in patients with B cell malignancies or autoimmune diseases. Clin. Infect. Dis. 58, 1716–1722. Grankvist, A., Moore, E.R., Svensson Stadler, L., Pekova, S., Bogdan, C., Geissdorfer, W., Grip-Linden, J., Brandstrom, K., Marsal, J., Andreasson, K., Lewerin, C., WelinderOlsson, C., Wenneras, C., 2015a. Multilocus sequence analysis of clinical “Candidatus Neoehrlichia mikurensis” strains from Europe. J. Clin. Microbiol. 53, 3126–3132. Grankvist, A., Sandelin, L.L., Andersson, J., Fryland, L., Wilhelmsson, P., Lindgren, P.E., Forsberg, P., Wenneras, C., 2015b. Infections with Candidatus Neoehrlichia mikurensis and cytokine responses in 2 persons bitten by ticks, Sweden. Emerg. Infect. Dis. 21, 1462–1465. Jahfari, S., Hofhuis, A., Fonville, M., van der Giessen, J., van Pelt, W., Sprong, H., 2016. Molecular detection of tick-borne pathogens in humans with tick bites and erythema migrans, in the Netherlands. PLoS Negl. Trop. Dis. 10, e0005042. Jenkins, A., Kristiansen, B.E., 2013. Neoehrlichia—a new tick-borne bacterium. Tidsskr. Nor. Laegeforen. 133, 1058–1059. Jore, S., Viljugrein, H., Hofshagen, M., Brun-Hansen, H., Kristoffersen, A.B., Nygard, K., Brun, E., Ottesen, P., Saevik, B.K., Ytrehus, B., 2011. Multi-source analysis reveals latitudinal and altitudinal shifts in range of Ixodes ricinus at its northern distribution limit. Parasites Vectors 4, 84. Kawahara, M., Rikihisa, Y., Isogai, E., Takahashi, M., Misumi, H., Suto, C., Shibata, S., Zhang, C., Tsuji, M., 2004. Ultrastructure and phylogenetic analysis of ‘Candidatus Neoehrlichia mikurensis’ in the family Anaplasmataceae, isolated from wild rats and found in Ixodes ovatus ticks. Int. J. Syst. Evol. Microbiol. 54, 1837–1843. Kjelland, V., Rollum, R., Korslund, L., Slettan, A., Tveitnes, D., 2015. Borrelia miyamotoi is widespread in Ixodes ricinus ticks in southern Norway. Ticks Tick Borne Dis. 6, 516–521. Kjelland, V., Stuen, S., Skarpaas, T., Slettan, A., 2010. Prevalence and genotypes of Borrelia burgdorferi sensu lato infection in Ixodes ricinus ticks in southern Norway. Scand. J. Infect. Dis. 42, 579–585. Kristiansen, B.E., Jenkins, A., Tveten, Y., Karsten, B., Line, O., Bjoersdorff, A., 2001. Human granulocytic ehrlichiosis in Norway. Tidsskr. Nor. Laegeforen. 121, 805–806. Li, H., Jiang, J.F., Liu, W., Zheng, Y.C., Huo, Q.B., Tang, K., Zuo, S.Y., Liu, K., Jiang, B.G., Yang, H., Cao, W.C., 2012. Human infection with Candidatus Neoehrlichia mikurensis, China. Emerg. Infect. Dis. 18, 1636–1639. Morch, K., Holmaas, G., Frolander, P.S., Kristoffersen, E.K., 2015. Severe human Babesia divergens infection in Norway. Int. J. Infect. Dis. 33, 37–38.
5
Ticks and Tick-borne Diseases xxx (xxxx) xxx–xxx
H. Quarsten et al.
Th1-type inflammatory cytokine expression in the skin is associated with persisting symptoms after treatment of erythema migrans. PLoS ONE 6, e18220. Tsao, J.I., Wootton, J.T., Bunikis, J., Luna, M.G., Fish, D., Barbour, A.G., 2004. An ecological approach to preventing human infection: vaccinating wild mouse reservoirs intervenes in the Lyme disease cycle. Proc. Natl. Acad. Sci. U.S.A. 101, 18159–18164. von Loewenich, F.D., Geissdorfer, W., Disque, C., Matten, J., Schett, G., Sakka, S.G., Bogdan, C., 2010. Detection of “Candidatus Neoehrlichia mikurensis” in two patients with severe febrile illnesses: evidence for a European sequence variant. J. Clin. Microbiol. 48, 2630–2635. Welc-Faleciak, R., Sinski, E., Kowalec, M., Zajkowska, J., Pancewicz, S.A., 2014. Asymptomatic “Candidatus Neoehrlichia mikurensis” infections in immunocompetent humans. J. Clin. Microbiol. 52, 3072–3074. Welinder-Olsson, C., Kjellin, E., Vaht, K., Jacobsson, S., Wenneras, C., 2010. First case of human “Candidatus Neoehrlichia mikurensis” infection in a febrile patient with chronic lymphocytic leukemia. J. Clin. Microbiol. 48, 1956–1959. Wenneras, C., 2015. Infections with the tick-borne bacterium Candidatus Neoehrlichia mikurensis. Clin. Microbiol. Infect. 21, 621–630.
Nilsson, K., Lindquist, O., Pahlson, C., 1999. Association of Rickettsia helvetica with chronic perimyocarditis in sudden cardiac death. Lancet 354, 1169–1173. Oines, O., Radzijevskaja, J., Paulauskas, A., Rosef, O., 2012. Prevalence and diversity of Babesia spp. in questing Ixodes ricinus ticks from Norway. Parasites Vectors 5, 156. Platonov, A.E., Karan, L.S., Kolyasnikova, N.M., Makhneva, N.A., Toporkova, M.G., Maleev, V.V., Fish, D., Krause, P.J., 2011. Humans infected with relapsing fever spirochete Borrelia miyamotoi, Russia. Emerg. Infect. Dis. 17, 1816–1823. Pritt, B.S., Mead, P.S., Johnson, D.K., Neitzel, D.F., Respicio-Kingry, L.B., Davis, J.P., Schiffman, E., Sloan, L.M., Schriefer, M.E., Replogle, A.J., Paskewitz, S.M., Ray, J.A., Bjork, J., Steward, C.R., Deedon, A., Lee, X., Kingry, L.C., Miller, T.K., Feist, M.A., Theel, E.S., Patel, E.S., Irish, C.L., Petersen, J.M., 2016. Identification of a novel pathogenic Borrelia.species causing Lyme borreliosis with unusually high spirochaetaemia: a descriptive study. Lancet Infect. Dis. 16, 556–564. Quarsten, H., Skarpaas, T., Fajs, L., Noraas, S., Kjelland, V., 2015. Tick-borne bacteria in Ixodes ricinus collected in southern Norway evaluated by a commercial kit and established real-time PCR protocols. Ticks Tick Borne Dis. 6, 538–544. Sjowall, J., Fryland, L., Nordberg, M., Sjogren, F., Garpmo, U., Jansson, C., Carlsson, S.A., Bergstrom, S., Ernerudh, J., Nyman, D., Forsberg, P., Ekerfelt, C., 2011. Decreased
6
Contents lists available at ScienceDirect
Ticks and Tick-borne Diseases journal homepage: www.elsevier.com/locate/ttbdis
Original article
Candidatus Neoehrlichia mikurensis and Borrelia burgdorferi sensu lato detected in the blood of Norwegian patients with erythema migrans ⁎
H. Quarstena, , A. Grankvistb, L. Høyvollc, I.B. Myred, T. Skarpaasa, V. Kjellandd,e, C. Wennerasb, S. Noraasa a
Sørlandet Hospital Health Enterprise, Department of Medical Microbiology, Egsveien 100, PO-Box 416, NO-4604, Kristiansand, Norway University of Gothenburg, Department of Infectious Diseases, Guldhedsgatan 10, 413 46 Göteborg, Sweden c Sørlandet Hospital Health Enterprise, Medical Department, Egsveien 100, PO-Box 416, NO-4604 Kristiansand, Norway d University of Agder, Faculty of Engineering and Science, Department of Natural Sciences, Gimlemoen 25, PO-Box 422, NO-4604 Kristiansand, Norway e Sørlandet Hospital Health Enterprise, Research Unit, Egsveien 100, PO-Box 416, NO-4604 Kristiansand, Norway b
A R T I C L E I N F O
A B S T R A C T
Keywords: Tick-borne disease Candidatus Neoehrlichia mikurensis Borrelia burgdorferi sensu lato Lyme borreliosis Real-time PCR
The most common tick-borne human disease in Norway is Lyme borreliosis. Ticks in Norway also harbour less known disease-causing agents such as Candidatus Neoehrlichia mikurensis, Borrelia miyamotoi and Rickettsia helvetica. However, human infections caused by these pathogens have never been described in Norway. The main aims of the study were to evaluate the contribution of several tick-borne bacterial agents, other than Borrelia burgdorferi sensu lato, to zoonotic diseases in Norway and to determine their clinical pictures. Blood samples from 70 symptomatic tick-bitten adults from the Agder counties in southern Norway were screened for seven tick-borne pathogens by using a commercial multiplex PCR-based method and by singleplex real-time PCR protocols. Most patients (65/70) presented with a rash clinically diagnosed as erythema migrans (EM). The most frequently detected pathogen DNA was from Ca. N. mikurensis and was found in the blood of 10% (7/70) of the patients. The Ca. N. mikurensis-infected patients presented with an EM-like rash as the only symptom. B. burgdorferi s.l. DNA was present in the blood of 4% (3/70) of the study participants. None had detectable Anaplasma phagocytophilum, B. miyamotoi, Rickettsia typhus group or spotted fever group, Francisella tularensis, Coxiella burnetii or Bartonella spp. DNA in the blood. The commercially available multiplex PCR bacteria flow chip system failed to identify half of the infected patients detected by corresponding real-time PCR protocols. The recovery of Ca. N. mikurensis DNA was higher in the pellet/plasma fraction of blood than from whole blood. To conclude, Ca. N. mikurensis appeared to be the etiological agent in patients with EM in a surprisingly large fraction of tick-bitten persons in the southern part of Norway.
1. Introduction In Norway, the only frequently diagnosed human tick-borne disease is Lyme borreliosis (LB) with 300–400 cases of disseminated disease reported annually in a population of 5 million. Other diseases transmitted by ticks are rarely diagnosed; only 10–20 cases of tick-borne encephalitis (TBE) are reported yearly, and occasional cases of human granulocytic anaplasmosis and babesiosis have been described (Kristiansen et al., 2001; Morch et al., 2015). The incidence of human tick-borne disease in Norway is restricted by the distribution of Ixodes ricinus, the only significant tick vector of
human pathogens in the region. I. ricinus in Norway have been shown to harbour Borrelia burgdorferi sensu lato, Borrelia miyamotoi, Anaplasma phagocytophilum, Babesia spp., Candidatus Neoehrlichia mikurensis, Rickettsia helvetica and TBE-virus (Jenkins and Kristiansen, 2013; Kjelland et al., 2015; Oines et al., 2012; Quarsten et al., 2015). Ca. N. mikurensis, B. miyamotoi and R. helvetica are novel pathogens that have been associated with human disease in various European countries (Nilsson et al., 1999; Platonov et al., 2011; Welinder-Olsson et al., 2010), but not in Norway. It is unknown whether this is due to the existence of non-pathogenic strains, strains of low virulence giving rise to mild disease or due to low awareness in the healthcare sector leading
Abbreviations: LB, Lyme borreliosis; EM, erythema migrans; PCR-FCS, multiplex PCR bacteria flow chip system; SFG, spotted fever group; TG, typhus group; TBE, tick-borne encephalitis; Spp, species ⁎ Corresponding author. E-mail address: [email protected] (H. Quarsten). http://dx.doi.org/10.1016/j.ttbdis.2017.05.004 Received 26 January 2017; Received in revised form 9 May 2017; Accepted 10 May 2017 1877-959X/ © 2017 Elsevier GmbH. All rights reserved.
Please cite this article as: Quarsten, H., Ticks and Tick-borne Diseases (2017), http://dx.doi.org/10.1016/j.ttbdis.2017.05.004
Ticks and Tick-borne Diseases xxx (xxxx) xxx–xxx
H. Quarsten et al.
(< 10 weeks before blood sampling) tick bite. Out of 81 patients who were recruited to the study, 70 patients fulfilled the study criteria and were included in the study. Eleven patients did not meet the inclusion criteria; six did not have a recent tick bite, two patients were tick-bitten but did not contract any symptoms, and three patients either lacked written consent, critical information (about tick bite, symptoms and treatment) or the correct type of blood samples. Patients with characteristic EM diagnosed by a GP who did not recall tick bites (9/70) were accepted as study participants. The included patients answered questions regarding recent history of tick bite, EM, other symptoms and antibiotic treatment, but no patient histories were taken. 2.3. Ethical statements The study was approved by the Norwegian Regional Committee for Medical and Health Research Ethics, the South-Eastern region (2014/ 178), and all participants provided written informed consent to take part in the study. 2.4. DNA sample preparation Blood samples (2–8 ml EDTA blood and serum) were collected from the study participants by their GP and sent to the Department of Medical Microbiology (Kristiansand, Norway) by postal service. DNA was isolated from two different fractions of EDTA blood on the day of arrival in the laboratory: (1) Whole blood fraction; 200 μl blood was collected after inverting of the tube 5–6 times and used for extraction of total DNA by QIAamp DNA Mini Kit (Qiagen, Venlo, The Netherlands) as described by the manufacturer's instructions. (2) Pellet/plasma fraction; the remaining blood was centrifuged at 100 × g for 15 min to remove erythrocytes and all plasma/buffy coat was collected and centrifuged at 10,000 × g for 60 min in order to pellet bacteria together with leukocytes and platelets (Cerar et al., 2008). All the pelleted material was resuspended in 200 μl plasma and either processed immediately or stored at −70 °C until processing. The pellet/plasmasample was incubated in ATL lysis buffer (QIAamp DNA Mini kit) supplemented with proteinase K (1 mg/ml final concentration) for 1 h at 56 °C. Thereafter, the sample was processed as described by the manufacturer's instructions (QIAamp DNA Mini kit). All DNA extracts were stored at −20 °C for later analyses.
Fig. 1. The present distribution of I. ricinus in Norway depicted by a multi-source analysis (Jore et al., 2011). The distribution of ticks is mainly in coastal regions up to an altitude of 583 m above sea level and 66° north. The colour intensity represents the density of ticks from no ticks in the light yellow areas to high density in the darkest areas. The study patients were recruited from the coastal area in the south. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of the article.)
to under-diagnosis of certain tick-borne diseases. The focus of the study was (a) to evaluate to what extent tick-borne bacterial agents other than B. burgdorferi s.l. cause infectious disease in Norway, (b) to describe the clinical picture of non-B. burgdorferi s.l. tick-borne infections, (c) to evaluate a novel commercially available multiplex PCR bacteria flow chip system (PCR-FCS) for the detection of tick-borne bacteria by comparing performance with conventional established real-time PCR protocols, and (d) to evaluate which fraction of blood to use for molecular detection of tick-borne pathogens.
2.5. Tick-borne bacteria flow chip system A tick-borne bacteria flow chip system (PCR-FCS) from Master Diagnostica (Granada, Spain) was used for detection of DNA from tickborne bacteria by multiplex PCR, followed by automated reverse dot blot hybridisation with specific probes directed against Borrelia spp., A. phagocytophilum, Rickettsia typhus group (TG) and spotted fever group (SFG), Francisella tularensis, Bartonella spp., Coxiella burnetii and Ehrlichia (Ca. N. mikurensis, E. chaffeensis and E. ewingii). The samples were analysed according to the manufacturer's instructions. All samples were analysed in one replicate.
2. Materials and methods 2.1. Patient recruitment strategy Information regarding the study was disseminated to the public in the Agder counties (Fig. 1) through the internet, local newspapers, radio and posters set up at general practitioner (GP) offices in order to recruit patients with flu-like illness and/or EM after recent (within 10 weeks before inclusion in study) tick bite. The health workers at the general practices received information regarding the study by internet, email and ordinary postal service.
2.6. Real-time PCR All samples were tested by singleplex real-time PCR protocols for detection of B. burgdorferi s.l. (two independent assays targeting the ospA and 16S rRNA genes), B. miyamotoi (16S rRNA), A. phagocytophilum (groESL), Rickettsia TG and SFG (gltA), F. tularensis (fopA), Bartonella spp. (gltA), C. burnetii (icd) and Ca. N. mikurensis (groEL). Sequences, concentrations and thermocycling conditions of the TaqMan-based realtime PCR directed against a 169-bp fragment of the Ca. N. mikurensis groEL gene have been reported previously (Grankvist et al., 2015b). The sequences and final concentrations of all other primers and probes (except for B. miyamotoi) including the PCR thermocycling conditions used for the real-time PCR protocols have been published elsewhere
2.2. Patients From June 2014 through December 2015, patients were included in the study by their GPs. The inclusion criteria for participation in the study were (1) age 18 years or older, (2) ongoing symptoms after recent 2
Ticks and Tick-borne Diseases xxx (xxxx) xxx–xxx
H. Quarsten et al.
(Quarsten et al., 2015). The primers and probe for the B. miyamotoispecific real-time PCR (Tsao et al., 2004) were used at 0.5 μM and 0.2 μM, respectively, under the same thermocycling conditions employed in the B. burgdorferi s.l. protocols. All protocols were performed using 5 μl of DNA in a 15 μl reaction mixture consisting of 5 mM MgCl2, 0.5 U uracil DNA-glycosylase (Eurogentec S.A. Seraing, Belgium), and LightCycler FastStart DNA master mix (Roche) with primers and probe(s). All samples were analysed in one replicate by each real-time PCR protocol, except for the Ca. N. mikurensis protocol where samples were analysed in duplicate. All positive samples, except for one, were confirmed positive in a re-run. Samples positive for B. burgdorferi s.l. were confirmed and genotyped by direct sequencing of the rrs (16S)–rrlA (23S) intergenic spacer as previously described (Kjelland et al., 2010).
Table 1 Tick-borne bacteria detected in patient blood by two molecular methods. No. of patients with bacterial DNA in blood (%) Ca. N. mikurensis B. burgdorferi s.l. DNA DNA
Bacterial DNA (total)
Real-time singleplex PCR n = 70 PCR-FCS n = 69 n = 1 NAa
7 (10)
3 (4)
10 (14)
4 (6)
1 (1)
5 (7)
Both methods
4 (6)
1 (1)
5 (7)
Abbreviations: PCR-FCS multiplex PCR bacteria flow chip system (Master Diagnostica). a NA: not analysed; the sample was negative in all real-time protocols.
2.7. B. burgdorferi s.l. serology
blood. B. burgdorferi s.l. DNA was identified in 4% (3/70) of the patient samples by B. burgdorferi s.l.-specific real-time PCR protocols (ospA and/or 16S rRNA); 2/3 of the samples were successfully sequenced as B. garinii and B. afzelii, respectively. The third isolate could not be typed, possibly due to low amount of bacterial DNA in the sample. No A. phagocytophilum, Rickettsia spp., F. tularensis, C. burnetii or Bartonella spp. DNA were detected in any of the patient samples.
All patient serum samples collected at the start of the study, as well as follow-up serum samples from patients positive for bacterial DNA in the blood were analysed for antibodies against B. burgdorferi s.l. by using the Siemens Enzygnost Borreliosis/IgM and Lymelink VlsE/IgG ELISA kits (Marburg, Germany) with the manufacturer's cut-off levels and interpretation criteria. For detection of IgM and IgG in serum, the 98% specific diagnostic cut-offs were OD = 0.29 and 0.17, respectively.
3.3. Comparison of molecular tests and choice of blood fraction 2.8. Nucleotide sequence accession numbers Patient samples were additionally analysed by the multiplex PCRFCS kit (Table 1). Only 6% (4/69) Ca. N. mikurensis- and 1% (1/69) Borrelia spp.-infected samples were identified, i.e. the kit missed 3/7 Ca. N. mikurensis- and 2/3 B. burgdorferi s.l.-infected samples that were detected by the singleplex real-time PCR protocols. No samples were identified as infected by the PCR-FCS method only. Most of the samples with discrepant results had high cycle threshold values in real-time PCR, indicating that the real-time protocols have higher sensitivity than the PCR-FCS. DNA was isolated from two different fractions of patient blood: whole blood and a pellet/plasma fraction where extracellular and leukocyte-associated bacteria were expected to be concentrated by centrifugation. Among the Ca. N. mikurensis-positive patients, 6/7 had detectable Ca. N. mikurensis DNA in the pellet/plasma fraction, but only 3/7 were positive in the whole blood fraction (Table 2). Regarding B. burgdorferi s.l. DNA, one isolate was only recovered from the pellet/ plasma fraction, one isolate was only recovered from the whole blood fraction, and one isolate was recovered from both fractions (Table 2).
The sequences obtained in this study have been deposited in GenBank with accession numbers KY_782011 (B. afzelii) and KY_782012 (B. garinii). 2.9. Statistics Fisher's exact test was employed at a significance level of P < 0.05 (GraphPad Prism 6.0 software, San Diego, CA) in order to compare the frequency of symptoms other than EM in the Ca. N. mikurensis DNApositive (1/7) and pathogen DNA-negative patient groups (29/60). 3. Results 3.1. Patients The study comprised 70 patients presenting with the symptoms EM and/or flu-like illness after tick bite. The study cohort had a female to male ratio of 1.6:1 and a median age of 55 years. There were no restrictions regarding underlying diseases and/or current medication. Most study patients (61/70) reported a recent tick bite, less than ten weeks before the time of blood sampling. Nine patients were included with characteristic EM, diagnosed by a GP, without recalling a tick bite. Ten of the patients reported they had had antibiotics treatment within the previous three months. All except for five study participants developed EM. The ones without a rash experienced flu-like symptoms such as muscle pain, headache, fatigue, fever, and chills. The patients who had EM reported varying degrees of flu-like illness.
3.4. Patient symptoms Of the 70 patients included, 65 patients had EM alone or together Table 2 Detection of tick-borne bacterial DNA in blood sample fractions. PCR results
3.2. Detection of bacterial pathogen DNA in patient blood DNA from the whole blood and pellet/plasma fractions from each patient's blood sample was analysed by the real-time PCR methods for detection of the seven tick-borne pathogens. Patients from whom bacterial DNA was detected in either one or both of the blood fractions were considered infected. Bacterial DNA was demonstrated in 14% (10/ 70) of the patient samples in total (Table 1). Two out of the seven tested tick-borne bacterial pathogens were identified in blood of the symptomatic tick-bitten patients. Ca. N. mikurensis DNA was detected in 10% (7/70) of the patients and was the pathogen DNA most often detected in 3
Bacteria
Patient (no.)
Whole blood
Plasma/pellet
Ca. N. mikurensis
1 2 3 4 5 6 7 Positive fractions
Neg Neg Neg Pos Pos Pos Neg 3/7
Pos Pos Pos Neg Pos Pos Pos 6/7
B. burgdorferi s.l.
8 9 10 Positive fractions
Pos Neg Pos 2/3
Pos Pos Neg 2/3
Ticks and Tick-borne Diseases xxx (xxxx) xxx–xxx
H. Quarsten et al.
problems) during treatment but recovered completely after switching to doxycycline. Furthermore, B. burgdorferi s.l. DNA was not detected in the follow-up sample. Since there are no serological Ca. N. mikurensis assays available, it was not possible to screen Ca. N. mikurensis-positive patient samples for specific antibodies. However, no change in serological B. burgdorferi s.l. status was observed in the follow-up serum samples of Ca. N. mikurensis-patients, hence there was no serologic evidence of co-infections with B. burgdorferi s.l.
Table 3 Symptoms reported by the study participants. Number of patients Pathogen DNA detected in blood
Erythema migrans
Other symptoms
Ca. N. mikurensis B.burgdorferi s.l.
7/7 3/3
None
55/60
Fatigue 1/7 Myalgia 2/3 Unspecified ear problems 1/3 Flu-like symptoms 29/60a
Total
65/70
a
3.5. Patient follow-up The patients identified as infected (in total 10/70) by PCR, all presented with an EM rash at the time of inclusion and blood sampling. They were treated with antibiotics (penicillin or doxycycline) according to Norwegian guidelines for treatment of LB. New blood samples drawn after six weeks or later following the first detection of bacterial DNA in 9/10 patients were all negative for bacterial DNA. One patient did not provide a follow-up sample.
Muscle pain, headache, fatigue, fever, chills, pain.
with various degrees of flu-like illness and five patients had flu-like symptoms only after recent tick bite (Table 3). All of the Ca. N. mikurensis-positive patients presented with EM. Whereas 1/7 of the patients in the Ca. N. mikurensis DNA-positive group reported symptoms (fatigue) other than EM, flu-like symptoms were experienced by 29/60 in the pathogen DNA-negative patient group (Not significant; Fisher's exact test). None of the Ca. N. mikurensis patients presented with flu-like symptoms, in contrast, two out of three B. burgdorferi s.l. PCR-positive patients experienced muscle pain and one of them also reported unspecified ear problems.
4. Discussion In this study, Ca. N. mikurensis (10%) and B. burgdorferi s.l. (4%) DNA was detected in the blood of recently tick-bitten, symptomatic adult persons from the Agder counties in southern Norway. Among the 70 patients examined, none had detectable A. phagocytophilum, B. miyamotoi, Rickettsia TG or SFG, F. tularensis, C. burnetii or Bartonella spp. DNA in the blood. We provide the first record of human Ca. N. mikurensis infections in Norway. Ca. N. mikurensis is widespread among ticks in Europe (Wenneras, 2015). The pathogen has previously been detected in Norwegian ticks with a prevalence of 2–12% (Jenkins and Kristiansen, 2013). A high prevalence of Ca. N. mikurensis in ticks collected in southern Norway (17% (13/76); own unpublished data), may explain the relatively high infection rate documented in the study patients with 10% of them being infected by Ca. N. mikurensis. In comparison, 1.4% (7/291) of tick-bitten patients with EM in the Netherlands had Ca. N. mikurensis DNA in their blood, and it was estimated that the prevalence of Ca. N. mikurensis in ticks obtained from tick-bitten individuals was 5% (Jahfari et al., 2016). Establishment of a serological test for monitoring Ca. N. mikurensis antibodies,
3.4.1. B. burgdorferi s.l. serology All acute serum samples (except for one, due to lack of material) were analysed for B. burgdorferi s.l.-specific IgM and IgG antibodies. The B. burgdorferi s.l. IgG seroprevalence in this cohort was 39%: IgG alone was detected in close to 28% of the samples, whereas in nearly 12% of the samples both IgM and IgG were detected. Presence of IgM alone (12%) was considered to be unspecific. Two out of the three patients with B. burgdorferi s.l. DNA in blood at the time of inclusion had detectable B. burgdorferi s.l.-specific IgM or IgM/IgG in serum (Table 4). Their antibody level remained unchanged after antibiotic treatment. The third B. burgdorferi s.l. DNA-positive patient seroconverted for B. burgdorferi s.l. IgM and IgG during treatment with penicillin. This particular patient complained of a lack of improvement of symptoms (muscle pain and unspecified ear
Table 4 Borrelia antibody titers in acute and follow-up serum samples of patients with bacterial DNA in blood. Borrelia burgdorferi s.l. serum antibody titer (% cut off) Findings
Case no.
Sample type
IgM−/IgG−
Ca. N. mikurensis
1
Acute Follow-up Acute Follow-up Acute Follow-up Acute Follow-up Acute Follow-up Acute Follow-up Acute Follow-up
–/– nt/nt –/– –/– nt/nt –/–
Acute Follow-up Acute Follow-up Acute Follow-up
–/–
2 3 4 5 6 7 B. burgdorferi s.l.
8 9 10
IgM+/IgG−
IgM−/IgG+
IgM+/IgG+
Interpretation No change in antibody status
–/314 109/383 –/1503 –/1530 286/203 262/163 229/225 143/222 Seroconversion 657/837 –/284 –/201 222/– 166/–
Abbreviations: nt: not tested.
4
No change in antibody status
Ticks and Tick-borne Diseases xxx (xxxx) xxx–xxx
H. Quarsten et al.
an increased B. burgdorferi s.l. DNA yield compared to its recovery from whole blood. However, our data do not support this notion, probably due to small sample size of B. burgdorferi s.l.-positive findings. Consequently, we recommend isolating DNA from both the whole blood and the plasma/pellet fraction in order to increase the chance of detection of tick-borne pathogens. It should be emphasised that serology, not PCR analysis of blood samples, is the preferred method for diagnosing LB. However, a novel pathogenic Borrelia species, B. mayonii, causes LB with an unusually high bacteraemia and PCR analysis of blood may have a potential role for diagnosing infections caused by this pathogen (Pritt et al., 2016).
which is lacking, will in the future provide more information on the seroprevalence and risk of transmission of Ca. N. mikurensis to human beings after tick bites. The first reports of Ca. N. mikurensis as the cause of human disease were published in 2010 and concerned immunocompromised patients (Fehr et al., 2010; von Loewenich et al., 2010; Welinder-Olsson et al., 2010). Among this category of patients, symptoms such as remitting fever and pain in joints and muscles may be rather severe (Grankvist et al., 2014). Moreover, a high incidence of thromboembolic complications is seen among immunocompromised Ca. N. mikurensis-infected patients (Wenneras, 2015). However, Ca. N. mikurensis infections have also been demonstrated in immunocompetent, healthy subjects with varying clinical presentations ranging from no symptoms at all (WelcFaleciak et al., 2014) or only EM in Europe (Grankvist et al., 2015b) to febrile disease in China (Li et al., 2012). This seemingly lower virulence among European versus Asian strains of Ca. N. mikurensis may be due to the documented genotypic differences between Asian and European isolates (Grankvist et al., 2015a; Wenneras, 2015). Although no personal histories were taken, we assume that the majority, if not all patients in this study, were immunocompetent, including the Ca. N. mikurensis-infected patients. Thus, their lack of fever and other flu-like symptoms are in accordance with previous case histories of neoehrlichiosis among non-immunocompromised patients in Europe. Possible co-infection of B. burgdorferi s.l. and Ca. N. mikurensis in a patient has previously been reported in Sweden (Grankvist et al., 2015b). Whether any of the seven Ca. N. mikurensis-infected patients in the present study were co-infected with B. burgdorferi s.l. cannot be determined with certainty. It is estimated that up to 20% of skin biopsies collected from EM-like rashes are negative for B. burgdorferi s.l. DNA (Sjowall et al., 2011), which raises the question whether other microbes might give rise to similar rashes, Ca. N. mikurensis, for instance. It has been reported that prolonged Ca. N. mikurensis DNA carriage may occur in immunocompetent persons: two Ca. N. mikurensispositive patients with EM who were treated with penicillin, which does not eradicate Ca. N. mikurensis, due to suspected LB had detectable Ca. N. mikurensis DNA in follow-up blood samples collected 1–3 months later (Grankvist et al., 2015b). However, Ca. N. mikurensis DNA was not detected in the 6-week follow-up blood samples in the present study, indicating that all patients had either been successfully treated with antibiotics (the two who received doxycycline) or recovered spontaneously (the five who were treated with penicillin). Patient samples were analysed both by real-time PCR protocols and the commercial multiplex PCR-FCS method. The PCR-FCS has previously been demonstrated to have slightly lower sensitivity for the detection of pathogens in ticks compared with singleplex PCRs (Quarsten et al., 2015). In the present study, the PCR-FCS-method identified only half of the patients with tick-borne infections compared with the real-time PCR protocols. The most probable reason for the marked difference in performance is that the patient group in this study had low quantities of bacteria in the blood, which is to be expected in immunocompetent (Grankvist et al., 2015b) compared to immunocompromised individuals (Grankvist et al., 2014, 2015b). Thus, a small difference in sensitivity will have a major influence on the number of infected immunocompetent patients that may be identified. In patients with high-level bacteraemia, the discrepancy in the performance of the methods may not be as prominent. The tissue tropism of Ca. N. mikurensis is not known, though it is likely that infection of endothelial cells is involved (Kawahara et al., 2004). The pellet/plasma fraction was clearly superior for detection of Ca. N. mikurensis in blood in this study. Possible explanations are that Ca. N. mikurensis has invaded or been phagocytosed by white blood cells and/or that free bacteria derived from disrupted endothelial cells are more easily recovered from this fraction. Along this line, the B. burgdorferi s.l. spirochetes being extracellular organisms should be enriched by centrifugation of the pellet/plasma fraction, resulting in
5. Conclusions This study provides the first record of Ca. N. mikurensis infections in recently tick-bitten individuals in Norway. The Ca. N. mikurensisinfected patients all presented with an EM-like skin rash. Ca. N. mikurensis is optimally detected in the pellet/plasma fraction where the pathogen is likely to be concentrated. Real-time PCR methods identified twice as many Ca. N. mikurensis- and B. burgdorferi s.l.infected individuals as a commercial tick-borne bacteria flow chip kit (PCR-FCS). Whether a causal relationship exists between Ca. N. mikurensis and EM remains to be established. Acknowledgment This work was partly supported by The EU-Interreg ÖKS project ScandTick Innovation. References Cerar, T., Ogrinc, K., Cimperman, J., Lotric-Furlan, S., Strle, F., Ruzic-Sabljic, E., 2008. Validation of cultivation and PCR methods for diagnosis of Lyme neuroborreliosis. J. Clin. Microbiol. 46, 3375–3379. Fehr, J.S., Bloemberg, G.V., Ritter, C., Hombach, M., Luscher, T.F., Weber, R., Keller, P.M., 2010. Septicemia caused by tick-borne bacterial pathogen Candidatus Neoehrlichia mikurensis. Emerg. Infect. Dis. 16, 1127–1129. Grankvist, A., Andersson, P.O., Mattsson, M., Sender, M., Vaht, K., Hoper, L., Sakiniene, E., Trysberg, E., Stenson, M., Fehr, J., Pekova, S., Bogdan, C., Bloemberg, G., Wenneras, C., 2014. Infections with the tick-borne bacterium “Candidatus Neoehrlichia mikurensis” mimic noninfectious conditions in patients with B cell malignancies or autoimmune diseases. Clin. Infect. Dis. 58, 1716–1722. Grankvist, A., Moore, E.R., Svensson Stadler, L., Pekova, S., Bogdan, C., Geissdorfer, W., Grip-Linden, J., Brandstrom, K., Marsal, J., Andreasson, K., Lewerin, C., WelinderOlsson, C., Wenneras, C., 2015a. Multilocus sequence analysis of clinical “Candidatus Neoehrlichia mikurensis” strains from Europe. J. Clin. Microbiol. 53, 3126–3132. Grankvist, A., Sandelin, L.L., Andersson, J., Fryland, L., Wilhelmsson, P., Lindgren, P.E., Forsberg, P., Wenneras, C., 2015b. Infections with Candidatus Neoehrlichia mikurensis and cytokine responses in 2 persons bitten by ticks, Sweden. Emerg. Infect. Dis. 21, 1462–1465. Jahfari, S., Hofhuis, A., Fonville, M., van der Giessen, J., van Pelt, W., Sprong, H., 2016. Molecular detection of tick-borne pathogens in humans with tick bites and erythema migrans, in the Netherlands. PLoS Negl. Trop. Dis. 10, e0005042. Jenkins, A., Kristiansen, B.E., 2013. Neoehrlichia—a new tick-borne bacterium. Tidsskr. Nor. Laegeforen. 133, 1058–1059. Jore, S., Viljugrein, H., Hofshagen, M., Brun-Hansen, H., Kristoffersen, A.B., Nygard, K., Brun, E., Ottesen, P., Saevik, B.K., Ytrehus, B., 2011. Multi-source analysis reveals latitudinal and altitudinal shifts in range of Ixodes ricinus at its northern distribution limit. Parasites Vectors 4, 84. Kawahara, M., Rikihisa, Y., Isogai, E., Takahashi, M., Misumi, H., Suto, C., Shibata, S., Zhang, C., Tsuji, M., 2004. Ultrastructure and phylogenetic analysis of ‘Candidatus Neoehrlichia mikurensis’ in the family Anaplasmataceae, isolated from wild rats and found in Ixodes ovatus ticks. Int. J. Syst. Evol. Microbiol. 54, 1837–1843. Kjelland, V., Rollum, R., Korslund, L., Slettan, A., Tveitnes, D., 2015. Borrelia miyamotoi is widespread in Ixodes ricinus ticks in southern Norway. Ticks Tick Borne Dis. 6, 516–521. Kjelland, V., Stuen, S., Skarpaas, T., Slettan, A., 2010. Prevalence and genotypes of Borrelia burgdorferi sensu lato infection in Ixodes ricinus ticks in southern Norway. Scand. J. Infect. Dis. 42, 579–585. Kristiansen, B.E., Jenkins, A., Tveten, Y., Karsten, B., Line, O., Bjoersdorff, A., 2001. Human granulocytic ehrlichiosis in Norway. Tidsskr. Nor. Laegeforen. 121, 805–806. Li, H., Jiang, J.F., Liu, W., Zheng, Y.C., Huo, Q.B., Tang, K., Zuo, S.Y., Liu, K., Jiang, B.G., Yang, H., Cao, W.C., 2012. Human infection with Candidatus Neoehrlichia mikurensis, China. Emerg. Infect. Dis. 18, 1636–1639. Morch, K., Holmaas, G., Frolander, P.S., Kristoffersen, E.K., 2015. Severe human Babesia divergens infection in Norway. Int. J. Infect. Dis. 33, 37–38.
5
Ticks and Tick-borne Diseases xxx (xxxx) xxx–xxx
H. Quarsten et al.
Th1-type inflammatory cytokine expression in the skin is associated with persisting symptoms after treatment of erythema migrans. PLoS ONE 6, e18220. Tsao, J.I., Wootton, J.T., Bunikis, J., Luna, M.G., Fish, D., Barbour, A.G., 2004. An ecological approach to preventing human infection: vaccinating wild mouse reservoirs intervenes in the Lyme disease cycle. Proc. Natl. Acad. Sci. U.S.A. 101, 18159–18164. von Loewenich, F.D., Geissdorfer, W., Disque, C., Matten, J., Schett, G., Sakka, S.G., Bogdan, C., 2010. Detection of “Candidatus Neoehrlichia mikurensis” in two patients with severe febrile illnesses: evidence for a European sequence variant. J. Clin. Microbiol. 48, 2630–2635. Welc-Faleciak, R., Sinski, E., Kowalec, M., Zajkowska, J., Pancewicz, S.A., 2014. Asymptomatic “Candidatus Neoehrlichia mikurensis” infections in immunocompetent humans. J. Clin. Microbiol. 52, 3072–3074. Welinder-Olsson, C., Kjellin, E., Vaht, K., Jacobsson, S., Wenneras, C., 2010. First case of human “Candidatus Neoehrlichia mikurensis” infection in a febrile patient with chronic lymphocytic leukemia. J. Clin. Microbiol. 48, 1956–1959. Wenneras, C., 2015. Infections with the tick-borne bacterium Candidatus Neoehrlichia mikurensis. Clin. Microbiol. Infect. 21, 621–630.
Nilsson, K., Lindquist, O., Pahlson, C., 1999. Association of Rickettsia helvetica with chronic perimyocarditis in sudden cardiac death. Lancet 354, 1169–1173. Oines, O., Radzijevskaja, J., Paulauskas, A., Rosef, O., 2012. Prevalence and diversity of Babesia spp. in questing Ixodes ricinus ticks from Norway. Parasites Vectors 5, 156. Platonov, A.E., Karan, L.S., Kolyasnikova, N.M., Makhneva, N.A., Toporkova, M.G., Maleev, V.V., Fish, D., Krause, P.J., 2011. Humans infected with relapsing fever spirochete Borrelia miyamotoi, Russia. Emerg. Infect. Dis. 17, 1816–1823. Pritt, B.S., Mead, P.S., Johnson, D.K., Neitzel, D.F., Respicio-Kingry, L.B., Davis, J.P., Schiffman, E., Sloan, L.M., Schriefer, M.E., Replogle, A.J., Paskewitz, S.M., Ray, J.A., Bjork, J., Steward, C.R., Deedon, A., Lee, X., Kingry, L.C., Miller, T.K., Feist, M.A., Theel, E.S., Patel, E.S., Irish, C.L., Petersen, J.M., 2016. Identification of a novel pathogenic Borrelia.species causing Lyme borreliosis with unusually high spirochaetaemia: a descriptive study. Lancet Infect. Dis. 16, 556–564. Quarsten, H., Skarpaas, T., Fajs, L., Noraas, S., Kjelland, V., 2015. Tick-borne bacteria in Ixodes ricinus collected in southern Norway evaluated by a commercial kit and established real-time PCR protocols. Ticks Tick Borne Dis. 6, 538–544. Sjowall, J., Fryland, L., Nordberg, M., Sjogren, F., Garpmo, U., Jansson, C., Carlsson, S.A., Bergstrom, S., Ernerudh, J., Nyman, D., Forsberg, P., Ekerfelt, C., 2011. Decreased
6