Pyrazinamide resistance-conferring mutations in pncA and the transmission of multidrug resistant TB in Georgia

Abstract

BackgroundThe ongoing epidemic of multidrug-resistant tuberculosis (MDR-TB) in Georgia highlights the need for more effective control strategies. A new regimen to treat MDR-TB that includes pyrazinamide (PZA) is currently being evaluated and PZA resistance status will largely influence the success of current and future treatment strategies. PZA susceptibility testing was not routinely performed at the National Reference Laboratory (NRL) in Tbilisi between 2010 and September 2015. We here provide a first insight into the prevalence of PZA resistant TB in this region.MethodsPhenotypic susceptibility to PZA was determined in a convenience collection of well-characterised TB patient isolates collected at the NRL in Tbilisi between 2012 and 2013. In addition, the pncA gene was sequenced and whole genome sequencing was performed on two isolates.ResultsOut of 57 isolates tested 33 (57.9%) showed phenotypic drug resistance to PZA and had a single pncA mutation. All of these 33 isolates were MDR-TB strains. pncA mutations were absent in all but one of the 24 PZA susceptible isolate. In total we found 18 polymorphisms in the pncA gene. From the two major MDR-TB clusters represented (94–32 and 100–32), 10 of 15, 67.0% and 13 of 14, 93.0% strains, respectively were PZA resistant. We also identified a member of the potentially highly transmissive clade A strain carrying the characteristic I6L substitution in PncA. Another strain with the same MLVA type as the clade A strain acquired a different mutation in pncA and was genetically more distantly related suggesting that different branches of this particular lineage have been introduced into this region.ConclusionIn this high MDR-TB setting more than half of the tested MDR-TB isolates were resistant to PZA. As PZA is part of current and planned MDR-TB treatment regimens this is alarming and deserves the attention of health authorities. Based on our typing and sequence analysis results we conclude that PZA resistance is the result of primary transmission as well as acquisition within the patient and recommend prospective genotyping and PZA resistance testing in high MDR-TB settings. This is of utmost importance in order to preserve bacterial susceptibility to PZA to help protect (new) second line drugs in PZA containing regimens.

MODS-Wayne, a Colorimetric Adaptation of the Microscopic-Observation Drug Susceptibility (MODS) Assay for Detection of Mycobacterium tuberculosis Pyrazinamide Resistance from Sputum Samples

“…Using our composite reference standard (MGIT, classic Wayne test, and pncA sequencing), we found that around half of the selected MDR-TB strains were PZA resistant (30/57 samples [52.6%]), but few of the selected pansusceptible strains were PZA resistant (4/101 samples [4.0%]). These findings are in agreement with rates reported by other studies (3, 6, 20).…”

“…All sputum samples selected for this study were positive for TB according to the MODS assay. Given that MDR-TB isolates have a 50% chance of also being resistant to PZA (3, 14, 29), in order to select a high number of PZA-resistant sputum samples for evaluation, we selected approximately 50% of specimens that were MDR and monoresistant to isoniazid or rifampin and 50% of non-MDR specimens. Data concerning patient age, sex, and TB treatment were collected.…”

“…Pyrazinamide (PZA) is the only drug with different activity in vivo and in vitro (2). Its importance is based on its in vivo sterilizing effect that permits killing of persistent bacilli (3). PZA is frequently used in both first- and second-line treatment regimens (1, 4, 5).…”

Determining Genotypic Drug Resistance by Ion Semiconductor Sequencing With the Ion AmpliSeq™ TB Panel in Multidrug-Resistant Mycobacterium tuberculosis Isolates

“…PZA, a synthetic derivative of nicotinamide, is used as a first-line drug, and PZA resistance is attributed to mutations in pncA encoding a pyrazinamidase [ 27 ]. However, these resistance-conferring mutations are variable and widely dispersed [ 28 ].…”

Molecular Specific and Sensitive Detection of Pyrazinamide and Its Metabolite Pyrazinoic Acid by Means of Surface Enhanced Raman Spectroscopy Employing In Situ Prepared Colloids

“…PZA is the only drug with different activity in vivo and in vitro [17]. Its importance is based on its in vivo sterilizing effect that permits killing of persistent bacilli [18]. PZA is frequently used in both first-and second-line treatment regimens [19][20][21].…”