{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,2,26]],"date-time":"2026-02-26T08:20:39Z","timestamp":1772094039471,"version":"3.50.1"},"reference-count":46,"publisher":"MDPI AG","issue":"7","license":[{"start":{"date-parts":[[2014,6,25]],"date-time":"2014-06-25T00:00:00Z","timestamp":1403654400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/3.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>A highly conserved 357-bp segment of the 16S ribosomal RNA gene  (16S rDNA) of Borrelia burgdorferi sensu lato and the correspondent 358-bp segment of the Borrelia miyamotoi gene were amplified by a single pair of nested polymerase chain reaction (PCR) primers for detection, and the amplicons were used as the templates for direct Sanger DNA sequencing. Reliable molecular diagnosis of these borreliae was confirmed by sequence alignment analysis of the hypervariable regions of the PCR amplicon, using the Basic Local Alignment Search Tool (BLAST) provided by the GenBank. This methodology can detect and confirm B. burgdorferi and B. miyamotoi in blood samples of patients with off-season spirochetemia of low bacterial density. We  found four B. miyamotoi infections among 14 patients with spirochetemia, including one patient co-infected by both B. miyamotoi and B. burgdorferi in a winter month when human exposure to tick bites is very limited in the Northeast of the U.S.A. We conclude that sensitive and reliable tests for these two Borrelia species should be implemented in  the microbiology laboratory of hospitals located in the disease-endemic areas, for timely diagnosis and appropriate treatment of the patients at an early stage of the infection to prevent potential tissue damages.<\/jats:p>","DOI":"10.3390\/ijms150711364","type":"journal-article","created":{"date-parts":[[2014,6,26]],"date-time":"2014-06-26T02:28:30Z","timestamp":1403749710000},"page":"11364-11386","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":7,"title":["DNA Sequencing Diagnosis of Off-Season Spirochetemia  with Low Bacterial Density in Borrelia burgdorferi and  Borrelia miyamotoi Infections"],"prefix":"10.3390","volume":"15","author":[{"given":"Sin","family":"Lee","sequence":"first","affiliation":[{"name":"Department of Pathology, Milford Hospital, 300 Seaside Ave., Milford, CT 06460, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jessica","family":"Vigliotti","sequence":"additional","affiliation":[{"name":"Department of Pathology, Milford Hospital, 300 Seaside Ave., Milford, CT 06460, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Veronica","family":"Vigliotti","sequence":"additional","affiliation":[{"name":"Department of Pathology, Milford Hospital, 300 Seaside Ave., Milford, CT 06460, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"William","family":"Jones","sequence":"additional","affiliation":[{"name":"Department of Pathology, Milford Hospital, 300 Seaside Ave., Milford, CT 06460, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Thomas","family":"Moorcroft","sequence":"additional","affiliation":[{"name":"Origins of Health, LLC, 279 New Britain Road Berlin, CT 06037, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Katherine","family":"Lantsman","sequence":"additional","affiliation":[{"name":"My Path Medical, 20 Park Plaza, 804, Boston, MA 02116, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2014,6,25]]},"reference":[{"key":"ref_1","unstructured":"National Institute of Allergy and Infectious Diseases. Lyme Disease. Available online: http:\/\/www.niaid.nih.gov\/topics\/lymedisease\/research\/pages\/antibiotic.aspx."},{"key":"ref_2","unstructured":"Centers for Disease Control and Prevention: Lyme Disease. Available online: http:\/\/www.cdc.gov\/lyme\/."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"325","DOI":"10.1155\/2005\/278304","article-title":"Lyme disease: Is it or is it not?","volume":"16","author":"Conly","year":"2005","journal-title":"Can. J. Infect. Dis. Med. Microbiol."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"79","DOI":"10.1186\/1471-2334-9-79","article-title":"Diagnostic challenges of early Lyme disease: Lessons from a community case series","volume":"9","author":"Aucott","year":"2009","journal-title":"BMC Infect. Dis."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"2166","DOI":"10.1128\/JCM.00350-11","article-title":"Improving the yield of blood cultures from patients with early Lyme disease","volume":"49","author":"Liveris","year":"2011","journal-title":"J. Clin. Microbiol."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"6","DOI":"10.1016\/S0002-9343(99)80097-7","article-title":"Bloodstream invasion in early Lyme disease: Results from a prospective, controlled, blinded study using the polymerase chain reaction","volume":"99","author":"Goodman","year":"1995","journal-title":"Am. J. Med."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"1541","DOI":"10.1093\/infdis\/168.6.1541","article-title":"Circulating Borrelia burgdorferi in patients with acute Lyme disease: Results of blood cultures and serum DNA analysis","volume":"168","author":"Wallach","year":"1993","journal-title":"J. Infect. Dis."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"1107","DOI":"10.1086\/318138","article-title":"Death from inappropriate therapy for Lyme disease","volume":"31","author":"Patel","year":"2000","journal-title":"Clin. Infect. Dis."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"412","DOI":"10.1086\/321911","article-title":"False-positive results of PCR testing for Lyme disease","volume":"33","author":"Molloy","year":"2001","journal-title":"Clin. Infect. Dis."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"628","DOI":"10.1128\/jcm.30.3.628-632.1992","article-title":"Species-specific identification of and distinction between Borrelia burgdorferi genomic groups by using 16S rRNA\u2013directed oligonucleotide probes","volume":"30","author":"Marconi","year":"1992","journal-title":"J. Clin. Microbiol."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"2830","DOI":"10.1128\/jcm.30.11.2830-2834.1992","article-title":"Development of polymerase chain reaction primer sets for diagnosis of Lyme disease and for species-specific identification of Lyme disease isolates by 16S rRNA signature nucleotide analysis","volume":"30","author":"Marconi","year":"1992","journal-title":"J. Clin. Microbiol."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"962","DOI":"10.1111\/j.1365-2672.2005.02539.x","article-title":"Improving the specificity of 16S rDNA\u2013based polymerase chain reaction for detecting Borrelia burgdorferi sensu lato\u2013causative agents of human Lyme disease","volume":"98","author":"Cyr","year":"2005","journal-title":"J. Appl. Microbiol."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"569","DOI":"10.1309\/AJCPI72YAXRHYHEE","article-title":"Increased sensitivity and specificity of Borrelia burgdorferi 16S ribosomal DNA detection","volume":"133","author":"Lee","year":"2010","journal-title":"Am. J. Clin. Path."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"273","DOI":"10.1186\/1756-0500-3-273","article-title":"Early lyme disease with spirochetemia\u2014Diagnosed by DNA sequencing","volume":"3","author":"Lee","year":"2010","journal-title":"BMC Res. Notes"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"240","DOI":"10.1056\/NEJMoa1209039","article-title":"Meningoencephalitis from Borrelia miyamotoi in an Immunocompromised Patient","volume":"368","author":"Gugliotta","year":"2013","journal-title":"N. Engl. J. Med."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"21","DOI":"10.7326\/0003-4819-159-1-201307020-00005","article-title":"Borrelia miyamotoi infection presenting as human granulocytic anaplasmosis: A case report","volume":"159","author":"Chowdri","year":"2013","journal-title":"Ann. Intern. Med."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"1120","DOI":"10.4269\/ajtmh.2009.09-0208","article-title":"Niche partitioning of Borrelia burgdorferi and Borrelia miyamotoi in the same tick vector and mammalian reservoir species","volume":"81","author":"Barbour","year":"2009","journal-title":"Am. J. Trop. Med. Hyg."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"1816","DOI":"10.3201\/eid1710.101474","article-title":"Humans infected with relapsing fever spirochete Borrelia miyamotoi, Russia","volume":"17","author":"Platonov","year":"2011","journal-title":"Emerg. Infect. Dis."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"697","DOI":"10.3201\/eid0906.020459","article-title":"Relapsing fever-like spirochetes infecting European vector tick of Lyme disease agent","volume":"9","author":"Richter","year":"2003","journal-title":"Emerg. Infect. Dis."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"1070","DOI":"10.1086\/323424","article-title":"Yield of large-volume blood cultures in patients with early Lyme disease","volume":"184","author":"Wormser","year":"2001","journal-title":"J. Infect. Dis."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"3757","DOI":"10.1128\/jb.174.11.3757-3765.1992","article-title":"rRNA gene organization in the Lyme disease spirochete, Borrelia burgdorferi","volume":"174","author":"Schwartz","year":"1992","journal-title":"J. Bacteriol."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"483","DOI":"10.1099\/jmm.0.030387-0","article-title":"Detection and identification of bacteria in clinical samples by 16S rRNA gene sequencing: Comparison of two different approaches in clinical practice","volume":"61","author":"Jenkins","year":"2012","journal-title":"J. Med. Microbiol."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"330","DOI":"10.1016\/j.mimet.2007.02.005","article-title":"A detailed analysis of 16S ribosomal RNA gene segments for the diagnosis of pathogenic bacteria","volume":"69","author":"Chakravorty","year":"2007","journal-title":"J. Microbiol. Methods."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"30","DOI":"10.1111\/j.1574-6968.2008.01134.x","article-title":"Detection of Borrelia burgdorferi sensu lato DNA by PCR in serum of patients with clinical symptoms of Lyme borreliosis","volume":"283","author":"Santino","year":"2008","journal-title":"FEMS Microbiol. Lett."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"4284","DOI":"10.3390\/ijms15034284","article-title":"Detection of borreliae in archived sera from patients with clinically suspect lyme disease","volume":"15","author":"Lee","year":"2014","journal-title":"Int. J. Mol. Sci."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"980","DOI":"10.1128\/jcm.34.4.980-982.1996","article-title":"Host DNA can interfere with detection of Borrelia burgdorferi in skin biopsy specimens by PCR","volume":"34","author":"Cogswell","year":"1996","journal-title":"J. Clin. Microbiol."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"2443","DOI":"10.1128\/JCM.36.9.2443-2446.1998","article-title":"Strategy for optimizing DNA amplification in a peripheral blood PCR assay used for diagnosis of human brucellosis","volume":"36","author":"Morata","year":"1998","journal-title":"J. Clin. Microbiol."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"304","DOI":"10.1046\/j.1365-2133.1998.02080.x","article-title":"Lyme disease with facial nerve palsy: Rapid diagnosis using a nested polymerase chain reaction-restriction fragment length polymorphism analysis","volume":"138","author":"Hashimoto","year":"1998","journal-title":"Br. J. Dermatol."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"915","DOI":"10.7150\/ijms.6273","article-title":"Lyme borreliosis in human patients in Florida and Georgia, USA","volume":"10","author":"Clark","year":"2013","journal-title":"Int. J. Med. Sci."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"309","DOI":"10.1099\/jmm.0.013508-0","article-title":"A comparative analysis of molecular markers for the detection and identification of Borrelia spirochaetes in Ixodes ricinus","volume":"59","author":"Wodecka","year":"2010","journal-title":"J. Med. Microbiol."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"e36825","DOI":"10.1371\/journal.pone.0036825","article-title":"Direct molecular detection and genotyping of Borrelia burgdorferi from whole blood of patients with early Lyme disease","volume":"7","author":"Eshoo","year":"2012","journal-title":"PLoS One"},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"643","DOI":"10.1128\/AAC.00788-09","article-title":"Ineffectiveness of tigecycline against persistent Borrelia burgdorferi","volume":"54","author":"Barthold","year":"2010","journal-title":"Antimicrob. Agents Chemother."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"665","DOI":"10.1111\/j.1600-0463.2010.02615.x","article-title":"Persistence of borrelial DNA in the joints of Borrelia burgdorferi-infected mice after ceftriaxone treatment","volume":"118","author":"Hartiala","year":"2010","journal-title":"APMIS"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"e29914","DOI":"10.1371\/journal.pone.0029914","article-title":"Persistence of Borrelia burgdorferi in rhesus macaques following antibiotic treatment of disseminated infection","volume":"7","author":"Embers","year":"2012","journal-title":"PLoS One"},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"219","DOI":"10.3122\/jabfm.2009.02.080130","article-title":"Unusual presentation of Lyme disease: Horner syndrome with negative serology","volume":"22","author":"Morrison","year":"2009","journal-title":"J. Am. Board Fam. Med."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"961","DOI":"10.1073\/pnas.94.3.961","article-title":"Foreign (M13) DNA ingested by mice reaches peripheral leukocytes, spleen, and liver via the intestinal wall mucosa and can be covalently linked to mouse DNA","volume":"94","author":"Schubbert","year":"1997","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"18159","DOI":"10.1073\/pnas.0405763102","article-title":"An ecological approach to preventing human infection: Vaccinating wild mouse reservoirs intervenes in the Lyme disease cycle","volume":"101","author":"Tsao","year":"2004","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"2476","DOI":"10.1128\/AEM.02633-08","article-title":"Climate and tick seasonality predict Borrelia burgdorferi genotype distribution","volume":"75","author":"Gatewood","year":"2009","journal-title":"Appl. Environ. Microbiol."},{"key":"ref_39","doi-asserted-by":"crossref","unstructured":"Krause, P.J., Narasimhan, S., Wormser, G.P., Barbour, A.G., Platonov, A.E., Brancato, J., Lepore, T., Dardick, K., Mamula, M., and Rollend, L. Borrelia miyamotoi sensu lato seroreactivity and seroprevalence in the northeastern United States. Available online: http:\/\/dx.doi.org\/10.3201\/eid2007.131587.","DOI":"10.3201\/eid2007.131587"},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"12853","DOI":"10.3390\/ijms140612853","article-title":"A novel low temperature PCR assured high-fidelity DNA amplification","volume":"14","author":"Hong","year":"2013","journal-title":"Int. J. Mol. Sci."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"15","DOI":"10.1093\/nar\/28.1.15","article-title":"GenBank","volume":"28","author":"Benson","year":"2000","journal-title":"Nucleic Acids Res."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"2761","DOI":"10.1128\/JCM.01228-07","article-title":"16S rRNA gene sequencing for bacterial identification in the diagnostic laboratory: Pluses, perils, and pitfalls","volume":"45","author":"Janda","year":"2007","journal-title":"J. Clin. Microbiol."},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"1071","DOI":"10.1007\/s12275-012-2642-z","article-title":"Effects of PCR cycle number and DNA polymerase type on the 16S rRNA gene pyrosequencing analysis of bacterial communities","volume":"50","author":"Ahn","year":"2012","journal-title":"J. Microbiol."},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"880","DOI":"10.1128\/AEM.67.2.880-887.2001","article-title":"Evaluation of PCR-generated chimeras, mutations, and heteroduplexes with 16S rRNA gene-based cloning","volume":"67","author":"Qiu","year":"2001","journal-title":"Appl. Environ. Microbiol."},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"8966","DOI":"10.1128\/AEM.71.12.8966-8969.2005","article-title":"PCR-induced sequence artifacts and bias: Insights from comparison of two 16S rRNA clone libraries constructed from the same sample","volume":"71","author":"Acinas","year":"2005","journal-title":"Appl. Environ. Microbiol."},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"65","DOI":"10.1007\/978-1-61779-937-2_5","article-title":"Guidelines for the use of molecular tests for the detection and genotyping of human papilloma virus from clinical specimens","volume":"903","author":"Lee","year":"2012","journal-title":"Methods Mol. Biol."}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/15\/7\/11364\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T21:12:54Z","timestamp":1760217174000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/15\/7\/11364"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2014,6,25]]},"references-count":46,"journal-issue":{"issue":"7","published-online":{"date-parts":[[2014,7]]}},"alternative-id":["ijms150711364"],"URL":"https:\/\/doi.org\/10.3390\/ijms150711364","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2014,6,25]]}}}