Taxonomic identification and characterization of Leptospira from retrospective clinical samples in the Philippines
- 1Advanced Molecular Technologies Laboratory, Research Institute for Tropical Medicine, Department of Health, Muntinlupa City, Philippines
Abstract
Leptospirosis is a highly endemic neglected tropical disease in the Philippines, where conventional diagnostic methods such as the microscopic agglutination test (MAT) provide limited information on circulating Leptospira and are affected by serological cross-reactivity. This study evaluated the utility of shotgun metagenomic next-generation sequencing (mNGS) for pathogen detection and species-level characterization using 20 archived serum-derived total nucleic acid extracts collected from suspected leptospirosis cases. Samples were previously tested by quantitative real-time PCR (qPCR) and MAT, and sequenced using the Illumina MiSeq platform. Host-derived reads were removed using BWA and SAMtools prior to taxonomic profiling with KrakenUniq. Species-level Leptospira assignments were retained based on predefined KrakenUniq filtering criteria, including a minimum taxReads threshold and evidence of taxon-specific unique k-mers. Leptospira taxReads were detected in 58.8% (n=10/17) of qPCR-positive samples. Notably, mNGS detected pathogenic Leptospira in 60% (n=6/10) qPCR-positive but MAT-negative samples, demonstrating its potential to complement conventional serology for etiological characterization. MAT reactivity was predominantly against L. biflexa serovar Patoc (n=4/5), with one sample reacting to L. interrogans serovar Autumnalis in the MAT-positive samples. Meanwhile, mNGS identified a predominance of L. interrogans, with additional detection of L. borgpetersenii and L. kirschneri. The absence of detectable Leptospira in some qPCR-positive samples underscores the sensitivity limitations of mNGS in low-burden or archived samples. Overall, mNGS provided species-level information beyond conventional diagnostics and supports its potential application in Leptospira genomic surveillance in the Philippines.
IMPACT STATEMENTLeptospirosis remains an important public health concern in the Philippines, but genomic information on human Leptospira infections is limited. Shotgun metagenomic sequencing provided species-level information beyond routine qPCR and MAT. Leptospira interrogans predominated, alongside other pathogenic species. These findings establish an initial genomic baseline for Leptospira in Philippine clinical specimens and support future surveillance of strain and serovar diversity and transmission.
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BibTeX
@misc{realingo2026taxonomic,
title = {{Taxonomic identification and characterization of Leptospira from retrospective clinical samples in the Philippines}},
author = {A. M. L. Realingo and J. I. G. Manalo and L. L. M. Dancel and K. Alvarado-Dela Cruz and A. N. A. Digman and C. B. A. Roxas and D. D. Argana and R. A. Baterna and E. J. G. Medina and A. D. Nicolasora and T. J. R. Dizon and F. G. M. Polotan and A. A. P. Larano},
howpublished = {medRxiv},
year = {2026},
month = sep,
doi = {10.64898/2026.09.16.26363189},
url = {https://www.medrxiv.org/content/10.64898/2026.09.16.26363189v1},
note = {CC BY-NC-ND},
}
RIS
TY - UNPB
TI - Taxonomic identification and characterization of Leptospira from retrospective clinical samples in the Philippines
AU - A. M. L. Realingo
AU - J. I. G. Manalo
AU - L. L. M. Dancel
AU - K. Alvarado-Dela Cruz
AU - A. N. A. Digman
AU - C. B. A. Roxas
AU - D. D. Argana
AU - R. A. Baterna
AU - E. J. G. Medina
AU - A. D. Nicolasora
AU - T. J. R. Dizon
AU - F. G. M. Polotan
AU - A. A. P. Larano
PY - 2026
DA - 2026/09/16
PB - medRxiv
DO - 10.64898/2026.09.16.26363189
UR - https://www.medrxiv.org/content/10.64898/2026.09.16.26363189v1
AB - Leptospirosis is a highly endemic neglected tropical disease in the Philippines, where conventional diagnostic methods such as the microscopic agglutination test (MAT) provide limited information on circulating Leptospira and are affected by serological cross-reactivity. This study evaluated the utility of shotgun metagenomic next-generation sequencing (mNGS) for pathogen detection and species-level characterization using 20 archived serum-derived total nucleic acid extracts collected from suspected leptospirosis cases. Samples were previously tested by quantitative real-time PCR (qPCR) and MAT, and sequenced using the Illumina MiSeq platform. Host-derived reads were removed using BWA and SAMtools prior to taxonomic profiling with KrakenUniq. Species-level Leptospira assignments were retained based on predefined KrakenUniq filtering criteria, including a minimum taxReads threshold and evidence of taxon-specific unique k-mers. Leptospira taxReads were detected in 58.8% (n=10/17) of qPCR-positive samples. Notably, mNGS detected pathogenic Leptospira in 60% (n=6/10) qPCR-positive but MAT-negative samples, demonstrating its potential to complement conventional serology for etiological characterization. MAT reactivity was predominantly against L. biflexa serovar Patoc (n=4/5), with one sample reacting to L. interrogans serovar Autumnalis in the MAT-positive samples. Meanwhile, mNGS identified a predominance of L. interrogans, with additional detection of L. borgpetersenii and L. kirschneri. The absence of detectable Leptospira in some qPCR-positive samples underscores the sensitivity limitations of mNGS in low-burden or archived samples. Overall, mNGS provided species-level information beyond conventional diagnostics and supports its potential application in Leptospira genomic surveillance in the Philippines. IMPACT STATEMENTLeptospirosis remains an important public health concern in the Philippines, but genomic information on human Leptospira infections is limited. Shotgun metagenomic sequencing provided species-level information beyond routine qPCR and MAT. Leptospira interrogans predominated, alongside other pathogenic species. These findings establish an initial genomic baseline for Leptospira in Philippine clinical specimens and support future surveillance of strain and serovar diversity and transmission.
ER -