{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,6,19]],"date-time":"2026-06-19T05:54:58Z","timestamp":1781848498273,"version":"3.54.5"},"reference-count":40,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2026,6,19]],"date-time":"2026-06-19T00:00:00Z","timestamp":1781827200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Public Health"],"abstract":"\n Background<\/jats:title>\n Leptospiral isolation and whole-genome sequencing are often unfeasible for genotyping; therefore, single-locus culture-independent sequencing is widely used. However, primer sets remain broadly applied without comprehensive quantitative or qualitative performance evaluation.<\/jats:p>\n <\/jats:sec>\n \n Objective<\/jats:title>\n \n This study integrated\n in silico<\/jats:italic>\n primer performance, phylogenetic benchmarking, and barcoding gap analysis to identify\n Leptospira<\/jats:italic>\n markers that best delimit species, support diagnostics, and enable standardized BLAST identity cut-off thresholds.\n <\/jats:p>\n <\/jats:sec>\n \n Methods<\/jats:title>\n \n An integrative analysis evaluated 10 primer pairs targeting seven loci:\n secY<\/jats:italic>\n inner,\n secY<\/jats:italic>\n outer,\n lfb1, flaB, rpoB, gyrB,<\/jats:italic>\n and\n lipL32<\/jats:italic>\n . New primers for 16S rRNA and\n flaB<\/jats:italic>\n were designed and included in the framework. Primer hybridization and theoretical amplification were assessed\n in silico<\/jats:italic>\n in genomes of five pathogenic\n Leptospira<\/jats:italic>\n species. The best-performing markers were then tested for congruence with core-genome and full-\n ppk<\/jats:italic>\n phylogenies. Global and species-specific barcoding gap analyses assessed genetic distances, and BLAST identity cut-offs were proposed for markers performing consistently across all steps.\n <\/jats:p>\n <\/jats:sec>\n \n Results<\/jats:title>\n \n Only five markers showed good theoretical amplification performance: 16S rRNA,\n secY<\/jats:italic>\n outer,\n flaB, rpoB,<\/jats:italic>\n and\n lipL32<\/jats:italic>\n . However, only the phylogenies generated from\n secY<\/jats:italic>\n outer, redesigned\n flaB,<\/jats:italic>\n and\n lipL32<\/jats:italic>\n were similar with core-genome and\n ppk<\/jats:italic>\n trees. Barcoding analyses further showed that only\n secY<\/jats:italic>\n outer and redesigned\n flaB<\/jats:italic>\n could discriminate both species and intraspecific strains, with proposed BLAST identity cut-offs of 96 and 95%, respectively.\n <\/jats:p>\n <\/jats:sec>\n \n Discussion<\/jats:title>\n \n The present study extends barcoding gap analysis to bacterial marker evaluation within an integrative framework, an approach previously applied mainly to Metazoa. It also highlights an important limitation in the widespread use of genotyping markers that lack reliable\n in silico<\/jats:italic>\n amplification performance and sufficient intra- and interspecific discriminatory power. Together, these findings support the combined use of\n secY<\/jats:italic>\n outer and redesigned\n flaB<\/jats:italic>\n for molecular surveillance of the main pathogenic leptospires, although further\n in vitro<\/jats:italic>\n validation remains needed.\n <\/jats:p>\n <\/jats:sec>","DOI":"10.3389\/fpubh.2026.1837984","type":"journal-article","created":{"date-parts":[[2026,6,19]],"date-time":"2026-06-19T05:48:25Z","timestamp":1781848105000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":0,"title":["Integrative assessment of species-level genetic markers for the diagnosis of pathogenic Leptospira"],"prefix":"10.3389","volume":"14","author":[{"given":"Luiza","family":"Aym\u00e9e","sequence":"first","affiliation":[{"name":"Laboratory of Veterinary Bacteriology, Biomedical Institute of Fluminense Federal University","place":["Niteroi, Brazil"]},{"name":"Department of Microbiology, Immunology and Parasitology, Faculty of Medical Sciences, Rio de Janeiro State University","place":["Rio de Janeiro, Brazil"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Vinicius de Carvalho","family":"Moura","sequence":"additional","affiliation":[{"name":"Laboratory of Investigation in Medical Microbiology, Institute of Microbiology, Federal University of Rio de Janeiro","place":["Rio de Janeiro, Brazil"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Myllena dos Santos","family":"Pereira","sequence":"additional","affiliation":[{"name":"Laboratory of Investigation in Medical Microbiology, Institute of Microbiology, Federal University of Rio de Janeiro","place":["Rio de Janeiro, Brazil"]},{"name":"National Reference Laboratory for Leptospirosis, Fiocruz","place":["Rio de Janeiro, Brazil"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Walter","family":"Lilenbaum","sequence":"additional","affiliation":[{"name":"Laboratory of Veterinary Bacteriology, Biomedical Institute of Fluminense Federal University","place":["Niteroi, Brazil"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Maria Isabel Nogueira Di","family":"Azevedo","sequence":"additional","affiliation":[{"name":"Laboratory of Investigation in Medical Microbiology, Institute of Microbiology, Federal University of Rio de Janeiro","place":["Rio de Janeiro, Brazil"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1965","published-online":{"date-parts":[[2026,6,19]]},"reference":[{"key":"ref1","doi-asserted-by":"publisher","first-page":"835","DOI":"10.1007\/s10096-019-03797-4","article-title":"Leptospirosis: a neglected tropical zoonotic infection of public health importance - an updated review","volume":"39","author":"Karpagam","year":"2020","journal-title":"Eur J Clin Microbiol Infect Dis"},{"key":"ref2","doi-asserted-by":"publisher","first-page":"1589","DOI":"10.2460\/javma.22.06.0258","article-title":"A global one health perspective on leptospirosis in humans and 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