A-Z Index × Submit A-Z Index × Submit A-Z Index Search Dropdown × Submit Facebook Twitter LinkedIn Syndicate Emerging Infectious Disease journal ISSN: 1080-6059 Disclaimer: Early release articles are not considered as final versions. Any changes will be reflected in the online version in the month the article is officially released.
Whole-genome sequencing strengthens tuberculosis surveillance, but incomplete lineage schemes can misclassify isolates. We developed Pathotypr, an alignment-free tool for assemblies and reads, using 2,145 markers derived from 26,813 genomes. Across 88,071 samples, Pathotypr improved concordance, expanded uncommon lineage coverage, and detected resistance markers, supporting molecular epidemiology and public health surveillance.
According to the World Health Organization, tuberculosis caused an estimated 1.23 million deaths in 2024 ( https://www.who.int/teams/global-programme-on-tuberculosis-and-lung-health/tb-reports/global-tuberculosis-report-2025 ). Whole-genome sequencing can strengthen disease control by linking cases, detecting resistance, and distinguishing local transmission from repeated introduction; however, those benefits depend on results that are both rapid and comparable. The Mycobacterium tuberculosis complex (MTBC) currently includes 10 human-adapted lineages (L1–L10) and 4 animal-adapted clades (A1–A4), and several uncommon or recently described groups remain sparsely sampled or absent from some typing schemes ( 2 – 6 ). When a scheme does not represent that diversity, isolates may remain unclassified or be assigned to the nearest available label. Differences in sequencing access and reporting remain broader challenges across programs in Europe, but a shared and current lineage framework is still essential wherever genomic surveillance is used ( 7 , 8 ).
We developed Pathotypr as an open-source, alignment-free software that accepts assembled genomes or raw short reads and returns lineage assignments, World Health Organization–cataloged resistance-associated variants, and a closely related reference genome for downstream analysis. Assemblies can be classified from phylogenetic markers or an independent k -mer model, whereas reads are screened directly for lineage and resistance markers and can return > 1 supported lineage when mixed signals are present. We evaluated those functions in independent datasets, an 88,071-sample benchmark, and 7,148 genomes from the Comprehensive Resistance Prediction for Tuberculosis: an International Consortium (CRyPTIC) dataset ( Appendix ).
Figure 1 . Development and implementation of the lineage framework for Pathotypr, an alignment-free tool for assemblies and reads in genomic surveillance of tuberculosis. A) Maximum-likelihood phylogeny of 26,813 Mycobacterium tuberculosis complex...
To extract lineage phylomarkers, we obtained a maximum-likelihood phylogeny of 26,813 MTBC genomes and 609,003 polymorphic sites, which placed L1–L10 and A1–A4 within 1 evolutionary backbone and exposed the marked underrepresentation of certain lineages ( Figure 1 , panel A). We then mapped substitutions to branches and selected single-nucleotide polymorphisms (SNPs) on the stem branch leading to each monophyletic lineage or clade. After removing 3,039 missense variants and 454 homoplastic nonmissense variants, 2,145 of 5,638 candidates remained as lineage-defining markers. A variable overlap of lineage markers occurred with other tools, ranging from no overlap to 698 (62.8…