Pyrazinamide-resistant Tuberculosis Obscured From Common Targeted Molecular Diagnostics

Abstract

Here, we describe a clinical case of pyrazinamide-resistant (PZA-R) tuberculosis (TB) reported as PZA-susceptible (PZA-S) by common molecular diagnostics. Phenotypic susceptibility testing (pDST) indicated PZA-R TB. Targeted Sanger sequencing reported wild-type PncA, indicating PZA-S TB. Whole Genome Sequencing (WGS) by PacBio and IonTorrent both detected deletion of a large portion of pncA, indicating PZA-R. Importantly, both WGS methods showed deletion of part of the primer region targeted by Sanger sequencing. Repeating Sanger sequencing from a culture in presence of PZA returned no result, revealing that 1) two minority susceptible subpopulations had vanished, 2) the PZA-R majority subpopulation harboring the pncA deletion could not be amplified by Sanger primers, and was thus obscured by amplification process. This case demonstrates how a small susceptible subpopulation can entirely obscure majority resistant populations from targeted molecular diagnostics and falsely imply homogenous susceptibility, leading to incorrect diagnosis. To our knowledge, this is the first report of a minority susceptible subpopulation masking a majority resistant population, causing targeted molecular diagnostics to call false susceptibility. The consequence of such genomic events is not limited to PZA. This phenomenon can impact molecular diagnostics' sensitivity whenever the resistance-conferring mutation is not fully within primer-targeted regions. This can be caused by structural changes of genomic context with phenotypic consequence as we report here, or by uncommon mechanisms of resistance. Such false susceptibility calls promote suboptimal treatment and spread of strains that challenge targeted molecular diagnostics. This motivates development of molecular diagnostics unreliant on primer conservation, and impels frequent WGS surveillance for variants that evade prevailing molecular diagnostics.

Keywords: Heteroresistance; Molecular diagnostics; Pyrazinamide; Tuberculosis.

Fig. 1.

Model for initial composition of subpopulations that give rise to the gDST and pDST observed in this study. The fraction of each subpopulation we estimate was present in the original sample (left) contrasted with the composition of the three subpopulations’ DNA that would ultimately be sequenced (right) given the original composition.

Fig. 2.

Undetected pyrazinamide resistance (PZA-R) conferring deletion with respect to primer schemes for targeted PZA-R molecular diagnostics. Primer sequence boundaries from previously published studies using PCR schemes for molecular investigation of pncA genotype. Dots indicate the inner boundary of primers in each scheme, which need to both be outside of the deleted region for the intervening sequences to be amplified and subsequently sequenced to identify the presence of mutations that confer PZA resistance. Location of the resistance conferring deletion and primer sequence boundaries are shown with respect to pncA.