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Pyrazinamide Drug susceptibility testing Heteroresistance A B S T R A C T Aim: Pyrazinamide (PZA) is a first-line key agent in the effective treatment of tuberculosis (TB), including PZA susceptible multidrug-resistant tuberculosis (MDR-TB). Occasionally, TB patients might have mixed infections with both drug-sensitive and -resistant strains. This is termed heteroresistance. If 10% of the bacterial population is resistant to PZA, there is an increased risk for poor treatment outcome. The aim of this study is to evaluate the ability of the three established drug susceptibility testing (DST) techniques -BACTEC MGIT 960, Wayne's pyrazinamidase test and Sanger sequencing of the pncA gene -to detect 10% PZA heteroresistance.Methods: Mixed cultures of the fully drug susceptible Mycobacterium tuberculosis H37Rv reference strain and two laboratory-generated isogenic H37Rv mutants (with C475G and T254C pncA mutations, respectively) were made in proportions of 100%, 10%, 5% and 1% of the PZA-resistant (PZA-R) strain. Corresponding mixed cultures were also made using one drug-susceptible and one PZA-resistant MDR clinical isolate with the T62G pncA mutation, both belonging to one specific MIRU cluster. Additional mixes of 50%, 75%, 90% and 99% of the PZA-R strains were prepared for the Wayne's test. Tests were for all methods performed in duplicates at two separate occasions.Results: Using the MGIT system, the in vitro-generated PZA-R strains were generally detected at a 5% proportion while the clinical PZA-R isolate only was detected at the critical 10% proportion, except for one test occasion. Sanger sequencing was unable to detect 10% PZA heteroresistance. Wayne's test also failed to detect the critical level of 10% PZA resistance; instead it displayed misguiding results determining highly resistant samples as sus- ceptible.Conclusion: Heteroresistance is caused by present drug-resistance development and/or dual infections with one resistant and one susceptible strain. Mixed infections with resistant strains may occur in up to 20% of all TB cases in high burden areas, according to limited data. This study showed that only the phenotypic BACTEC MGIT system was capable in determining the critical proportion of 10% PZA resistance, whereas neither the Sanger nor the Wayne's test were successful in this respect. This indicates a need for diagnostic tools with increased sensitivity to determine heteroresistance in M. tuberculosis. I n t e r n a t i o n a l J o u r n a l o f M y c o b a c t e r i o l o g y 4 ( 2 0 1 5 ) 2 9 H O S T E D BY A v ai l abl e a t w w w . s c i e n c e d i r e c t. c o m ScienceDirect j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / I J M Y C O

Heteroresistance is defined as the coexistence of both susceptible and resistant bacteria in a bacterial population. Previously published data show that it may occur in 9-57% of Mycobacterium tuberculosis isolates for various drugs. Pyrazinamide (PZA) is an important first-line drug used for treatment of both drug-susceptible and PZA-susceptible multidrug-resistant TB. Clinical PZA resistance is defined as a proportion of resistant bacteria in the isolate exceeding 10%, when the drug is no longer considered clinically effective. The capability of traditional drug susceptibility testing techniques to detect PZA heteroresistance has not yet been evaluated. The aim of this study was to compare the capacity of BACTEC MGIT 960, Wayne’s test and whole genome sequencing (WGS) to detect PZA resistant subpopulations in bacterial suspensions prepared with different proportions of mutant strains. Both BACTEC MGIT 960 and WGS were able to detect the critical level of 10% PZA heteroresistance whereas Wayne’s test failed to do so, with the latter falsely reporting highly resistant samples as PZA susceptible. Failure to detect drug resistant subpopulations may lead to inadvertently weak treatment regimens if ineffective drugs are included, with the risk of treatment failure with the selective growth of resistant subpopulations. We need clinical awareness of heteroresistance as well as evaluation of new diagnostic tools in their capacity in detecting heteroresistance in TB.