Farkas, Róbert

Földvári, Gábor

Lakos, András

Emerging Infectious Diseases

English

PubMed

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LETTERSBorrelia spielmaniiErythema Migrans,HungaryTo the Editor: Lyme disease is themost frequent tickborne human infec-tion in the northern hemisphere. Atleast 5 species of the Borreliaburgdorferi sensu lato complex, B.burgdorferi sensu stricto, B. afzelii, B.garinii, B. bissettii, and B. lusitaniae,have a pathogenic role in humanLyme disease in central Europe (1–3).A sixth pathogenic strain, A14S, hasbeen isolated from 1 Dutch (4) and 2German patients with erythemamigrans (5). This strain was alsodetected in 4 questing Ixodes ricinusticks in Germany (6,7) and 1 in theCzech Republic (8). A14S has recent-ly been described as a new species, B.spielmanii (9); its main reservoir hostis probably the garden dormouse(Eliomys quercinus), but B. spielmaniicould not be detected in mice or voles.Richter et al. (9) could not find ticksharboring B. spielmanii in 3 of 5examined areas in Germany. Theywere present almost exclusively in asingle area where the prevalence ofinfection with this genotype was 15(6%) of 251. We describe the isolationof this novel Lyme disease spirochetefrom a human patient with erythemamigrans in Hungary.Since 1999, we have regularly iso-lated Borrelia burgdorferi sensu latofrom skin biopsy specimens of erythe-ma migrans and acrodermatitis chron-ica atrophicans taken from patients atthe Center for Tick-borne Diseases,Budapest, Hungary. To identify theBorrelia species occurring inHungarian Lyme disease patients, wehave started to molecularly analyzecultured isolates that originate fromerythema migrans of differentpatients. DNA was isolated from 8bacterial pellets by using QIAampDNA mini kit (Qiagen, Hilden,Germany). Primers BSL-F and BSL-R were used; these amplify an ≈250-bp region of the outer surface protein(osp) A gene from all Lyme diseasespirochetes (10). We added 2 µLextracted DNA to a 20-µL reactionmixture composed of 1.0 U HotStart-Taq DNA polymerase, 200 µmol/L ofeach dNTP, 25 pmol of each primer,and 1.5 mmol/L MgCl2 (HotStartTaqMaster Mix, Qiagen). An initialdenaturation step at 94°C for 15 minwas followed by 40 cycles of denatu-ration at 94°C for 30 s, annealing at58°C for 30 s, and extension at 72°Cfor 30 s. Final extension was done at72°C for 5 min. Amplified DNA wassubjected to electrophoresis in a 1.5%agarose gel that was prestained withethidium bromide and viewed underUV light. After purification, thedideoxy chain termination (AppliedBiosystems Division, Foster City, CA,USA) was used for sequencing.Obtained sequences were checkedwith Chromas v.1.45 and compared tosequence data available fromGenBank by using BLAST (http://www.ncbi.nlm.nih.gov/BLAST/).New sequences were submitted toGenBank.Six sequences (DQ007298, DQ00-7299, DQ007300, DQ007301, DQ00-7302, DQ007303) showed 100%homology to B. afzelii Khab 625strain (AY502599). One (DQ007297)of the remaining 2 samples showed99.6% similarity with B. burgdorferiB31 (AE000790), and the other(AY995900) showed 99.21% similar-ity with B. spielmanii (AF102057).The patient whose culture showedB. spielmanii was a 42-year-oldwoman with a homogenous erythemamigrans, diagnosed on September 24,1999. The erythema was 10 cm indiameter on the front surface of theknee at the first visit (see onlineFigure, available at http://www.cdc.gov/ncidod/EID/vol11no11/05-0542-G.htm). The immunoglobulin M(IgM) and IgG Borrelia immunoblotthat applied B. afzelii (ACA1) antigenwas negative in serum drawn on theseventh day after the appearance oferythema migrans. The patient did notremember a tick bite and had not trav-eled abroad during the previous 6months. She complained of an“extremely unusual,” intense, serousnasal discharge that started 3 weeksbefore the appearance of erythemamigrans and of a moderate headache;both disappeared spontaneously 2weeks before treatment.Our results show at least 3 distinctspecies of B. burgdorferi sensu lato inHungary. In addition to B. burgdorferisensu stricto and B. afzelii, knownthroughout Europe, we detected therecently described species B. spiel-manii among randomly selected sam-ples. Together with 2 previous publi-cations (4,5), our observation alsosuggest that B. spielmanii has a path-ogenic role in human Lyme disease.Although B. spielmanii is distributedmore focally than other species of theB. burgdorferi sensu lato complex (9),it occurs from the Netherlandsthrough Germany and CzechRepublic to Hungary (4,5,7,8).AcknowledgmentWe acknowledge the advice and thepositive control samples provided byMartin J. Kenny.Gábor Földvári,* Róbert Farkas,*and András Lakos†*St. István University Faculty of VeterinaryScience, Budapest, Hungary; and †Centerfor Tick-borne Disease, Budapest, HungaryReferences1. Postic D, Ras NM, Lane RS, Hendson M,Baranton G. Expanded diversity amongCalifornian Borrelia isolates and descrip-tion of Borrelia bissettii sp. nov. (formerlyBorrelia group DN127). J Clin Microbiol.1998;36:3497–504.2. Maraspin V, Cimperman J, Lotric-Furlan S,Ruzic-Sabljic E, Jurca T, Picken RN, et al.Solitary borrelial lymphocytoma in adultpatients. Wien Klin Wochenschr.2002;114:515–23.3. Collares-Pereira M, Couceiro S, Franca I,Kurtenbach K, Schafer SM, Vitorino L, etal. First isolation of Borrelia lusitaniaefrom a human patient. J Clin Microbiol.2004;42:1313–8.1794 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 11, No. 11, November 2005LETTERS4. Wang G, van Dam AP, Dankert J.Phenotypic and genetic characterization ofa novel Borrelia burgdorferi sensu lato iso-late from a patient with Lyme borreliosis. JClin Microbiol. 1999;37:3025–8.5. Fingerle V, Michel H, Schulte-Spechtel U,Göttner G, Hizo-Teufel C, Hofmann H, etal. A14S—a new Borrelia burgdorferi s.l.genospecies as relevant cause of humandisease [abstract]. Int J Med Microbiol.2004;294(Suppl 1):207.6. Rauter C, Oehme R, Dietrich I, Engele M,Hartung T. Distribution of clinically rele-vant Borrelia genospecies in ticks assessedby a novel, single-run real-time PCR. J ClinMicrobiol. 2002;40:36–43.7. Michel HB, Wilske B, Hettche G, GöttnerG, Heimerl C, Reischl U, et al. An ospA-polymerase chain reaction/restriction frag-ment length polymorphism–based methodfor sensitive detection and reliable differen-tiation of all European Borrelia burgdorferisensu lato species and OspA types. MedMicrobiol Immunol. 2003;193:219–26.8. Derdáková M, Beati L, Pet’ko B, Stanko M,Fish D. Genetic variability within Borreliaburgdorferi sensu lato genospecies estab-lished by PCR-single-strand conformationpolymorphism analysis of the rrfA-rrlBintergenic spacer in Ixodes ricinus ticksfrom the Czech Republic. Appl EnvironMicrobiol. 2003;69:509–16.9. Richter D, Schlee DB, Allgöwer R,Matuschka FR. Relationships of a novelLyme disease spirochete, Borrelia spiel-mani sp. nov., with its hosts in centralEurope. Appl Environ Microbiol.2004;70:6414–9.10. Demaerschalck I, Benmessaoud A,Dekesel, M, Hoyois B, Lobet Y, Hoet P, etal. Simultaneous presence of differentBorrelia burgdorferi genospecies in biolog-ical fluids of Lyme-disease patients. J ClinMicrobiol. 1995;33:602–8.Address for correspondence: András Lakos,Center for Tick-borne Diseases, Visegrádi u.14. H-1132 Budapest, Hungary; fax: 36-1-329-3898; email: alakos@t-online.huProfilingMycobacteriumulcerans withhsp65To the Editor: Mycobacteriumulcerans is an emerging humanpathogen responsible for Buruli ulcer,a necrotizing skin disease most com-monly found in West Africa, but out-breaks have also been reported in theAmericas, Australia, and Asia (1).Environmental sources of infectionand mode of transmission are notcompletely known. M. ulceransgrows slowly at 32°C, requiring 6–8weeks for colonies to be visible in pri-mary culture. Differentiation from M.marinum, which also causes skininfections, is important, since M. mar-inum can usually be treated withantimicrobial agents, whereas M.ulcerans most often does not respondfavorably to drug therapy, and treat-ment is usually by surgical excision(2). M. shinshuense, initially isolatedfrom a child in Japan, is phenotypical-ly and genetically related but bio-chemically distinct from M. ulcerans(3). In the last decade, several DNA-based techniques for mycobacterialidentification have been developed.Rapid molecular detection and differ-entiation of organisms that cause skininfections directly from tissue or exu-dates could be of great value for earlytreatment. Some techniques, especial-ly those that include nucleic acidamplification, could be used directlyon clinical samples. The acceptedstandard for molecular identificationof mycobacteria is sequencing analy-sis of 2 hypervariable regions identi-fied in 16S rRNA gene. M. marinumand M. ulcerans share identical 5′-16S rDNA and 16S-23S rRNA genespacer sequences (4). Polymerasechain reaction (PCR)-dependentmethods are based on the 16S rRNAgene (5), the hsp65 gene (6) or theinsertion sequence IS2404 (7).Recently, a novel category of variablenumber tandem repeats that could dis-tinguish M. marinum and M. ulceransgenotypes has been described (8).Polymorphisms in the 3′-16SrDNA region discriminate M. ulcer-ans from M. marinum and M. shin-shuense (5). These polymorphismsalso allow the separation of M. ulcer-ans into 3 subgroups according togeographic origin and variable pheno-typic differences. IS2404 discrimi-nates M. ulcerans from M. marinum(7). It has been used in restrictionfragment length polymorphism analy-sis applied to a comparable number ofM. ulcerans and M. marinum strains,confirming that this sequence is pres-ent in high copy numbers in M. ulcer-ans but absent in M. marinum.Nevertheless, an unusual mycobac-terium was recently isolated that isclosely related to M. marinum by phe-notypic tests, lipid pattern, and partial16S rDNA sequencing but presentslow copy numbers of this element (9).PCR-restriction enzyme analysis(PRA) of a 441-bp fragment of thehsp65 gene is a rapid, easy, and inex-pensive method for identifyingmycobacteria (10). Devallois et al. (6)described the PRA-hsp65 pattern of 1M. ulcerans strain ATCC 33728 thatoriginated in Japan. This isolate wasconsidered a new species that resem-bled M. ulcerans and was named M.shinshuense (3). We report here the usefulness ofPRA-hsp65 to differentiate M. ulcer-ans strains from different geographicareas. Since Buruli ulcer cases havebeen reported on 5 continents, westudied 33 M. ulcerans strains thatoriginated from Africa (Benin, Zaire,Ghana, Congo, Angola, Côte d’Ivoire,Togo), Asia (China, Malaysia),Australia (Papua New Guinea,Australia), the Caribbean (Mexico,Surinam, French Guiana), 1 M. shin-shuense from Japan, 1 M. marinumisolate and 1 IS2404-positive M. mar-inum isolate from France (9). Allstrains were identified at the InstituteEmerging Infectious Diseases • www.cdc.gov/eid • Vol. 11, No. 11, November 2005 1795

A14S—a new Borrelia burgdorferi s.l. genospecies as relevant cause of human disease [abstract].

An ospApolymerase chain reaction/restriction fragment length polymorphism–based method for sensitive detection and reliable differentiation of all European Borrelia burgdorferi sensu lato species and OspA types. Med Microbiol Immunol.

Distribution of clinically relevant Borrelia genospecies in ticks assessed by a novel, single-run real-time PCR.

Expanded diversity among Californian Borrelia isolates and description of Borrelia bissettii sp. nov. (formerly Borrelia group DN127).

First isolation of Borrelia lusitaniae from a human patient.

Genetic variability within Borrelia burgdorferi sensu lato genospecies established by PCR-single-strand conformation polymorphism analysis of the rrfA-rrlB intergenic spacer in Ixodes ricinus ticks from the Czech Republic. Appl Environ Microbiol.

Relationships of a novel Lyme disease spirochete, Borrelia spielmani sp. nov., with its hosts in central Europe. Appl Environ Microbiol.

Simultaneous presence of different Borrelia burgdorferi genospecies in biological fluids of Lyme-disease patients.

Borrelia spielmanii erythema migrans, Hungary

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