P14

Molecular diagnostics inform clinical decisions for treating infectious disease. A key assumption of molecular diagnostics is that assayed genetic material faithfully captures the genomes of the cell populations driving disease. Here, we describe a pyrazinamide-resistant (PZA-R) tuberculosis infection appearing susceptible to targeted (primer-based) molecular platforms despite the susceptible subpopulation comprising a small minority among a resistant majority subpopulation. The PZA-R subpopulation had a deletion spanning primary PZA-resistance gene pncA and its promoter. The major subpopulation was identified by IonTorrent performed on PZA MGIT culture, phenotypic testing in standard media, and by PacBio WGS. However, targeted Sanger sequencing, a common PZA-R molecular diagnostic method, instead identified two small minority subpopulations with functional pncA. Most pncA amplification schemes that interrogate PZA-resistance via genotyping pncA also fail to capture sequences flanking the deletion. This exemplifies a drawback of targeted molecular diagnostics: when resistance is caused by mutations outside the amplified/targeted region, the presence of a susceptible subpopulation—even if comparatively small—gives the false impression of homogenous susceptibility. This issue persists for Line Probe Assays, as no mutated pncA subsequence would be present to positively identify resistance, while minority WT-pncA subpopulations would read-out as susceptible. Such erroneous PZA susceptibility calls would negatively impact clinical decision-making, motivating development of more direct molecular diagnostics for drug-resistant tuberculosis. More broadly, it underlines the clinical imperative to remain mindful of molecular diagnostics’ tacit assumptions and impels development of programmatic genomic surveillance using long-read sequencing technologies to monitor for variants that evade detection by prevailing molecular diagnostics.