Close Geographic Association of Human Neoehrlichiosis and TickPopulations Carrying “Candidatus Neoehrlichia mikurensis” inEastern Switzerland

Florian P. Maurer,a Peter M. Keller,a* Christian Beuret,e Cornelia Joha,b Yvonne Achermann,c Jacques Gubler,d Daniela Bircher,d

Urs Karrer,d Jan Fehr,c Lukas Zimmerli,b Guido V. Bloemberga

Institute of Medical Microbiology, University of Zurich, Zurich, Switzerlanda; Division of Internal Medicine, University Hospital Zurich, University of Zurich, Zurich,Switzerlandb; Division of Infectious Diseases and Hospital Epidemiology, University Hospital Zurich, University of Zurich, Zurich, Switzerlandc; Division of InfectiousDiseases and Outpatient Clinic, Department of Medicine, Kantonsspital Winterthur, Winterthur, Switzerlandd; Spiez Laboratory, Federal Department of Defence, CivilProtection and Sports, Federal Office for Civil Protection, Spiez, Switzerlande

Neoehrlichiosis caused by “Candidatus Neoehrlichia mikurensis” is an emerging zoonotic disease. In total, six patients havebeen described in Europe, with the first case detected in 2007. In addition, seven patients from China were described in a reportpublished in October 2012. In 2009, we diagnosed the first human case of “Ca. Neoehrlichia mikurensis” infection in the Zuricharea (Switzerland). Here, we report two additional human cases from the same region, which were identified by broad-range 16SrRNA gene PCR. Both patients were immunocompromised and presented with similar clinical syndromes, including fever, mal-aise, and weight loss. A diagnostic multiplex real-time PCR was developed for specific detection of “Ca. Neoehrlichia mikuren-sis” infections. The assay is based on the signature sequence of a 280-bp fragment of the “Ca. Neoehrlichia mikurensis” 16SrRNA gene and incorporates a “Ca. Neoehrlichia mikurensis” species, a “Ca. Neoehrlichia” genus, and an Anaplasmataceae fam-ily probe for simultaneous screening. The analytical sensitivity was determined to be below five copies of the “Ca. Neoehrlichiamikurensis” 16S rRNA gene. Our results show that the assay is suitable for the direct detection of “Ca. Neoehrlichia mikurensis”DNA in clinical samples from, for example, blood and bone marrow. In addition, it allows for monitoring treatment responseduring antibiotic therapy. Using the same assay, DNA extracts from 1,916 ticks collected in four forests in close proximity to thepatients’ residences (<3 km) were screened. At all sampling sites, the minimal prevalence of “Ca. Neoehrlichia mikurensis” wasbetween 3.5 to 8% in pools of either nymphs, males, or females, showing a strong geographic association between the three pa-tients and the assumed vector.

Detection of the “Candidatus Neoehrlichia mikurensis” 16SrRNA gene was first described in 1999 from a tick isolated in

The Netherlands and initially named the “Schottii variant” (1).After further sequence detections in ticks from Italy (2, 3), “Ca.Neoehrlichia mikurensis” was validly described as a novel intra-cellular pathogen in 2004 by a Japanese group investigating infec-tions caused by members of the Anaplasmataceae in rats on thesmall island of Mikura, 60 km east of Tokyo, Japan (4). Since then,several groups reported the discovery of highly similar DNA se-quences in potential arthropod vectors such as Ixodes ovatus,Ixodes persulcatus, and Ixodes ricinus ticks in eastern Asia (5), theNetherlands (1, 6–8), Belgium (6), Germany (9), Denmark (10),the Czech Republic (11), Slovakia (12), Russia (13), Italy (3, 14,15), and recently Switzerland (16). In addition, “Ca. Neoehrlichiamikurensis” was found in rodent populations in China (17), Japan(18), Sweden (19), and the Netherlands (6). Moreover, an infec-tion was described in a dog in Germany with symptoms similar toehrlichiosis (20).

By mid-2012, a total of six European human cases of “Ca. Neo-ehrlichia mikurensis” bacteremia had been described in Sweden,Germany, Switzerland, and the Czech Republic (9, 21–23). “Ca.Neoehrlichia mikurensis” has also been detected in several parts ofAsia (4, 5, 17). Only very recently (October 2012) seven humaninfections in China were described (24). Specific serological testshave not been established, and cross-reactivity with serologies forEhrlichia spp., Anaplasma spp., and Rickettsia spp. has not beenobserved (24; also unpublished data). So far, all human infections

were detected by 16S rRNA broad-range PCR followed by se-quencing of the amplicon. The infection was fatal in one case andcharacterized by an unspecific sepsis syndrome with fever, mal-aise, and weight loss in the other cases. Cure was achieved byantibiotic treatment covering intracellular pathogens (tetracy-clines alone or in combination with rifampin). Ticks are consid-ered the most probable vector although formal proof for transmis-sion from ticks to humans has not yet been provided.

This study describes two new human cases of “Ca. Neoehrli-chia mikurensis” infections in the Zurich area. In this context, weaimed at (i) the development of a diagnostic multiplex real-timePCR for the rapid and accurate detection of “Ca. Neoehrlichiamikurensis” in clinical and environmental samples and (ii) thedetection of “Ca. Neoehrlichia mikurensis” in tick populationscollected in the patients’ residential areas.

Received 26 July 2012 Returned for modification 31 August 2012Accepted 18 October 2012

Published ahead of print 31 October 2012

Address correspondence to Guido V. Bloemberg, bloemberg@imm.uzh.ch.

* Present address: Peter M. Keller, University Hospital Jena, Institute of MedicalMicrobiology, Jena, Germany.

F.P.M. and P.M.K. contributed equally to this article.

Copyright © 2013, American Society for Microbiology. All Rights Reserved.

doi:10.1128/JCM.01955-12

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CASE REPORTS

Between October 2011 and January 2012, two cases of humanneoehrlichiosis were diagnosed in our molecular diagnostic labo-ratory. Patient 1, a 68-year-old Swiss locksmith with a history ofchronic lymphocytic leukemia (CLL), was referred to the Univer-sity Hospital Zurich because of fever of unknown origin (FUO).He had a 4-week history of recurrent fever attended with chills,night sweats, weight loss, and pain in his left temporomandibularjoint. The history of the patient was remarkable for clinicallyasymptomatic tick bites years ago and extensive travel all over theworld. He was a dog owner and went for regular walks in the areasurrounding his home. On admission, he was in reduced generalcondition, with a body temperature of 39.7°C. Laboratory find-ings showed normochromic anemia with a hemoglobin of 106g/liter, leukocytosis of 19.1 � 109 cells/liter (94% neutrophils), anelevated C-reactive protein (CrP) level of 65 mg/liter (normalvalue, �5 mg/liter), and a procalcitonin level of 20.2 �g/liter (nor-mal value, �0.5 �g/liter). A progression of CLL or therapy-asso-ciated myelodysplastic syndrome had been excluded by bone mar-row biopsy specimen and splenectomy prior to hospitalization.The patient did not respond to empirical antibiotic therapy withmeropenem. Repeated microbiological and immunological anal-yses as well as various imaging procedures (echocardiography,computed tomography [CT] scans of thorax and abdomen, andpositron emission tomography [PET]) failed to reveal an infec-tious, malignant, or autoimmune cause of the fever.

Patient 2, a 58-year-old computer expert, was referred to theoutpatient clinic at the Kantonsspital in Winterthur, Switzerland,in January 2012 because of progressive deterioration of his generalcondition for 3 months. He complained of pronounced fatigue,daily fever up to 40°C, chills, night sweats, and weight loss of 5 kg.He was under maintenance treatment with rituximab every 3months for lymphoma and was taking a vitamin K antagonist(phenprocoumon) for recurrent deep vein thrombosis. Follicularlymphoma stage IV with involvement of the bone marrow andseveral lymph node regions on both sides of the diaphragm hadbeen diagnosed 1 year earlier and was treated with six cycles ofR-CHOP (rituximab, cyclophosphamide, doxorubicin, vincris-tine, and prednisone) chemotherapy until June 2011. He hadreached complete clinical, biochemical, and radiological remis-sion by August 2011 and thus had resumed his usual full-timework. He had no exposure to pets and did not recall any tick bites.However, he frequently went for outdoor walks in nearby forests.Prior to referral, blood cultures were negative, an echocardiogramwas normal, serologies for HIV, Epstein-Barr virus (EBV), andcytomegalovirus (CMV) were negative or indicated past infection,and a PET-CT scan was unremarkable beside some diffuse butunspecific glucose uptake of the bone marrow. In particular, thePET-CT scan showed no signs for recurrent lymphoma. On ad-mission, the patient was in a markedly reduced general condition.He was underweight, with a body mass index of 19 kg/m2; histemperature was normal (36.7°C), his blood pressure was low(95/60 mmHg), his pulse was regular (100 beats/min), and therewere no cardiac murmurs. Lung, abdomen, joints, neurology, andskin were unremarkable. There was oral thrush and an enlargedinguinal lymph node on the left. His blood tests revealed normo-chromic, normocytic anemia (hemoglobin, 93 g/liter), normalplatelet and leukocyte counts (6.2 Gpt/liter), profound lymphope-nia (0.22 Gpt/liter), reduced sodium of 127 mmol/liter, substan-

tial inflammation (erythrocyte sedimentation rate, 94 mm/h; CrP,92 mg/liter), reduced IgM and IgG levels, and a slightly elevatedlactate dehydrogenase of 481 U/liter. Hepatic and renal parame-ters were within normal limits, and his prothrombin time was ontarget (international normalized ratio [INR], 2.48). Additionalblood cultures remained negative. A bone marrow biopsy speci-men was compatible with chronic inflammation or regenerationafter R-CHOP chemotherapy but without any signs of recurrentlymphoma.

In both cases, broad-range PCR targeting the bacterial 16SrRNA gene followed by sequence analysis of the amplificationproduct detected “Ca. Neoehrlichia mikurensis” DNA in periph-eral blood and bone marrow samples (Fig. 1) (sequence homologyanalysis showing 0/444-bp and 0/457-bp mismatches with the“Ca. Neoehrlichia mikurensis” 16S rRNA gene, respectively).Upon diagnosis, both patients received oral antibiotic treatmentwith doxycycline (100 mg twice daily [BD]) for 6 weeks. Patient 1became afebrile within 2 days, and clinical symptoms improvedrapidly. On day 28 after diagnosis, bacterial broad-range PCR of ablood sample was negative for the first time and has remainednegative ever since (Fig. 1). In patient 2, fever subsided within 5days, and after 2 weeks, when the PCR was already negative, thepatient had gained 6 kg. Antibiotic treatment was continued, andthe patient made a full clinical recovery without any further signsof recurrence of neoehrlichiosis despite reinitiation of rituximabtreatment.

MATERIALS AND METHODSClinical samples and DNA extraction. Blood and bone marrow samplescollected from three patients suffering from symptomatic neoehrlichiosiswere used to establish the real-time PCR. One of these patients has beendescribed previously (21). In parallel, all samples were routinely tested bybroad-range PCR targeting the 16S rRNA gene (25). DNA was extractedfrom 1 ml of blood or bone marrow aspirate (stored at �20°C) with anEZ1 DNA Tissue Kit (Qiagen, Hombrechtikon, Switzerland) followingthe manufacturer’s instructions. DNA extracts were eluted in 50 �l ofPCR-grade water (Limulus amebocyte lysate [LAL] water; Lonza, Walk-ersville, MD) of which 5 �l was tested undiluted and in a 1:5 (broad-rangePCR) or 1:1,000 dilution (species-specific assay) in order to reduce resid-ual inhibitors of DNA polymerase activity.

All patients gave their written informed consent for the study.Tick populations. A total of 1,916 questing ticks were collected in

2009 at four collection sites in the greater Zurich area (Switzerland) byflagging low vegetation (see Fig. 4). The collection sites were Winterthur(�47°30=51�, �8°43=44�; altitude, 480 m), Bassersdorf (�47°26=20�,�8°36=40�; 470 m), Rümlang (�47°26=20�, �8°31=1�; 490 m), and RütiZH (�47°15=40�, �8°52=60�; 600 m). Ticks were identified based on mor-phological characteristics and immediately stored at �80°C. Subse-quently, ticks were washed once in 75% ethanol and twice in deionizedwater, dried on paper towels, and sorted into pools of 10 nymphs or of 5adult male or female ticks. DNA extracts from these pools were preparedby adding 600 �l of phosphate-buffered saline (PBS) and one 3-mm tung-sten carbide bead (Qiagen) to each frozen tick pool, followed by homog-enization for 4 min at 30 Hz using a TissueLyser system (Qiagen). After ashort centrifugation step (5 s at 3,220 � g), the supernatants were col-lected in separate collection microtubes for further use. Nucleic acid ex-traction was performed using a QIAsymphony Virus/Bacteria Midi Kit(Qiagen) and a specially adapted protocol (CP Complex 920 FIX, version1; Qiagen). DNA was eluted in a final volume of 60 �l and either directlyused for downstream applications or stored at �80°C for further use.

Minimal prevalence of “Ca. Neoehrlichia mikurensis” was determinedfor pools of nymphs and female and male ticks based on the assumptionthat at least one tick in each positive pool was infected with “Ca. Neoeh-

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rlichia mikurensis.” The minimal total prevalence per collection site wasdetermined by calculating the weighted means of the respective preva-lence rates for males, females, and nymphs.

Primer design and TaqMan hydrolysis probes. 16S rRNA sequencesavailable from the GenBank database and representing all known generaof the Anaplasmataceae family were aligned using Lasergene MegAlignsoftware (DNAstar, Madison, WI) (Fig. 2). Real-time PCR primers andTaqMan hydrolysis probes were chosen using PrimerExpress software,version 3.0 (Life Technologies, Zug, Switzerland) following visual inspec-tion of the aligned target sequences: Ana_for (5=-ATC CTG GCT CAGAAC GAA CG-3=), Neo_rev (5=-TGA TCG TCC TCT CAG ACC AGC-3=), Neo_spec (5=-6FAM-ACC CAT AGT AAA CTA CAG CTA CA-MGB-3=, where FAM is 6-carboxyfluorescein and MGB is minor groove binder),Neo_genus (5=-Cy5-CTA GTA GTA TGG AAT AGC TGT TAG A-BBQ-3=, where BBQ is BlackBerry quencher), and Ana_family (5=-NED-TAACAC ATG CAA GTC GAA C-MGB-3=, where NED is 2,7=,8=-benzo-5=-fluoro-2=,4,7-trichloro-5-carboxyfluorescein). The forward primerAna_for was designed by modification of the previously published broad-range Anaplasmataceae family amplification primer EE1 (26) in order tooptimize its melting temperature for the reaction conditions of the “Ca.Neoehrlichia mikurensis” real-time PCR. For the same purpose, the Neo-_genus probe was modified with four locked nucleic acid (LNA) bases(Exiqon, Vedbaek, Denmark) at the 3= end. The Anaplasmataceae fam-ily probe (Ana_family) was used as previously published except for MGBmodification (27). All primers and probes were checked for cross-reactiv-ity with other published DNA sequences using the NCBI BLASTN algo-rithm.

Positive-control plasmid. The positive-control plasmid pCNM1 con-taining a 400-bp segment of the 5= end of the 16S rRNA gene (Escherichiacoli 16S rRNA gene positions 14 to 414) was constructed using in silicodesign and de novo synthesis and subcloning (Genscript, CA). PlasmidDNA was purified from transformed Invitrogen Escherichia coli XL-1 blue(Life Technologies) using Wizard Plus Midiprep (Promega, Basel, Swit-zerland) and quantified by spectrophotometric analysis on the basis ofplasmid size and the corresponding DNA mass using both a NanoDrop2000 instrument (Thermo Fisher Scientific, Wohlen, Switzerland) andInvitrogen Quant-iT PicoGreen chemistry (Life Technologies).

Multiplex real-time PCR. Real-time PCR was performed on an Ap-plied Bioystems 7500 fast instrument with 7500 System software (version2.0.4). Each 25.5-�l PCR mixture contained 12.5 �l of 2� PCR Master-mix (Roche Diagnostics, Rotkreuz, Switzerland), 2.5 �l of 10� exogenousinternal positive-control primer and probe mix (VIC-labeled), 0.5 �l of50� exogenous internal positive-control target DNA (both, Life Technol-ogies), 1.0 �l of each primer (stock concentration, 10 �M) and probe(stock concentration, 2.5 �M), and a 5.0-�l sample of DNA extract. Theexogenous internal positive-control reagents were added to distinguishtruly negative from falsely negative results due to PCR inhibition. PCRconditions were 120 s at 50°C and 10 min at 95°C, followed by 40 cycles of15 s at 95°C and 60 s at 60°C.

The analytical sensitivity of the assay and reproducibility of the testresults were determined by repeated testing of 10-fold dilutions of theplasmid positive control ranging from 5 � 105 to 5 � 10�1 copies in 10independent runs. In addition, PCR-grade water (LAL water) was used asa negative control (Fig. 3). Specificity was evaluated by testing DNA ex-

FIG 1 Detection of “Ca. Neoehrlichia mikurensis” in clinical samples of two patients with symptomatic neoehrlichiosis diagnosed in November 2011 andJanuary 2012, respectively. Shown are polyacrylamide gel electrophoresis analyses of broad-range 16S rRNA gene PCR products (insets) and the amplificationplots for the “Ca. Neoehrlichia mikurensis” TaqMan species probe (Neo_spec) obtained from the same samples. Day 0 represents the start of antibiotic therapy.(A) Patient 1, blood and bone marrow samples taken on days �30 (1.48 � 104 16S rRNA gene copies/ml bone marrow), �8 (3.53 � 107 16S rRNA gene copies/mlbone marrow), 1 (1.32 � 105 16S rRNA gene copies/ml blood), 6 (8.67 � 106 16S rRNA gene copies/ml blood), 14 (4.53 � 105 16S rRNA gene copies/ml blood),28, and 49. (B) Patient 2, blood and bone marrow samples obtained on days �12, 1, 15, 29, and 58. The CT values for samples taken on days �12 and 1 correspondto 1.05 � 106 16S rRNA gene copies/ml bone marrow and 7.01 � 106 16S rRNA gene copies/ml blood, respectively. Samples were tested undiluted and in a 1:5(16S rRNA gene broad-range PCR) or 1:1,000 (multiplex real-time PCR) dilution to reduce PCR inhibition. M, molecular mass standard; Rn, normalizedreporter dye fluorescence; d, day. E. coli chromosomal DNA was used as a positive control for the broad-range 16S rRNA PCR assay. Dilutions were tested todetect possible PCR inhibitions.

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tracts of four closely related members of the Anaplasmataceae, two Rick-ettsia spp., Francisella tularensis, Borrelia burgdorferi, and human DNA.To further assess the assay specificity, amplification products from ninetick pools tested either positive or negative with the “Ca. Neoehrlichiamikurensis” probe were sequenced and compared to known sequencesusing the NCBI BLAST tool.

RESULTSDevelopment of a real-time PCR for the detection of “Ca. Neo-ehrlichia mikurensis,” “Ca. Neoehrlichia” genus, and Anaplas-mataceae family members. A TaqMan-based real-time PCR al-lowing for highly specific and sensitive detection of both “Ca.Neoehrlichia mikurensis” and other bacteria belonging to theAnaplasmataceae family in clinical and environmental sampleswas developed based on homology analysis of the 16S rRNA genespresent in public databases. A 280-bp fragment was selected at the5= end of the 16S rRNA gene, which allowed for the design of a“Ca. Neoehrlichia mikurensis”-specific probe (Neo_spec), a “Ca.Neoehrlichia” genus probe (Neo_genus), and an Anaplasmata-ceae family probe (Ana_family). Using different fluorescent labelswith emission maxima in the blue (FAM), yellow (NED), or far-red (Cy5) part of the visible spectrum, all three targets can simul-taneously be detected in one reaction (Fig. 2).

The specificity of the probes was analyzed by testing chromo-somal DNA of Ehrlichia canis, Ehrlichia ruminantium, Anaplasmaphagocytophilum, Wolbachia pipientis, Rickettsia helvetica, Rickett-sia monacensis, Borrelia burgdorferi, and Francisella tularensis. No

cross-reactions were observed for the “Ca. Neoehrlichia mikuren-sis” probe. The “Ca. Neoehrlichia” genus probe showed onlycross-reactivity with Ehrlichia ruminantium. The Anaplasmata-ceae family probe detected all Anaplasmataceae family memberstested and in addition members of the closely related Rickettsi-aceae family, as shown for Rickettsia helvetica and Rickettsia mo-nacensis. None of the probes reacted with Borrelia burgdorferi orFrancisella tularensis.

The analytical sensitivity of the assay was determined usingplasmid pCNM1, which contains a partial 16S rRNA gene of “Ca.Neoehrlichia mikurensis” including the 280-bp region targeted bythe real-time PCR. Repeated testing of 10-fold serial dilutions ofpurified pCNM1 DNA in 10 independent runs consistentlyshowed that the limit of detection for “Ca. Neoehrlichia mikuren-sis” by either the species or the genus probe was below 5 copies ofthe 16S rRNA gene. No amplification was detected for a templatedilution of 5 � 10�1 copies and the negative control (Table 1 andFig. 3).

Evaluation of the real-time PCR for detection of “Ca. Neoeh-rlichia mikurensis” DNA in clinical samples. To evaluate thesuitability of the “Ca. Neoehrlichia mikurensis” assay in the rou-tine diagnostic laboratory, patient specimens of the two new casesdescribed above were tested and compared with the results of thebroad-range 16S rRNA PCR assays. Testing of DNA extracts of allblood and bone marrow samples obtained from the two patientsshowed a complete concordance between TaqMan-based and

FIG 2 Homology analysis of two regions within a 280-bp fragment of the 16S rRNA gene including hypervariable regions V1 and V2 in members of theAnaplasmataceae family. Sequences were aligned using the ClustalW algorithm. The reference sequence (GenBank accession number GQ501090) was derivedfrom a human case of neoehrlichiosis diagnosed by broad-range PCR targeting the 16S rRNA gene (21). The lines immediately above the reference sequencedepict the positions of the forward (Ana_for) and reverse (Neo_rev) primers as well as of the TaqMan probes designed for “Ca. Neoehrlichia mikurensis” “Ca.Neoehrlichia” genus, and Anaplasmataceae family detection (Neo_spec, Neo_genus, and Ana_family, respectively).

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broad-range 16S rRNA gene PCR results (Fig. 1). By generating astandard curve from 10-fold serial dilutions of positive-controlplasmid pCNM1, an estimate was made of the number of 16SrRNA gene copies per ml of patient sample. Bacterial DNA loadsfor patient 1, diagnosed in November 2011 using bone marrowand blood specimens obtained at days �30, �8, 1, 6, 14, 28, and 49(as calculated from the initiation of antibiotic therapy) were de-termined based on the obtained threshold cycle (CT) values. Bac-

terial loads were estimated to be 1.48 � 104 16S rRNA gene cop-ies/ml bone marrow at day �30, 3.53 � 107 16S rRNA genecopies/ml bone marrow at day �8, 1.32 � 105 16S rRNA genecopies/ml blood at day 1, 8.67 � 106 16S rRNA gene copies/mlblood at day 6, and 4.53 � 105 16S rRNA gene copies/ml blood atday 14. Blood samples obtained at 28 and 49 days after initiation ofantibiotic treatment were negative using both tests. Similar calcu-lations were made for patient 2, showing bacterial loads of 1.05 �

FIG 3 Analytical sensitivity of the multiplex real-time PCR test for detection of “Ca. Neoehrlichia mikurensis,” the “Ca. Neoehrlichia,” genus, and members ofthe Anaplasmataceae family. (A) “Ca. Neoehrlichia mikurensis”-specific probe (Neo_spec). (B) “Ca. Neoehrlichia” genus probe (Neo_genus). (C) Anaplas-mataceae family probe (Ana_family). A dilution series ranging from 105 copies to 10�1 copies of the plasmid pCNM1 containing a partial 16S rRNA gene of “Ca.Neoehrlichia mikurensis” (one copy per plasmid) was used for determining the analytic sensitivity of the probes. The experiment was performed 10 times, ofwhich one representative run is shown. All runs showed comparable results. Rn, normalized reporter dye fluorescence; H2O, negative water control.

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106 16S rRNA gene copies/ml bone marrow at day �12 and 7.01 �106 16S rRNA gene copies/ml blood at day 1. Blood samples ob-tained on days 15, 29, and 58 after the start of antibiotic therapywere negative using both tests.

Detection of “Ca. Neoehrlichia mikurensis” in local I. rici-nus tick populations. Since all three patients pursued regular out-door activities and since ticks are considered to be the main zoo-notic vector of “Ca. Neoehrlichia mikurensis,” four forest sites in

close proximity to the patients’ residential areas (�3-km distance)were analyzed for the presence of questing ticks carrying “Ca.Neoehrlichia mikurensis” (Fig. 4). For this purpose, DNA from1,916 ticks initially collected in 2009 for viral pathogen screening(28) were tested using the “Ca. Neoehrlichia mikurensis” real-time PCR. To facilitate fast screening of large populations, tickswere separated into groups of nymphs, males, and females andpooled (5 to 10 per pool) before DNA extraction.

The weighted means for the prevalence of “Ca. Neoehrlichiamikurensis” were 0.9% (Winterthur), 2.4% (Bassersdorf), 1.9%(Rümlang), and 4.7% (Rüti). This calculation was made basedupon the assumption that at least one tick per positive pool wasinfected with “Ca. Neoehrlichia mikurensis.” Furthermore, testresults showed that at every sampling site, the prevalence was be-tween 3.5% and 8% in at least one of the three subpopulations(nymphs, females, or males) (Fig. 4).

PCR products from three tick pools which tested positive in the“Ca. Neoehrlichia mikurensis” TaqMan assay were sequenced.Furthermore, DNA from the same pools was separately analyzedby broad-range PCR targeting the 16S rRNA gene. Along the226-bp section of the 16S rRNA gene that could be compared, theelectropherograms obtained by either method showed completeidentity to the “Ca. Neoehrlichia mikurensis” sequences obtainedfrom the clinical specimens. In addition, several “Ca. Neoehrlichiamikurensis”-negative, but Anaplasmataceae family probe-positivepools showed 16S rRNA sequences of Rickettsia helvetica, Wolba-chia pipientis, and “Candidatus Hamiltonella defensa.”

DISCUSSION

“Ca. Neoehrlichia mikurensis” is an emerging human pathogencausing septicemia and clinical symptoms such as relapsing fever,malaise, and weight loss (21). So far, six human case reports havebeen published, in 2010 and 2011, from four different Europeancountries, including Germany (n � 2), Sweden (n � 1), Switzer-land (n � 1), and the Czech Republic (n � 2), showing that “Ca.Neoehrlichia mikurensis” is widespread over Europe (9, 21–23).This is supported by several reports of the detection of “Ca. Neo-ehrlichia mikurensis” in questing tick populations in different Eu-ropean countries, with an average prevalence between 5 and 8%(29). In this study, we describe two new human cases of neoehrli-chiosis in immunocompromised patients. Both were identified in

TABLE 1 Analytical sensitivity of the three TaqMan hydrolysis probesused in the “Ca. Neoehrlichia mikurensis” multiplex real-time PCR

ProbePlasmid copy no.tested or control Avg CT valuea

Neo_spec 105 22.43 � 0.18104 24.86 � 0.25103 27.01 � 0.30102 29.87 � 0.33101 33.14 � 0.31100 35.27 � 1.0410�1 NDWater ND

Neo_genus 105 24.35 � 0.10104 26.66 � 0.17103 28.72 � 0.22102 31.37 � 0.42101 33.86 � 0.17100 35.11 � 0.8010�1 NDWater ND

Ana_family 105 25.51 � 0.56104 27.95 � 0.37103 30.11 � 0.36102 32.22 � 0.44101 34.15 � 0.49100 ND10�1 NDWater ND

a Shown are the average CT values and standard deviations for serial dilutions of thepositive-control plasmid pCNM1, which contains a partial 16S rRNA gene of “Ca.Neoehrlichia mikurensis.” Each data point was measured in 10 separate PCRs. ND, notdetected.

FIG 4 Geographic association between three patients diagnosed with neoehrlichiosis in 2009 (Kloten area), 2011 (Rüti area), and 2012 (Winterthur area) andinfection rates of “Ca. Neoehrlichia mikurensis” in nymphal and adult Ixodes ricinus ticks (total n � 1,916) collected in four forests in the greater Zurich area inSeptember 2009 (black arrowheads). The gray circles depict areas (radius, 3 km) that include both tick collection sites and the patients’ homes. Of note, only CT

values of �35.0 were counted as positive for the Anaplasmataceae family probe (12 pools showed CT values of 35.0). The minimal percentage of positive tickswas calculated based on the fact that at least 1 tick per pool must have carried “Ca. Neoehrlichia mikurensis” DNA. The map in the figure is used with permissionfrom swisstopo.

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close geographic proximity to each other as well as to the firstpatient we diagnosed in 2009 (Fig. 4) (21). This indicates that thegreater Zurich region is a high-risk area for “Ca. Neoehrlichiamikurensis” infections, especially for immunocompromised indi-viduals. All three patients have in common that they frequentlywent for outdoor activities, e.g., gardening, hiking, and playinggolf. Two patients were dog owners, but blood samples from bothdogs tested negative for “Ca. Neoehrlichia mikurensis” DNA(data not shown). Although both patients did not recall any tickbites at least during the last 2 years, this is still the most likely modeof transmission given that (i) I. ricinus and other tick species arehighly prevalent in the area, (ii) a significant percentage of theinvestigated ticks tested positive for “Ca. Neoehrlichia mikuren-sis” DNA (Fig. 4), and (iii) only 50 to 70% of patients with Lymedisease remember a tick bite (30). In addition, none of the patientsreported having come into contact with rodents, another poten-tial source of infection.

Until now, in all patients infected with “Ca. Neoehrlichia mi-kurensis,” the pathogen has been detected by rather laboriousbroad-range 16S rRNA gene analysis followed by sequencing.However, human infections with “Ca. Neoehrlichia mikurensis”may have been underdiagnosed in the past due to a lack of diag-nostic techniques such as serology or species-specific PCR. In thiscontext, the apparent increase of neoehrlichiosis may also be ex-plained by increasing awareness among physicians.

Recently, a “Ca. Neoehrlichia mikurensis” reverse line blot hasbeen developed and used to test ticks, but it has not been appliedto the testing of human samples (16). In order to extend diagnos-tic tools for specific, sensitive, fast, and cost-efficient testing andscreening purposes, we developed a TaqMan-based real-time PCRtargeting the 16S rRNA gene sequence of “Ca. Neoehrlichia mi-kurensis” (Fig. 2). The amplification product of this assay is 280 bpin size, which allows for sequencing and specificity control. Re-sults show that the limit of detection is approximately five copiesof the “Ca. Neoehrlichia mikurensis” 16S rRNA gene per reaction(Fig. 3). Since chromosomes of members of the Anaplasmataceaefamily contain a single 16S rRNA gene (31, 32), we made the sameassumption for “Ca. Neoehrlichia mikurensis” and made the 16SrRNA gene count equal to the number of bacterial cells.

Homology analysis showed a certain degree of variability in the16S rRNA gene of different “Ca. Neoehrlichia mikurensis” strains(Fig. 2). To overcome this problem, which may lead to decreasedassay sensitivity, two TaqMan probes were constructed. The firstprobe (Neo_spec) was optimized for the detection of the “Ca.Neoehrlichia mikurensis” sequence which is typically found inSwitzerland (GenBank accession number GQ501090). All se-quences obtained from the three patients and various ticks fromSwitzerland showed identical 16S rRNA sequences. Cross-reactiv-ity of this probe was not observed. The second probe (Neo_genus)was designed to hybridize with all currently published sequencevariants of “Ca. Neoehrlichia spp.,” including “Candidatus Neo-ehrlichia lotoris” (33) and some of the most closely related speciessuch as Ehrlichia ruminantium. In case of negative Neo_spec andpositive Neo_genus signals, sequencing of the PCR product mayreveal new sequevars. In these cases, it may also be useful to per-form an additional 16S rRNA gene broad-range PCR. In thisstudy, no samples with negative Neo_spec probe and positiveNeo_genus probe signals were detected. The assay contains anadditional third probe, which detects members of the Rickettsialesorder (Fig. 2). This probe is considered an additional control and

can also detect other infectious agents of the Anaplasmataceae andRickettsiaceae families. Using this probe, we found that almost alltick pools analyzed were positive for DNA of Rickettsiales, for ex-ample, Anaplasma phagocytophilum and Rickettsia helvetica,which were identified by sequencing of the PCR amplicon.

The assay we describe here serves as a valuable complementa-tion of current diagnostic protocols since it facilitates both semi-quantitative detection of “Ca. Neoehrlichia mikurensis” DNA inhuman samples (Fig. 1 and 3) and rapid screening of tick popula-tions for epidemiological purposes (Fig. 4). Data show that resultsof the real-time PCR were fully consistent with the results ob-tained by the broad-range 16S rRNA gene PCR (Fig. 1). By com-paring CT values with a standard curve generated from serial di-lutions of the positive-control plasmid pCNM1, we were able toestimate for the first time the bacterial DNA load in clinical sam-ples before and during antibiotic treatment. In both patients pre-sented in this study, initial DNA concentrations were relativelyhigh (Fig. 1). However, bacterial DNA was completely clearedfrom the bloodstream at most by 4 weeks after initiation of anti-biotic treatment with doxycycline. In patient 2, both broad-rangeand real-time PCR were already negative after 2 weeks of antibi-otic treatment. In addition to the rapid improvement of clinicalsymptoms, these data prove the excellent therapeutic efficacy ofdoxycycline against “Ca. Neoehrlichia mikurensis.”

Some DNA extracts from bone marrow and blood specimensshowed inhibitory effects in the PCR (Fig. 1). This is a well-knownphenomenon, which can be overcome by parallel testing of (up to1,000-fold) dilutions of the DNA extracts although sensitivity willdecrease as a consequence. Further improvement of DNA extrac-tion methods from blood is therefore desirable.

After establishing the real-time PCR, we screened questing I.ricinus tick populations collected in forests located less than 3 kmfrom the patients’ residences. The assay proved to be a powerfultool for high-throughput screening of close to 2,000 ticks using96-well microtiter plates. The minimal prevalence rates for “Ca.Neoehrlichia mikurensis” DNA in I. ricinus ticks at the four sam-pling sites located in the Zurich area (eastern Switzerland) wereslightly lower than the prevalence data recently published forwestern Switzerland (6.4%) (16). However, since it was assumedthat only one tick of each positive pool carried DNA from “Ca.Neoehrlichia mikurensis,” the true prevalence in the tick popula-tions of the Zurich area may be higher. Together, these data sup-port the finding that ticks in Switzerland are commonly infectedwith “Ca. Neoehrlichia mikurensis” without a clear predomi-nance for nymphs or female or male ticks (Fig. 4).

In conclusion, the real-time multiplex PCR described hereprovides a highly sensitive and specific tool that can assist clinicalmicrobiologists and researchers in detection of “Ca. Neoehrlichiamikurensis” as an emerging pathogen in both human and animalsamples. The close geographic association of tick collection siteswith the affected patients’ homes strongly supports the paradigmthat neoehrlichiosis is a vector-borne disease transmitted by ticks.

ACKNOWLEDGMENTS

We thank Ken Kraaijeveld, Svea Sachse, and Erich Zweygarth for provid-ing DNA samples of various members of the Anaplasmataceae family.

This study was supported in part by the University of Zürich.We have no conflicts of interest to report.

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