DIAGNOSTICS Resistance to pyrazinamide in Russian Mycobacterium tuberculosis isolates: pncA sequencing versus Bactec MGIT 960 Dmitry A. Maslov a , Marina V. Zaĭchikova a , Larisa N. Chernousova b , Kirill V. Shur a , Olga B. Bekker a , Tatiana G. Smirnova b , Elena E. Larionova b , Sofya N. Andreevskaya b , Ying Zhang c , Valery N. Danilenko a, * a Laboratory of Bacterial Genetics, Department of Genetics and Biotechnology, Vavilov Institute of General Genetics, Russian Academy of Sciences, 3 Gubkina Str., 119991 Moscow, Russia b Department of Microbiology, Central TB Research Institute, Russian Academy of Medical Sciences, 2 Yauzskaya Alleya,107564 Moscow, Russia c W. Harry Feinstone Department of Molecular Microbiology & Immunology, Bloomberg School of Public Health, Johns Hopkins University, 615 N. Wolfe Street, Baltimore, MD 21205, USA article info Article history: Received 3 April 2015 Received in revised form 22 May 2015 Accepted 24 May 2015 Keywords: Pyrazinamide Resistance Tuberculosis pncA MGIT960 Drug susceptibility testing summary Resistance to pyrazinamide (PZA) may impact clinical outcome of anti-tuberculosis chemotherapy. PZA susceptibility testing using MGIT 960 is not reliable and little information is available on the prevalence of PZA resistance in Russia. A collection of 64 clinical isolates of Mycobacterium tuberculosis, including 35 multidrug resistant and extensively drug-resistant (MDR/XDR), was analyzed for PZA resistance using MGIT 960, Wayne test, and sequencing of PZA resistance genes pncA, rpsA and panD. In addition, we analyzed 519 MDR-TB strains for susceptibility to PZA by MGIT 960. Sequencing of pncA revealed 17 of 25 (68%) MDR strains and all 10 XDR strains harboring pncA mutations. A correlation of 4 ¼ 0.81 between MGIT 960 and pncA sequencing was observed. Mutations in rpsA and panD not associated with PZA resistance as defined by MGIT 960 were identified. We found 1 PZA-resistant strain without mutations in known PZA resistance genes. Almost 73% of MDR-TB strains isolated in Moscow, Russia, were PZA- resistant by MGIT 960 testing of 519 MDR-TB clinical isolates. Further studies are needed to determine the role of rpsA and panD mutations in possible low-level PZA resistance and to identify the molecular basis of new PZA resistance in the isolate without known PZA resistance mutations. © 2015 Elsevier Ltd. All rights reserved. 1. Introduction Tuberculosis (TB) is a major global health problem with an estimated 9.0 million new TB cases and 1.5 million TB deaths in 2013 [1]. Pyrazinamide (PZA) is a key first-line drug in TB chemo- therapy, which when introduced shortened modern anti-TB ther- apy to 6 months due to its sterilizing ability [2]. PZA is recommended for treatment of both drug-susceptible and drug- resistant TB cases [3]. PZA is a unique drug, lacking activity against replicating Myco- bacterium tuberculosis, but killing only non-growing persister bacilli [4]. PZA is a prodrug, which needs to be activated by M. tuberculosis enzyme pyrazinamidase (PZase) into its active form e pyrazinoic acid (POA) [5]. Mutations in gene pncA encoding PZase are the major mechanism of PZA resistance in M. tuberculosis [5e7]. One of the probable mechanisms of action of PZA, is the disruption of membrane potential by the protonated POA, leading to the in- hibition of major cell processes [4]. Recently, S1 ribosomal protein (RpsA) was identified as a molecular target for POA [8]. Mutations in rpsA gene were found in some PZA-resistant M. tuberculosis isolates lacking mutations in pncA [8,9]. RpsA is a key protein of trans-translation, a mechanism for saving stalled ribosomes or damaged mRNA, essential for dormant and persister cells [10,11]. Another gene involved in PZA resistance is panD, encoding an aspartate decarboxylase involved in pantothenate and coenzyme A biosynthesis [12]. The phenotypic testing for PZA susceptibility can show both false-positive and false-negative results [13]. Up to 40% of false- positive resistance can be obtained on Bactec MGIT 960 [14]. Abbreviations: H, isoniazid; R, rifampicin; E, ethambutol; PZA, Z, pyrazinamide; Eto, ethionamide; Am, amikacin; Cm, capreomycin; Lfx, levofloxacin; PZase, pyr- azinamidase; POA, pyrazinoic acid. * Corresponding author. Tel.: þ7 4991354194. E-mail address: valerid@vigg.ru (V.N. Danilenko). Contents lists available at ScienceDirect Tuberculosis journal homepage: http://intl.elsevierhealth.com/journals/tube http://dx.doi.org/10.1016/j.tube.2015.05.013 1472-9792/© 2015 Elsevier Ltd. All rights reserved. Tuberculosis xxx (2015) 1e5 Please cite this article in press as: Maslov DA, et al., Resistance to pyrazinamide in Russian Mycobacterium tuberculosis isolates: pncA sequencing versus Bactec MGIT 960, Tuberculosis (2015), http://dx.doi.org/10.1016/j.tube.2015.05.013