Summary
A medRxiv preprint reports that shotgun metagenomic sequencing identified Leptospira genetic material and provided species-level assignments in archived samples from suspected Philippine leptospirosis cases. The method detected pathogenic Leptospira in 60% of qPCR-positive samples that were negative by the microscopic agglutination test.
A retrospective laboratory study from the Philippines used shotgun metagenomic next-generation sequencing to identify Leptospira in archived clinical samples and assign the bacteria to species. The medRxiv preprint found that sequencing detected Leptospira genetic material in 10 of 17 samples that had previously tested positive by quantitative real-time PCR (qPCR), or 58.8%.
The sequencing results also added information in samples that were negative by the microscopic agglutination test (MAT), a conventional serological method. Among 10 qPCR-positive but MAT-negative samples, metagenomic sequencing detected pathogenic Leptospira in six, or 60%.
Contents
What the study examined
The researchers analysed 20 archived serum-derived total nucleic acid extracts collected from suspected leptospirosis cases. The samples had already been assessed using qPCR and MAT before being sequenced on an Illumina MiSeq platform.
Shotgun metagenomic sequencing examines genetic material in a sample rather than targeting only one predetermined organism. For this analysis, human-derived sequence reads were removed with the BWA and SAMtools tools. The remaining data were taxonomically profiled with KrakenUniq, which uses short sequence signatures called unique k-mers to help distinguish organisms. Species-level Leptospira assignments were retained only when they met predefined read-count and taxon-specific evidence thresholds.
Leptospirosis is caused by Leptospira bacteria. qPCR looks for targeted genetic material from the pathogen, while MAT measures antibody-mediated agglutination against Leptospira antigens. The authors note that MAT can provide limited information about the organisms circulating in a population and can be affected by serological cross-reactivity.
What sequencing added
The metagenomic analysis produced a different level of information from the conventional tests. It identified a predominance of L. interrogans, along with additional detection of L. borgpetersenii and L. kirschneri.
The MAT results showed a different pattern. Four of the five MAT-positive samples reacted predominantly against L. biflexa serovar Patoc, while one reacted against L. interrogans serovar Autumnalis. This contrast illustrates how antibody reactivity and direct genetic detection can provide different views of a suspected infection.
The study did not position sequencing as a replacement for qPCR or MAT. Instead, the results support its potential as a complementary approach that can add species-level information and help characterise the organisms detected in clinical material.
Why the findings matter
Leptospirosis is highly endemic in the Philippines, and understanding which Leptospira species are present can support genomic surveillance. Species-level information may help laboratories and public-health programmes describe the pathogen population more precisely than serological testing alone.
The evidence is from a small retrospective study using archived material, and the sequencing method did not detect Leptospira in every qPCR-positive sample. The authors link these missed detections to the sensitivity challenges of metagenomic sequencing when pathogen levels are low or samples have been stored before analysis.
The work is a medRxiv preprint posted on September 16, 2026. It evaluates pathogen detection and taxonomic characterisation rather than treatment or patient outcomes. The findings support further assessment of metagenomic sequencing in larger, prospective sample sets as a complementary tool for leptospirosis diagnosis and surveillance.