A rationalized molecular and genomic strategy to get a snapshot of the tuberculosis peri-pandemic transmission in Madrid

Genomic epidemiology has enhanced our comprehension of tuberculosis (TB) transmission, surpassing the lower discriminatory MIRU-VNTR analysis. Nevertheless, universal long-term genomic analysis is required to adequately identify transmission clusters, a task which is not feasible in many settings. The objective of this study is to evaluate an alternative strategy for obtaining a rapid snapshot of TB transmission in instances where systematic molecular/genomic surveillance is not in operation. The study utilised Madrid as a model population, examining the period before and after the emergence of the COVD-19 pandemic. A rationalized use of MIRU-VNTR and whole genome sequencing (WGS) is proposed, with the implementation of a three-sequential step analytical pipeline: firstly, a preliminary screening of potential clusters [U1] is conducted by a 6 loci-MIRU reduced panel (MIRU-6); secondly, extended MIRU-24 genotyping is performed exclusively on the MIRU-6-defined clusters; and thirdly, WGS is applied solely for the confirmation of the MIRU-24-defined clusters. The application of the strategy to 454 isolates (from the years 2019 and 2021) by progressively ruling out the orphan cases limited the application of WGS to 59 cases (13% of the initial total cases), who were part of 25 MIRU-24 clusters and genomic confirmation was ultimately achieved for seventeen of these clusters. The genomic data from six of these clusters were utilised for the identification of strain marker single-nucleotide polymorphisms (SNPs). A multiplex polymerase chain reaction (PCR) was designed to amplify the regions containing 11 marker SNPs for the selected clustered strains and the amplicons were then analysed by targeted nanopore sequencing. This approach enabled us to swiftly eliminate their involvement in 202 new incident cases and 261 retrospective cases, obviating the necessity for WGS. Our strategy based on the identification of clusters by a retrospective sequential application of progressively enhanced discriminatory molecular/genomic methods, coupled with targeted sequencing of amplicons harbouring strain-marker SNPs means a rationalised, cost-effective usage of resources to facilitate the rapid update of knowledge on TB transmission, where universal long-term WGS cannot be assured.