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Mycobacterium tuberculosis Pyrazinamide Resistance Determinants: a Multicenter Study

Mycobacterium tuberculosis Pyrazinamide Resistance Determinants: a Multicenter Study RESEARCH ARTICLE crossmark Mycobacterium tuberculosis Pyrazinamide Resistance Determinants: a Multicenter Study a a b,c d,e d,e f g Paolo Miotto, Andrea M. Cabibbe, Silke Feuerriegel, Nicola Casali, Francis Drobniewski, Yulia Rodionova, Daiva Bakonyte, g h i j k l l Petras Stakenas, Edita Pimkina, Ewa Augustynowicz-Kopec´, Massimo Degano, Alessandro Ambrosi, Sven Hoffner, Mikael Mansjö, l m b,c a Jim Werngren, Sabine Rüsch-Gerdes, Stefan Niemann, Daniela M. Cirillo Emerging Bacterial Pathogens Unit, Division of Immunology, Transplantation and Infectious Diseases, IRCCS San Raffaele Scientific Institute, Milan, Italy ; Molecular b c d Mycobacteriology, German Centre for Infection Research (DZIF), Borstel, Germany; Infectious Diseases, Imperial College, London, United Kingdom ; Clinical TB and HIV Group, Public Health England (PHE) National Mycobacterium Reference Laboratory, Blizard Institute, Queen Mary University of London, London, United Kingdom ; f g Samara TB Service, Samara, Russian Federation ; Department of Immunology and Cell Biology, Institute of Biotechnology, Vilnius University, Vilnius, Lithuania ; National Tuberculosis Reference Laboratory, Infectious Diseases and Tuberculosis Hospital, Vilnius University Hospital Santariskiu Klinikos, Vilnius, Lithuania ; Department of Microbiology, National Tuberculosis and Lung Diseases Research Institute, Warsaw, Poland ; Biocrystallography Unit, Division of Immunology, Transplantation and j k Infectious Diseases, IRCCS San Raffaele Scientific Institute, Milan Italy ; Vita-Salute San Raffaele University, Milan, Italy ; Public Health Agency of Sweden, Solna, and Department of Microbiology, Tumor and Cell Biology (MTC), Karolinska Institute, Stockholm, Sweden ; National Reference Center for Mycobacteria, Research Center Borstel, Borstel, Germany S.N. and D.M.C. contributed equally to this work. ABSTRACT Pyrazinamide (PZA) is a prodrug that is converted to pyrazinoic acid by the enzyme pyrazinamidase, encoded by the pncA gene in Mycobacterium tuberculosis. Molecular identification of mutations in pncA offers the potential for rapid detection of pyrazinamide resistance (PZA ). However, the genetic variants are highly variable and scattered over the full length of pncA, complicating the development of a molecular test. We performed a large multicenter study assessing pncA sequence variations in 1,950 clinical isolates, including 1,142 multidrug-resistant (MDR) strains and 483 fully susceptible strains. The results of pncA sequencing were correlated with phenotype, enzymatic activity, and structural and phylogenetic data. We identified 280 genetic variants which were divided into four classes: (i) very high confidence resistance mutations that were found only in PZA strains (85%), (ii) high-confidence resistance mutations found in more than 70% of PZA strains, (iii) mutations with an unclear role found in less than 70% of PZA strains, and (iv) mutations not associated with phenotypic resistance (10%). Any future molecu- lar diagnostic assay should be able to target and identify at least the very high and high-confidence genetic variant markers of PZA ; the diagnostic accuracy of such an assay would be in the range of 89.5 to 98.8%. IMPORTANCE Conventional phenotypic testing for pyrazinamide resistance in Mycobacterium tuberculosis is technically chal- lenging and often unreliable. The development of a molecular assay for detecting pyrazinamide resistance would be a break- through, directly overcoming both the limitations of conventional testing and its related biosafety issues. Although the main mechanism of pyrazinamide resistance involves mutations inactivating the pncA enzyme, the highly diverse genetic variants scattered over the full length of the pncA gene and the lack of a reliable phenotypic gold standard hamper the development of molecular diagnostic assays. By analyzing a large number of strains collected worldwide, we have classified the different genetic variants based on their predictive value for resistance which should lead to more rapid diagnostic tests. This would assist clini- cians in improving treatment regimens for patients. Received 20 August 2014 Accepted 26 September 2014 Published 21 October 2014 Citation Miotto P, Cabibbe AM, Feuerriegel S, Casali N, Drobniewski F, Rodionova Y, Bakonyte D, Stakenas P, Pimkina E, Augustynowicz-Kopec´ E, Degano M, Ambrosi A, Hoffner S, Mansjö M, Werngren J, Rüsch-Gerdes S, Niemann S, Cirillo DM. 2014. Mycobacterium tuberculosis pyrazinamide resistance determinants: a multicenter study. mBio 5(5): e01819-14. doi:10.1128/mBio.01819-14. Editor Carol A. Nacy, Sequella, Inc. Copyright © 2014 Miotto et al. This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited. Address correspondence to Paolo Miotto, [email protected]. yrazinamide (PZA) is a key drug in current and future tuber- on the success rates of MDR treatment and may allow a shortening Pculosis (TB) treatment regimens. It has a high sterilizing ca- of current MDR therapy (5). Finally, PZA is the only first-line pacity in vivo, but it is not active against Mycobacterium tubercu- drug that will be maintained in all regimens in the near future (6). losis complex (MTBC) strains growing at neutral pH (1–4). These new regimens aim at reducing the treatment duration of In addition to its crucial role in the standard short-course reg- susceptible, drug-resistant (especially MDR TB and extensively imen for TB treatment, PZA is used in the treatment of patients resistant) strain variants. infected with strains that are multidrug resistant (MDR) (resistant The essential role of PZA underlines the need for accurate and to at least isoniazid and rifampin). Here PZA has a strong impact rapid detection of PZA resistance that is very difficult with current September/October 2014 Volume 5 Issue 5 e01819-14 mbio.asm.org 1 Miotto et al. phenotypic tests (7). The difficulties with culture-based PZA sus- ceptibility testing result from several factors, including subopti- mal test media with unreliable pH and larger inocula that reduce the activity of PZA (8, 9). Furthermore, the critical concentration itself may result in inconsistent results for isolates with a PZA MIC close to this concentration (10). While for isoniazid and rifampin, highly reliable culture-based drug susceptibility testing (DST) techniques and rapid molecular assays such as the line probe assay MTBDRplus (Hain Lifescience GmbH, Nehren, Germany) and the cartridge-based Xpert MTB/RIF assay (Cepheid, Sunnyvale, CA) are available (11), no commercial molecular assays are cur- rently marketed for PZA. Great efforts have been made in understanding molecular re- sistance mechanisms. PZA is a prodrug that needs to be converted to an active compound, pyrazinoic acid, by the bacterial pyrazi- namidase (PZase) (encoded by pncA). Mutations/variations in pncA leading to the loss of PZase activity are the major mechanism leading to PZA resistance (PZA ) (4, 12). However, while high numbers of PZA cases can be related to inactivation of the PZase, the genetic variants, including single nucleotide polymorphisms (SNPs) and small deletions, are highly diverse and scattered over the full length of the 561 bp of the pncA gene (4, 12). This compli- cates the development of molecular tests, as no “hot spot region” comprising the majority of mutations is present in the pncA gene, as is present in rpoB for rifampin resistance. Accordingly, future molecular approaches to detect PZA in clinical isolates need to cover at least a significant number of pos- sible variants, if not the complete gene, to reach a high sensitivity (e.g., using approaches based on classical Sanger sequencing or next-generation genome sequencing). These techniques must be combined with an appropriate interpretation algorithm/database that distinguishes SNPs clearly associated with drug resistance from those for which the impact for developing PZA is unclear, e.g., due to phylogenic variants found in members of the MTBC (13, 14). In-depth knowledge of the variants found in PZA strains combined with evidence-based correlation with resistance pheno- types are needed to develop large-scale databases ensuring valid FIG 1 Pie chart reporting percentages of lineages for isolates included in the data interpretation. The fact that such a valid data basis is cur- study. rently lacking represents a substantial limitation for molecular PZA DST. To tackle this question, we performed a large multicenter study assessing pncA sequence variations in 1,950 MTBC pan- rium bovis, H37Rv, UZB_H37Rv_like, New-1, X, CAS, TUR,and susceptible strains and PZA strains. The strains were classified in Mycobacterium microti vole) each represented less than 5% of iso- phylogenetic lineages to identify variants that are phylogenetically lates (Fig. 1). Six percent were classified as EuroAmerican strains informative but not likely to be involved in PZA and those that not belonging to a valid lineage described previously (“other un- are occurring in strains from different groups and are obviously defined,” as described in reference 15), and 5.2% of strains were under positive selection. Using this comprehensive approach, we classified as “unknown,” because it was not possible to assign a could catalog 239 high-confidence PZA mutations and a number defined lineage. of pncA variants most likely not involved in PZA . Considering the sequencing results, 1,062 (54.5%) isolates were found to be wild type (WT) for the pncA gene, whereas 888 RESULTS harbored variations in the pncA gene that amounted to a total of We studied 1,950 clinical isolates, including 1,142 MDR strains 280 genetic variants comprising 67 insertions or deletions (indels) and 483 fully susceptible strains (see Table S1 in the supplemental and 213 SNPs (see Table S2 in the supplemental material). The material). By phenotypic DST, 1,107 clinical isolates were suscep- PZase enzymatic activity was available for 251 clinical isolates ac- tible to PZA, whereas 843 were classified as PZA . Genotyping data counting for 90 different genetic variants. Considering the distri- were available for 1,853 isolates (95.0%). Predominant lineages bution of the mutations across the entire gene, 73 (39.0%) codons among the strains investigated were Beijing (47.8%), LAM were not affected by mutations, whereas the remaining 114 (9.0%), Ural (7.7%), and Haarlem (5.0%). Other lineages found codons presented one or more mutations (Fig. 2). Only 50 codons (Ghana, EAI, Delhi/CAS, H37Rv_like, Uganda I and II, West Af- showed a frequency of mutation over the mean value of 0.5%, but rican 1 and 2, S, Cameroon, Sierra Leone 1 and 2, Mycobacte- despite this, a clear hot spot region could not be found; the most 2 mbio.asm.org September/October 2014 Volume 5 Issue 5 e01819-14 Molecular Detection of PZA Resistance FIG 2 Number of different mutations found at each codon. Note that multiple mutations and IS6110 are not included. The broken line indicates mean value. s r frequently affected regions (representing more than 70% of mu- isolates originally reported as PZA were reclassified as PZA (Ta- tated strains) were found at the promoter (positions 13 to 3) ble S1). The final distribution of mutations among revised PZA and at codons 6 to 15, 50 to 70, 90 to 100, 130 to 145, and 170 to and PZA isolates is summarized in Table 1. To further validate the 175 (Fig. 3). classification data, we analyzed the homoplastic occurrence of s r For mutations found in both PZA-sensitive (PZA ) and PZA particular mutations (e.g., the emergence in strains of two phylo- isolates, enzymatic activity and structural analysis results were genetic lineages [16]). As the homoplasy level is rather low in used to adjust for possible errors in phenotypic DST whenever MTBC genomes, this confirms that these mutations are most possible and to obtain a “revised DST” (included as “DST rev” in likely under positive selection and involved in the development of Table S1 in the supplemental material). Accordingly, 56 clinical PZA . FIG 3 Frequency of mutations found at each codon (calculated with 888 mutated isolates). Note that multiple mutations and IS6110 are not included. The dotted line indicates mean value. September/October 2014 Volume 5 Issue 5 e01819-14 mbio.asm.org 3 Miotto et al. s r r TABLE 1 Distribution of mutations among PZA and PZA clinical Mutations conferring PZA at very high confidence. Out of isolates the 280 sequence variants identified in pncA, 239 (85.4%) muta- tions found in 644 clinical isol ates (644/1,950 [33.0%]) were clas- pncA gene No. of isolates (%) sified as very high confidence variants associated with phenotypic s r PZA (n  1,051) PZA (n  899) PZA (category A) (see Table S2 in the supplemental material). WT 893 (85.0) 158 (15.0) Several mutations affect the catalytic residues and amino acids Mutant 200 (22.2) 699 (77.8) recruited in the scaffold of the active site or directly/indirectly Includes 19 isolates harboring silent mutations or mutations at the distal region of the involved in the coordination of the Fe ion (Asp8Gly/Ala/Glu/ promoter (100 nucleotides upstream of the start codon). Asn, His51Gln/Tyr, His71Arg, Asp49Glu/Asn/Ala, His57Arg/Tyr/ Gln/Pro, Trp68Arg/Gly/Cys/Stop/Leu, Gln10Pro/Arg, and His137Pro/Arg/Asp) or residues engaged in the hydrophobic core Using this procedure, four classes of genetic variants were (Ile6Thr, Val44Gly, Val139Gly/Leu, Met175Thr/Val, and identified: (i) very high confidence resistance mutations that were Phe94Cys/Ser/Leu). Out of the 90 variants tested, 87 variants, in- found only in PZA strains (category A), (ii) high-confidence re- cluding nucleotide substitutions at position 11, were also asso- sistance mutations found in more than 70% of PZA strains (cat- ciated with negative PZase activity, and 55 genetic variants egory E), (iii) mutations with an unclear role found in less than 70% in PZA strains (category D), and (iv) genetic variants (including the (detected in 332 isolates) were found in strains of at least 2 differ- wild type) not involved in phenotypic resistance (category B). Ta- ent lineages, indicating homoplasy (data not shown). Table 2 ble S2 in the supplemental material summarizes these clinically reports the mutations mapping in the most frequently affected relevant categories; a graphical overview is provided in Fig. 4. regions. FIG 4 Distribution of genetic variants across the four categories identified: (i) very high confidence resistance mutations, (ii) high-confidence resistance mutations, (iii) mutations with an unclear role, and (iv) mutations not involved in phenotypic resistance. The number of isolates belonging to each category is also reported. The inner ring shows the percentages of mutations affecting the structure of the enzyme for each category of genetic variants. PZA-R, PZA resistance. *, including wild-type isolates for the pncA gene. 4 mbio.asm.org September/October 2014 Volume 5 Issue 5 e01819-14 Molecular Detection of PZA Resistance TABLE 2 Mutations for PZA affecting the most frequently affected regions of pncA gene and representing more than 70% of mutated cases a b c d Nucleotide change Result of the mutation p.S p.R No. of cases A-11C Promoter 11 0.01497006 0.98502994 5 A-11G Promoter 11 0.01497006 0.98502994 35 A-11T Promoter 11 0.01497006 0.98502994 1 T-7C Promoter 7 0.01497006 0.98502994 4 T-7G Promoter 7 0.01497006 0.98502994 1 Del-5¡ G Promoter (del) 0.01497006 0.98502994 2 ATC6ACC Ile6Thr 0.01497006 0.98502994 3 Del14¡ TCATCG FSC 6 (del) 0.01497006 0.98502994 1 GTC7GGC Val7Gly 0.01497006 0.98502994 9 GTC7TTC Val7Phe 0.01497006 0.98502994 2 WT  GTC7GGC WT  Val7Gly 0.01497006 0.98502994 1 GAC8AAC Asp8Asn 0.01497006 0.98502994 3 GAC8GAA Asp8Glu 0.01497006 0.98502994 6 GAC8GCC Asp8Ala 0.01497006 0.98502994 1 GAC8GGC Asp8Gly 0.01497006 0.98502994 9 GTG9GGG Val9Gly 0.01497006 0.98502994 1 CAG10AAG Gln10Lys 0.01497006 0.98502994 2 CAG10CCG Gln10Pro 0.01497006 0.98502994 21 CAG10CGG Gln10Arg 0.01497006 0.98502994 6 GAC12AAC Asp12Asn 0.01497006 0.98502994 GAC12GAG Asp12Glu 0.01497006 0.98502994 2 GAC12GCC Asp12Ala 0.01497006 0.98502994 6 Ins37¡ GACT FSC 13 (ins) 0.01497006 0.98502994 1 TTC13TCC Phe13Ser 0.01497006 0.98502994 2 TTC13TTG Phe13Leu 0.01497006 0.98502994 3 TGC14CGC Cys14Arg 0.01497006 0.98502994 1 TGC14TGA Cys14Stop 0.01497006 0.98502994 2 WT  TGC14CGC WT  Cys14Arg 0.01497006 0.98502994 1 Ins44¡ C FSC 15 (ins) 0.01497006 0.98502994 1 Del150¡ T FSC 50 (del) 0.01497006 0.98502994 1 CAC51CAA His51Gln 0.01497006 0.98502994 7 CAC51CCC His51Pro 0.01497006 0.98502994 2 CAC51CGC His51Arg 0.01497006 0.98502994 4 CAC51TAC His51Tyr 0.01497006 0.98502994 3 CCG54CAG Pro54Gln 0.01497006 0.98502994 4 CCG54CGG Pro54Arg 0.01497006 0.98502994 1 CCG54CTG Pro54Leu 0.01497006 0.98502994 4 CCG54TCG Pro54Ser 0.01497006 0.98502994 1 CAC57CAG His57Gln 0.01497006 0.98502994 1 CAC57CCC His57Pro 0.01497006 0.98502994 1 CAC57CGC His57Arg 0.01497006 0.98502994 14 CAC57GAC His57Asp 0.01497006 0.98502994 10 CAC57TAC His57Tyr 0.01497006 0.98502994 5 WT  CAC57CGC WT  His57Arg 0.01497006 0.98502994 2 TTC58CTC Phe58Leu 0.01497006 0.98502994 7 CCG62CTG Pro62Leu 0.01497006 0.98502994 3 Del186¡ C FSC 62 (del) 0.01497006 0.98502994 3 Ins185¡ 4 nt FSC 62 (ins) 0.01497006 0.98502994 1 Ins186¡ A FSC 62 (ins) 0.01497006 0.98502994 1 GAC63GGC Asp63Gly 0.01497006 0.98502994 4 Ins192¡ A FSC 64 (ins) 0.01497006 0.98502994 1 TAT64TAG Tyr64stop 0.01497006 0.98502994 3 Ins193¡ A FSC 65 (ins) 0.01497006 0.98502994 1 Ins193¡ TATCAGG FSC 65 (ins) 0.01497006 0.98502994 1 TCG67CCG Ser67Pro 0.01497006 0.98502994 2 TGG68CGG Trp68Arg 0.01497006 0.98502994 7 TGG68GGG Trp68Gly 0.01497006 0.98502994 16 TGG68TAG Trp68stop 0.01497006 0.98502994 1 TGG68TGC Trp68Cys 0.01497006 0.98502994 5 TGG68TGT Trp68Cys 0.01497006 0.98502994 1 GAG91TAG Glu91Stop 0.01497006 0.98502994 1 TTC94CTC Phe94Leu 0.01497006 0.98502994 8 TTC94TCC Phe94Ser 0.01497006 0.98502994 3 TTC94TGC Phe94Cys 0.01497006 0.98502994 6 TTC94TTA Phe94Leu 0.01497006 0.98502994 2 (Continued on following page) September/October 2014 Volume 5 Issue 5 e01819-14 mbio.asm.org 5 Miotto et al. TABLE 2 (Continued) a b c d Nucleotide change Result of the mutation p.S p.R No. of cases TTC94TTG Phe94Leu 0.01497006 0.98502994 1 WT  TTC94CTC WT  Phe94Leu 0.01497006 0.98502994 1 TAC95TAG Tyr95stop 0.01497006 0.98502994 1 AAG96AAC Lys96Asn 0.01497006 0.98502994 1 AAG96ACG Lys96Thr 0.01497006 0.98502994 2 AAG96AGG Lys96Arg 0.01497006 0.98502994 1 AAG96CAG Lys96Gln 0.01497006 0.98502994 1 AAG96GAC Lys96Glu 0.01497006 0.98502994 6 Ins288¡ T FSC 96 (ins) 0.01497006 0.98502994 2 Ins288¡ 33 nt FSC 96 (ins) 0.01497006 0.98502994 4 Del291¡ T FSC 97 (del) 0.01497006 0.98502994 1 GGT97AGT Gly97Ser 0.01497006 0.98502994 6 GGT97GAT Gly97Asp 0.01497006 0.98502994 4 GGT97GCT Gly97Ala 0.01497006 0.98502994 1 TAC99TAA Tyr99stop 0.01497006 0.98502994 2 ACC100CCC Thr100Pro 0.01497006 0.98502994 2 ACC100GCC Thr100Ala 0.01497006 0.98502994 1 GTG130GCG Val130Ala 0.01497006 0.98502994 1 GTG130GGG Val130Gly 0.01497006 0.98502994 1 Ins391¡ G FSC 131 (ins) 0.01497006 0.98502994 3 Ins391¡ GG FSC 131 (ins) 0.01497006 0.98502994 2 Ins392¡ G FSC 131 (ins) 0.01497006 0.98502994 2 Ins392¡ GG FSC 131 (ins) 0.01497006 0.98502994 4 Ins393¡ G FSC 131 (ins) 0.01497006 0.98502994 2 Ins393¡ GG FSC 131 (ins) 0.01497006 0.98502994 1 Ins394¡ ATGTGGTCG FSC 131 (ins) 0.01497006 0.98502994 1 TGC131GGTGC FSC 131 (ins) 0.01497006 0.98502994 1 GGT132AGT Gly132Ser 0.01497006 0.98502994 1 GGT132GAT Gly132Asp 0.01497006 0.98502994 1 GGT132GCT Gly132Ala 0.01497006 0.98502994 1 GGT132TGT Gly132Cys 0.01497006 0.98502994 2 ATT133ACT Ile133Thr 0.01497006 0.98502994 17 Del398¡ T FSC 133 (del) 0.01497006 0,98502994 1 GCC134GTC Ala134Val 0.01497006 0.98502994 2 ACC135AAC Thr135Asn 0.01497006 0.98502994 3 ACC135CCC Thr135Pro 0.01497006 0.98502994 4 GAT136TAT Asp136Tyr 0.01497006 0.98502994 3 Ins408¡ A FSC 136 (ins) 0.01497006 0.98502994 4 CAT137CCT His137Pro 0.01497006 0.98502994 1 CAT137CGT His137Arg 0.01497006 0.98502994 1 CAT137GAT His137Asp 0.01497006 0.98502994 1 TGT138CGT Cys138Arg 0.01497006 0.98502994 3 TGT138TGG Cys138Trp 0.01497006 0.98502994 1 Del417¡ G FSC 139 (del) 0.01497006 0.98502994 1 GTG139CTG Val139Leu 0.01497006 0.98502994 3 GTG139GGG Val139Gly 0.01497006 0.98502994 5 CGC140CCC Arg140Pro 0.01497006 0.98502994 1 CAG141CCG Gln141Pro 0.01497006 0.98502994 11 CAG141TAG Gln141stop 0.01497006 0.98502994 1 Ins423¡ CAGACGGCGCCAG FSC 141 (ins) 0.01497006 0.98502994 1 ACG142AAG Thr142Lys 0.01497006 0.98502994 1 ACG142ATG Thr142Met 0.01497006 0.98502994 3 ACG142GCG Thr142Ala 0.01497006 0.98502994 3 GCC143GGC Ala143Gly 0.01497006 0.98502994 1 CTG172CCG Leu172Pro 0.01497006 0.98502994 9 Del514¡ C FSC 172 (del) 0.01497006 0.98502994 1 Ins516¡ CG FSC 172 (ins) 0.01497006 0.98502994 1 ATG175ACG Met175Thr 0.01497006 0.98502994 1 ATG175ATA Met175Ile 0.01497006 0.98502994 10 ATG175GTG Met175Val 0.01497006 0.98502994 6 A-11C, nucleotide change A to C in position 11; Del-5¡ G, deletion of nucleotide G in position 5; ATC6AAC, ATC at codon 7 changed to AAC; WT  GTC7GGC, double pattern wild-type  GTC at codon 7 changed to GGC; Ins37¡ GACT, GACT inserted at codon 37. Promoter 11, nucleotidic mutation affecting the promoter region at position 11; del, deletion; FSC, frameshift codon; ins, insertion. p.S, probability associated with the susceptible phenotype. p.R, probability associated with the resistant phenotype. 6 mbio.asm.org September/October 2014 Volume 5 Issue 5 e01819-14 Molecular Detection of PZA Resistance Mutations conferring PZA at high confidence. Nine genetic tients. Phenotypic tests, however, have a long turnaround time, variants (32 strains, category E) were found in both PZA and are expensive, and are considered poorly reliable. As a conse- s r r PZA isolates, but with a proportion higher than 70% in PZA quence, the design of a molecular test for predicting PZA is a strains. These mutations were mainly associated with an increase priority. The development of a rapid molecular PZA DST is ham- in free energy and/or structural constraints and were most fre- pered by the diverse nature of resistance-associated mutations quently associated with PZA (93.5% of cases). We confirmed a mainly scattered over the full length of the pncA gene, and by the reduced but still present PZase activity for some of these variants fact that the impact of individual mutations has not been system- as a development of faint color during the enzymatic assay. atically investigated (4, 12). Therefore, we performed a large-scale Whereas Leu172Pro was found to be associated with homoplasy, study linking pncA sequence diversity with phenotypic, structural for other substitutions, the number of cases was too low to con- biology and population biology data to develop the first encyclo- r s sider convergent evolution in different lineages. pedia of pncA sequence variations linked to either a PZA or PZA Mutations with an unclear role in conferring PZA . Five ge- phenotype. This is likely to pave the way for application of new r r netic variants (21 cases, category D) were found in both PZA and genome-based sequencing technologies for predicting PZA ,al- s r PZA isolates but at a proportion less than 70% in PZA strains. lowing for personalized treatment algorithms. Two genetic variants (15 cases) showed borderline behavior in Strikingly, nearly 85% of the genetic variants identified in the terms of structure/free energy variation and enzyme activity. Ho- pncA gene were associated with phenotypic resistance to PZA and moplasy was found for the Val139Ala mutation, thus suggesting a were classified as “high-confidence” PZA resistance mutations. All putative role in phenotypic resistance or at least in increasing the in-frame and frameshift indel mutations within the coding region MIC. Pro62Arg, Asp63Ala, and Ser65Pro substitutions (6 cases) were included in this group. We found that nearly 90% of ob- represent another group of mutations belonging to this ambigu- served mutations are associated with protein structural destabili- ous category. Further characterization of these mutations is zation that causes loss of enzymatic activity. Equally importantly, needed to better understand their correlation with the PZA phe- we described 27 mutations most likely not involved in PZA resis- notype. tance that should be “filtered out” in future molecular tests and Mutations not involved in phenotypic resistance. Twenty- labeled as not “clinically relevant” (Fig. 4). Only five mutations seven genetic variants were not associated with PZA according to cannot be classified by our approach and remain without clear our classification. Eighteen mutations (163 cases, category C) association with a resistance or susceptible phenotype. These mu- s r were most frequently associated with PZA (91.4% of cases). It tations need further validation for association with PZA and/or should be noted that the Val21Ala mutation was also found asso- with a specific genetic background by an allelic exchange proce- ciated with other mutations in category A responsible for PZA / dure as performed for other drugs (19). PZase negativity. Interestingly, all these mutations were found to Discrepancies between molecular and phenotypic DST are be associated with single lineages; thus, no homoplasy was ob- confusing for clinicians managing patients; 180 isolates investi- served. Further characterization of these mutations is needed to gated here showed discrepant results between phenotypic and ge- better understand their role (if any) in PZA susceptibility. The notypic tests (WT pncA gene sequence and resistance by Bactec remaining genetic variants (27 cases, category B) did not affect the MGIT 960 DST). It has been reported that the Bactec MGIT 960 amino acid sequence of the PZase enzyme. We observed two silent mycobacterial detection system may overestimate resistance even mutations: TCC65TCT (Ser65Ser), GCG38GCC (Ala38Ala). The in the best laboratory settings (due to changes in the medium pH Ser65Ser silent mutation was found associated with the Delhi/CAS and/or variability in the inoculum size). Alternatively, a different lineage. In some cases, sequencing of the upstream region of pncA mechanism of resistance, such as mutations in rpsA, could also be allowed the identification of a deletion at position 125 or an hypothesized for a few cases, although these were not clearly con- insertion at nucleotide 3; however, isolates harboring these ge- firmed in clinical isolates (data not shown) (20–23). In Fig. S1 in netic variants were found associated with both phenotypic resis- the supplemental material, we modeled the impact of these “dis- tance and susceptibility. According to these data, and supported crepant cases” in different hypothetical diagnostic scenarios to by the lack of homoplasy for these mutations, the indels detected provide worst and best performances of pncA sequencing-based do not represent a marker for PZA . assay as follows. If all 180 cases were truly susceptible, the diag- A total of 1,062 clinical isolates (1,062/1,950 [55.4%]) showed nostic accuracy of a molecular test for PZA based on sequence a WT sequence for the PZase enzyme (included in category B), would be 98.77% (95% confidence interval [95% CI], 98.18 to and the sequence was associated with PZA in more than 80% of 99.17%) (Fig. S1C) in the range of the rifampin and isoniazid test cases. Enzymatic assay results were not available for all: 17 isolates results (11). If the 180 cases were truly PZA strains (wrongly (out of 138 tested; 12.3%) gave a negative PZase enzymatic activ- predicted as PZA by pncA gene sequence), the diagnostic accuracy ity, indicating that a WT PZase does not exclude phenotypic re- of pncA sequencing in detecting PZA would be 89.54% (95% CI, sistance a priori. 89.21 to 90.82%), in the range of isoniazid resistance (Fig. S1B) (11). DISCUSSION Based on our findings, any future molecular test for PZA resis- PZA DST is crucial for successful management of patients with tance should be able not only to detect the absence of the wild-type susceptible and drug-resistant TB, especially with MDR TB. Fur- sequence but also to identify the specific SNPs. We found, indeed, thermore, future shorter regimens for both drug-resistant and a relevant number (10%) of mutations previously not reported as drug-susceptible TB will include PZA as a key drug for achieving associated with drug resistance (DR) and the degree of variability both sterilization and prevention of the development of drug re- in terms of indel mutations. In addition, we found mutations not sistance to new drugs (17, 18). Thus, reliable PZA data for clinical associated with DR, including the previously reported lineage- isolates are crucial for guiding the clinical management of pa- specific genetic variants (e.g., TCC65TCT in Delhi/CAS) (14). Ac- September/October 2014 Volume 5 Issue 5 e01819-14 mbio.asm.org 7 Miotto et al. cordingly, only an assay with the capacity to provide in-depth PZase assay. PZase activity was evaluated as described by Singh et al. (30). Briefly, a Middlebrook 7H9 (BD, Franklin Lakes, NJ, USA) 1.5% sequence information could comply with the minimal require- agarose containing PZA (Sigma-Aldrich Corporation, Saint Louis, MO, ments for a new molecular PZA test. Fully automated, low-cost g/ml was prepared. Melted PZA USA) at a final concentration of 400 medium-density arrays and user-friendly whole-gene/whole- agar was distributed in glass tubes by using an agarose base to obtain a genome sequencing-based approaches will become a reality in the semitransparent medium allowing the detection of a faint pink band very near future and will be the most suitable assays to fulfill this against a white background. A heavy loopful of actively growing culture task. In particular, new next-generation sequencing (NGS)-based was carefully inoculated on the surface of the PZA agar medium and diagnostics could represent innovative tools to reduce false PZA incubated at 37°C for 4 days. One milliliter of ferrous ammonium sulfate cases and to improve safe and fast detection of drug resistances by (1%) was added to each tube after incubation and observed for 4 h for the molecular DST (24). Our work has generated the minimum sets of appearance of a pink band (positive) in the subsurface agar. PZA-resistant mutations that should be included in any molecular test for PZA isolates of M. bovis (negative by the PZase test) were used as negative controls, and the PZA-susceptible strain M. tuberculosis H37Rv was used and provide a start point for a pncA genetic variation encyclopedia as a positive control. All isolates showing discrepant results (namely, pncA needed for the valid interpretation of data generated by massive mutant and PZase positive or WT pncA and PZase negative) were retested sequencing approaches. at 4 and 10 days (31). An additional aspect that is highlighted by our study is the great PZase structure. For each amino acid substitution, we performed an advantage in sharing large data sets generated by several groups. in silico analysis of the free energy variation associated with the specific The establishment of a common database combining data from mutation taking into account an acidic environmental pH (6.0), very close clinical isolates collected in a large number of settings was crucial to the one required for PZA activity. The crystal structure of the PZase to improve our understanding the role of pncA gene mutations in enzyme determined to 2.2-Å resolution (PDB code 3PL1) (32) was used in determining the PZA susceptibility phenotype of M. tuberculosis. conjunction with the program FoldX (33). Mean free energy variation was The high number of samples providing sufficient reiteration of calculated for triplicates of predicted structures, and based on statistical analysis, a free energy variation greater than 2 kJ/mol was considered to less frequent mutations together with the inclusion of different destabilize the enzyme. Frameshifts and mutations affecting the promoter parameters (phenotype, genotype, enzymatic activity, structure, region were not considered. Free energy variation was then integrated and free energy analyses) in a decision tree allowed us to define with a visual structural analysis in order to identify substitutions tolerated specific operational categories of mutations relevant from a clin- by the free energy term but detrimental for the specific activity of the ical point of view. This enabled us to build a user-friendly diag- enzyme. nostic algorithm through the classification of specific SNPs in a Statistical analysis. For understanding the significance of each muta- shared database collecting more-complex information. These tion, we predicted the DST by fitting a conditional inference tree model large shared databases of mutations involved in drug resistance considering results of sequencing, activity, and the combination of struc- could contribute to a better understanding of molecular mecha- ture and energy analyses as predictors. In the model, we applied recursive nisms of resistance, improved molecular diagnostics, new diag- partitioning based on conditional permutation tests. Furthermore, at each step, P values were adjusted for multiplicity by the procedure of nostic algorithms, and better public health control of drug- Benjamini and Yekutiely (34). The majority of recursive partitioning al- sensitive and drug-resistant TB. gorithms introduced since 1963 (35), such as CHAID and CART, yield trees with too many branches and can also fail to pursue branches which MATERIALS AND METHODS can add significantly to the overall fit. This leads to potential drawbacks: Strain selection. Strains were made available by six TB National/Supra- overfitting and a selection bias toward covariates with many possible splits national Reference and partner laboratories within the TB-PANNET or missing values (36, 37). Consortium to provide wide coverage for most of the lineages observed This approach is able to address missing data, since it uses surrogate for the M. tuberculosis complex. Strains were tested for PZA susceptibility splits to determine the daughter node where the observations with miss- and included in the study regardless of testing for other antitubercular ing values in the primary split variable are sent (for further details, see drugs. PZA drug susceptibility testing (DST) was performed by using a references 38 and 39). As output of the model, given an isolate’s profile, a Bactec MGIT 960 mycobacterial detection system and MGIT 960 PZA kits conditional probability of being PZA resistant is given. As a general rule, (BD, Franklin Lakes, NJ, USA) according to the manufacturer’s instruc- adjusted P values of less than 0.05 were considered significant. In the tions. A total of 1,950 clinical isolates were incorporated in the database. model, we applied recursive partitioning based on conditional permuta- Whenever available, genotyping information (spoligotyping and/or my- tion tests. In fact, when splitting, the use of the conditional distribution of cobacterial interspersed repetitive-unitvariable-number tandem-repeat the statistics ensures an unbiased selection of the covariates. This statisti- [MIRU-VNTR] typing [25]) were collected. The MIRU-VNTRplus web cal approach prevented overfitting and overgrown trees, and no further tool (26, 27) was used to define lineage information (similarity search pruning or cross-validation was needed. settings for identification: 0.17; distance measure for MIRU-VNTR: cate- Further details on the rationale used for the analysis is available in the gorical, weighting 1; distance measure for spoligotyping: categorical, supplemental material. weighting 1). SUPPLEMENTAL MATERIAL pncA gene sequencing. DNA was extracted as described elsewhere (28). The pncA gene (Rv2043c, NCBI gene identifier [ID] 888260), includ- Supplemental material for this article may be found at http://mbio.asm.org/ ing the proximal promoter region, was amplified. On a subset of samples, lookup/suppl/doi:10.1128/mBio.01819-14/-/DCSupplemental. the distal promoter region (100 bp upstream of the start codon) was also Table S1, XLSX file, 0.2 MB. included in the amplified region according to the protocol described in Table S2, XLSX file, 0.04 MB. Figure S1, DOCX file, 0.3 MB. reference 29. Amplicons were sequenced with an automated DNA se- Text S1, DOCX file, 0.01 MB. quencer. The pncA gene sequence of isolates from Samara, Russian Fed- eration, was determined from whole-genome sequencing data as previ- ACKNOWLEDGMENTS ously described (15). Mutations in the pncA gene were identified by comparison with the wild-type M. tuberculosis H37Rv pncA gene se- We thank Federica Piana (S. Croce and Carle Hospital, Cuneo, Italy) for quence. providing additional M. tuberculosis complex isolates. We thank the fol- 8 mbio.asm.org September/October 2014 Volume 5 Issue 5 e01819-14 Molecular Detection of PZA Resistance lowing for helpful discussions: Vladyslav Nikolayevskyy, Yanina Bal- Kontsevaya I, Corander J, Bryant J, Parkhill J, Nejentsev S, Horstmann RD, Brown T, Drobniewski F. 2014. Evolution and transmission of abanova, Irina Kontsevaya, and Olga Ignatyeva. drug-resistant tuberculosis in a Russian population. Nat. Genet. 46: This work was supported by the European Community’s Seventh 279 –286. http://dx.doi.org/10.1038/ng.2878. Framework Programme (EU FP7/2007–2013) under grant agreement 16. Comas I, Coscolla M, Luo T, Borrell S, Holt KE, Kato-Maeda M, http://www.tbpannet.org) and the TB-PANNET FP7-223681 to D.M.C. 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Mycobacterium tuberculosis Pyrazinamide Resistance Determinants: a Multicenter Study

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RESEARCH ARTICLE crossmark Mycobacterium tuberculosis Pyrazinamide Resistance Determinants: a Multicenter Study a a b,c d,e d,e f g Paolo Miotto, Andrea M. Cabibbe, Silke Feuerriegel, Nicola Casali, Francis Drobniewski, Yulia Rodionova, Daiva Bakonyte, g h i j k l l Petras Stakenas, Edita Pimkina, Ewa Augustynowicz-Kopec´, Massimo Degano, Alessandro Ambrosi, Sven Hoffner, Mikael Mansjö, l m b,c a Jim Werngren, Sabine Rüsch-Gerdes, Stefan Niemann, Daniela M. Cirillo Emerging Bacterial Pathogens Unit, Division of Immunology, Transplantation and Infectious Diseases, IRCCS San Raffaele Scientific Institute, Milan, Italy ; Molecular b c d Mycobacteriology, German Centre for Infection Research (DZIF), Borstel, Germany; Infectious Diseases, Imperial College, London, United Kingdom ; Clinical TB and HIV Group, Public Health England (PHE) National Mycobacterium Reference Laboratory, Blizard Institute, Queen Mary University of London, London, United Kingdom ; f g Samara TB Service, Samara, Russian Federation ; Department of Immunology and Cell Biology, Institute of Biotechnology, Vilnius University, Vilnius, Lithuania ; National Tuberculosis Reference Laboratory, Infectious Diseases and Tuberculosis Hospital, Vilnius University Hospital Santariskiu Klinikos, Vilnius, Lithuania ; Department of Microbiology, National Tuberculosis and Lung Diseases Research Institute, Warsaw, Poland ; Biocrystallography Unit, Division of Immunology, Transplantation and j k Infectious Diseases, IRCCS San Raffaele Scientific Institute, Milan Italy ; Vita-Salute San Raffaele University, Milan, Italy ; Public Health Agency of Sweden, Solna, and Department of Microbiology, Tumor and Cell Biology (MTC), Karolinska Institute, Stockholm, Sweden ; National Reference Center for Mycobacteria, Research Center Borstel, Borstel, Germany S.N. and D.M.C. contributed equally to this work. ABSTRACT Pyrazinamide (PZA) is a prodrug that is converted to pyrazinoic acid by the enzyme pyrazinamidase, encoded by the pncA gene in Mycobacterium tuberculosis. Molecular identification of mutations in pncA offers the potential for rapid detection of pyrazinamide resistance (PZA ). However, the genetic variants are highly variable and scattered over the full length of pncA, complicating the development of a molecular test. We performed a large multicenter study assessing pncA sequence variations in 1,950 clinical isolates, including 1,142 multidrug-resistant (MDR) strains and 483 fully susceptible strains. The results of pncA sequencing were correlated with phenotype, enzymatic activity, and structural and phylogenetic data. We identified 280 genetic variants which were divided into four classes: (i) very high confidence resistance mutations that were found only in PZA strains (85%), (ii) high-confidence resistance mutations found in more than 70% of PZA strains, (iii) mutations with an unclear role found in less than 70% of PZA strains, and (iv) mutations not associated with phenotypic resistance (10%). Any future molecu- lar diagnostic assay should be able to target and identify at least the very high and high-confidence genetic variant markers of PZA ; the diagnostic accuracy of such an assay would be in the range of 89.5 to 98.8%. IMPORTANCE Conventional phenotypic testing for pyrazinamide resistance in Mycobacterium tuberculosis is technically chal- lenging and often unreliable. The development of a molecular assay for detecting pyrazinamide resistance would be a break- through, directly overcoming both the limitations of conventional testing and its related biosafety issues. Although the main mechanism of pyrazinamide resistance involves mutations inactivating the pncA enzyme, the highly diverse genetic variants scattered over the full length of the pncA gene and the lack of a reliable phenotypic gold standard hamper the development of molecular diagnostic assays. By analyzing a large number of strains collected worldwide, we have classified the different genetic variants based on their predictive value for resistance which should lead to more rapid diagnostic tests. This would assist clini- cians in improving treatment regimens for patients. Received 20 August 2014 Accepted 26 September 2014 Published 21 October 2014 Citation Miotto P, Cabibbe AM, Feuerriegel S, Casali N, Drobniewski F, Rodionova Y, Bakonyte D, Stakenas P, Pimkina E, Augustynowicz-Kopec´ E, Degano M, Ambrosi A, Hoffner S, Mansjö M, Werngren J, Rüsch-Gerdes S, Niemann S, Cirillo DM. 2014. Mycobacterium tuberculosis pyrazinamide resistance determinants: a multicenter study. mBio 5(5): e01819-14. doi:10.1128/mBio.01819-14. Editor Carol A. Nacy, Sequella, Inc. Copyright © 2014 Miotto et al. This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited. Address correspondence to Paolo Miotto, [email protected]. yrazinamide (PZA) is a key drug in current and future tuber- on the success rates of MDR treatment and may allow a shortening Pculosis (TB) treatment regimens. It has a high sterilizing ca- of current MDR therapy (5). Finally, PZA is the only first-line pacity in vivo, but it is not active against Mycobacterium tubercu- drug that will be maintained in all regimens in the near future (6). losis complex (MTBC) strains growing at neutral pH (1–4). These new regimens aim at reducing the treatment duration of In addition to its crucial role in the standard short-course reg- susceptible, drug-resistant (especially MDR TB and extensively imen for TB treatment, PZA is used in the treatment of patients resistant) strain variants. infected with strains that are multidrug resistant (MDR) (resistant The essential role of PZA underlines the need for accurate and to at least isoniazid and rifampin). Here PZA has a strong impact rapid detection of PZA resistance that is very difficult with current September/October 2014 Volume 5 Issue 5 e01819-14 mbio.asm.org 1 Miotto et al. phenotypic tests (7). The difficulties with culture-based PZA sus- ceptibility testing result from several factors, including subopti- mal test media with unreliable pH and larger inocula that reduce the activity of PZA (8, 9). Furthermore, the critical concentration itself may result in inconsistent results for isolates with a PZA MIC close to this concentration (10). While for isoniazid and rifampin, highly reliable culture-based drug susceptibility testing (DST) techniques and rapid molecular assays such as the line probe assay MTBDRplus (Hain Lifescience GmbH, Nehren, Germany) and the cartridge-based Xpert MTB/RIF assay (Cepheid, Sunnyvale, CA) are available (11), no commercial molecular assays are cur- rently marketed for PZA. Great efforts have been made in understanding molecular re- sistance mechanisms. PZA is a prodrug that needs to be converted to an active compound, pyrazinoic acid, by the bacterial pyrazi- namidase (PZase) (encoded by pncA). Mutations/variations in pncA leading to the loss of PZase activity are the major mechanism leading to PZA resistance (PZA ) (4, 12). However, while high numbers of PZA cases can be related to inactivation of the PZase, the genetic variants, including single nucleotide polymorphisms (SNPs) and small deletions, are highly diverse and scattered over the full length of the 561 bp of the pncA gene (4, 12). This compli- cates the development of molecular tests, as no “hot spot region” comprising the majority of mutations is present in the pncA gene, as is present in rpoB for rifampin resistance. Accordingly, future molecular approaches to detect PZA in clinical isolates need to cover at least a significant number of pos- sible variants, if not the complete gene, to reach a high sensitivity (e.g., using approaches based on classical Sanger sequencing or next-generation genome sequencing). These techniques must be combined with an appropriate interpretation algorithm/database that distinguishes SNPs clearly associated with drug resistance from those for which the impact for developing PZA is unclear, e.g., due to phylogenic variants found in members of the MTBC (13, 14). In-depth knowledge of the variants found in PZA strains combined with evidence-based correlation with resistance pheno- types are needed to develop large-scale databases ensuring valid FIG 1 Pie chart reporting percentages of lineages for isolates included in the data interpretation. The fact that such a valid data basis is cur- study. rently lacking represents a substantial limitation for molecular PZA DST. To tackle this question, we performed a large multicenter study assessing pncA sequence variations in 1,950 MTBC pan- rium bovis, H37Rv, UZB_H37Rv_like, New-1, X, CAS, TUR,and susceptible strains and PZA strains. The strains were classified in Mycobacterium microti vole) each represented less than 5% of iso- phylogenetic lineages to identify variants that are phylogenetically lates (Fig. 1). Six percent were classified as EuroAmerican strains informative but not likely to be involved in PZA and those that not belonging to a valid lineage described previously (“other un- are occurring in strains from different groups and are obviously defined,” as described in reference 15), and 5.2% of strains were under positive selection. Using this comprehensive approach, we classified as “unknown,” because it was not possible to assign a could catalog 239 high-confidence PZA mutations and a number defined lineage. of pncA variants most likely not involved in PZA . Considering the sequencing results, 1,062 (54.5%) isolates were found to be wild type (WT) for the pncA gene, whereas 888 RESULTS harbored variations in the pncA gene that amounted to a total of We studied 1,950 clinical isolates, including 1,142 MDR strains 280 genetic variants comprising 67 insertions or deletions (indels) and 483 fully susceptible strains (see Table S1 in the supplemental and 213 SNPs (see Table S2 in the supplemental material). The material). By phenotypic DST, 1,107 clinical isolates were suscep- PZase enzymatic activity was available for 251 clinical isolates ac- tible to PZA, whereas 843 were classified as PZA . Genotyping data counting for 90 different genetic variants. Considering the distri- were available for 1,853 isolates (95.0%). Predominant lineages bution of the mutations across the entire gene, 73 (39.0%) codons among the strains investigated were Beijing (47.8%), LAM were not affected by mutations, whereas the remaining 114 (9.0%), Ural (7.7%), and Haarlem (5.0%). Other lineages found codons presented one or more mutations (Fig. 2). Only 50 codons (Ghana, EAI, Delhi/CAS, H37Rv_like, Uganda I and II, West Af- showed a frequency of mutation over the mean value of 0.5%, but rican 1 and 2, S, Cameroon, Sierra Leone 1 and 2, Mycobacte- despite this, a clear hot spot region could not be found; the most 2 mbio.asm.org September/October 2014 Volume 5 Issue 5 e01819-14 Molecular Detection of PZA Resistance FIG 2 Number of different mutations found at each codon. Note that multiple mutations and IS6110 are not included. The broken line indicates mean value. s r frequently affected regions (representing more than 70% of mu- isolates originally reported as PZA were reclassified as PZA (Ta- tated strains) were found at the promoter (positions 13 to 3) ble S1). The final distribution of mutations among revised PZA and at codons 6 to 15, 50 to 70, 90 to 100, 130 to 145, and 170 to and PZA isolates is summarized in Table 1. To further validate the 175 (Fig. 3). classification data, we analyzed the homoplastic occurrence of s r For mutations found in both PZA-sensitive (PZA ) and PZA particular mutations (e.g., the emergence in strains of two phylo- isolates, enzymatic activity and structural analysis results were genetic lineages [16]). As the homoplasy level is rather low in used to adjust for possible errors in phenotypic DST whenever MTBC genomes, this confirms that these mutations are most possible and to obtain a “revised DST” (included as “DST rev” in likely under positive selection and involved in the development of Table S1 in the supplemental material). Accordingly, 56 clinical PZA . FIG 3 Frequency of mutations found at each codon (calculated with 888 mutated isolates). Note that multiple mutations and IS6110 are not included. The dotted line indicates mean value. September/October 2014 Volume 5 Issue 5 e01819-14 mbio.asm.org 3 Miotto et al. s r r TABLE 1 Distribution of mutations among PZA and PZA clinical Mutations conferring PZA at very high confidence. Out of isolates the 280 sequence variants identified in pncA, 239 (85.4%) muta- tions found in 644 clinical isol ates (644/1,950 [33.0%]) were clas- pncA gene No. of isolates (%) sified as very high confidence variants associated with phenotypic s r PZA (n  1,051) PZA (n  899) PZA (category A) (see Table S2 in the supplemental material). WT 893 (85.0) 158 (15.0) Several mutations affect the catalytic residues and amino acids Mutant 200 (22.2) 699 (77.8) recruited in the scaffold of the active site or directly/indirectly Includes 19 isolates harboring silent mutations or mutations at the distal region of the involved in the coordination of the Fe ion (Asp8Gly/Ala/Glu/ promoter (100 nucleotides upstream of the start codon). Asn, His51Gln/Tyr, His71Arg, Asp49Glu/Asn/Ala, His57Arg/Tyr/ Gln/Pro, Trp68Arg/Gly/Cys/Stop/Leu, Gln10Pro/Arg, and His137Pro/Arg/Asp) or residues engaged in the hydrophobic core Using this procedure, four classes of genetic variants were (Ile6Thr, Val44Gly, Val139Gly/Leu, Met175Thr/Val, and identified: (i) very high confidence resistance mutations that were Phe94Cys/Ser/Leu). Out of the 90 variants tested, 87 variants, in- found only in PZA strains (category A), (ii) high-confidence re- cluding nucleotide substitutions at position 11, were also asso- sistance mutations found in more than 70% of PZA strains (cat- ciated with negative PZase activity, and 55 genetic variants egory E), (iii) mutations with an unclear role found in less than 70% in PZA strains (category D), and (iv) genetic variants (including the (detected in 332 isolates) were found in strains of at least 2 differ- wild type) not involved in phenotypic resistance (category B). Ta- ent lineages, indicating homoplasy (data not shown). Table 2 ble S2 in the supplemental material summarizes these clinically reports the mutations mapping in the most frequently affected relevant categories; a graphical overview is provided in Fig. 4. regions. FIG 4 Distribution of genetic variants across the four categories identified: (i) very high confidence resistance mutations, (ii) high-confidence resistance mutations, (iii) mutations with an unclear role, and (iv) mutations not involved in phenotypic resistance. The number of isolates belonging to each category is also reported. The inner ring shows the percentages of mutations affecting the structure of the enzyme for each category of genetic variants. PZA-R, PZA resistance. *, including wild-type isolates for the pncA gene. 4 mbio.asm.org September/October 2014 Volume 5 Issue 5 e01819-14 Molecular Detection of PZA Resistance TABLE 2 Mutations for PZA affecting the most frequently affected regions of pncA gene and representing more than 70% of mutated cases a b c d Nucleotide change Result of the mutation p.S p.R No. of cases A-11C Promoter 11 0.01497006 0.98502994 5 A-11G Promoter 11 0.01497006 0.98502994 35 A-11T Promoter 11 0.01497006 0.98502994 1 T-7C Promoter 7 0.01497006 0.98502994 4 T-7G Promoter 7 0.01497006 0.98502994 1 Del-5¡ G Promoter (del) 0.01497006 0.98502994 2 ATC6ACC Ile6Thr 0.01497006 0.98502994 3 Del14¡ TCATCG FSC 6 (del) 0.01497006 0.98502994 1 GTC7GGC Val7Gly 0.01497006 0.98502994 9 GTC7TTC Val7Phe 0.01497006 0.98502994 2 WT  GTC7GGC WT  Val7Gly 0.01497006 0.98502994 1 GAC8AAC Asp8Asn 0.01497006 0.98502994 3 GAC8GAA Asp8Glu 0.01497006 0.98502994 6 GAC8GCC Asp8Ala 0.01497006 0.98502994 1 GAC8GGC Asp8Gly 0.01497006 0.98502994 9 GTG9GGG Val9Gly 0.01497006 0.98502994 1 CAG10AAG Gln10Lys 0.01497006 0.98502994 2 CAG10CCG Gln10Pro 0.01497006 0.98502994 21 CAG10CGG Gln10Arg 0.01497006 0.98502994 6 GAC12AAC Asp12Asn 0.01497006 0.98502994 GAC12GAG Asp12Glu 0.01497006 0.98502994 2 GAC12GCC Asp12Ala 0.01497006 0.98502994 6 Ins37¡ GACT FSC 13 (ins) 0.01497006 0.98502994 1 TTC13TCC Phe13Ser 0.01497006 0.98502994 2 TTC13TTG Phe13Leu 0.01497006 0.98502994 3 TGC14CGC Cys14Arg 0.01497006 0.98502994 1 TGC14TGA Cys14Stop 0.01497006 0.98502994 2 WT  TGC14CGC WT  Cys14Arg 0.01497006 0.98502994 1 Ins44¡ C FSC 15 (ins) 0.01497006 0.98502994 1 Del150¡ T FSC 50 (del) 0.01497006 0.98502994 1 CAC51CAA His51Gln 0.01497006 0.98502994 7 CAC51CCC His51Pro 0.01497006 0.98502994 2 CAC51CGC His51Arg 0.01497006 0.98502994 4 CAC51TAC His51Tyr 0.01497006 0.98502994 3 CCG54CAG Pro54Gln 0.01497006 0.98502994 4 CCG54CGG Pro54Arg 0.01497006 0.98502994 1 CCG54CTG Pro54Leu 0.01497006 0.98502994 4 CCG54TCG Pro54Ser 0.01497006 0.98502994 1 CAC57CAG His57Gln 0.01497006 0.98502994 1 CAC57CCC His57Pro 0.01497006 0.98502994 1 CAC57CGC His57Arg 0.01497006 0.98502994 14 CAC57GAC His57Asp 0.01497006 0.98502994 10 CAC57TAC His57Tyr 0.01497006 0.98502994 5 WT  CAC57CGC WT  His57Arg 0.01497006 0.98502994 2 TTC58CTC Phe58Leu 0.01497006 0.98502994 7 CCG62CTG Pro62Leu 0.01497006 0.98502994 3 Del186¡ C FSC 62 (del) 0.01497006 0.98502994 3 Ins185¡ 4 nt FSC 62 (ins) 0.01497006 0.98502994 1 Ins186¡ A FSC 62 (ins) 0.01497006 0.98502994 1 GAC63GGC Asp63Gly 0.01497006 0.98502994 4 Ins192¡ A FSC 64 (ins) 0.01497006 0.98502994 1 TAT64TAG Tyr64stop 0.01497006 0.98502994 3 Ins193¡ A FSC 65 (ins) 0.01497006 0.98502994 1 Ins193¡ TATCAGG FSC 65 (ins) 0.01497006 0.98502994 1 TCG67CCG Ser67Pro 0.01497006 0.98502994 2 TGG68CGG Trp68Arg 0.01497006 0.98502994 7 TGG68GGG Trp68Gly 0.01497006 0.98502994 16 TGG68TAG Trp68stop 0.01497006 0.98502994 1 TGG68TGC Trp68Cys 0.01497006 0.98502994 5 TGG68TGT Trp68Cys 0.01497006 0.98502994 1 GAG91TAG Glu91Stop 0.01497006 0.98502994 1 TTC94CTC Phe94Leu 0.01497006 0.98502994 8 TTC94TCC Phe94Ser 0.01497006 0.98502994 3 TTC94TGC Phe94Cys 0.01497006 0.98502994 6 TTC94TTA Phe94Leu 0.01497006 0.98502994 2 (Continued on following page) September/October 2014 Volume 5 Issue 5 e01819-14 mbio.asm.org 5 Miotto et al. TABLE 2 (Continued) a b c d Nucleotide change Result of the mutation p.S p.R No. of cases TTC94TTG Phe94Leu 0.01497006 0.98502994 1 WT  TTC94CTC WT  Phe94Leu 0.01497006 0.98502994 1 TAC95TAG Tyr95stop 0.01497006 0.98502994 1 AAG96AAC Lys96Asn 0.01497006 0.98502994 1 AAG96ACG Lys96Thr 0.01497006 0.98502994 2 AAG96AGG Lys96Arg 0.01497006 0.98502994 1 AAG96CAG Lys96Gln 0.01497006 0.98502994 1 AAG96GAC Lys96Glu 0.01497006 0.98502994 6 Ins288¡ T FSC 96 (ins) 0.01497006 0.98502994 2 Ins288¡ 33 nt FSC 96 (ins) 0.01497006 0.98502994 4 Del291¡ T FSC 97 (del) 0.01497006 0.98502994 1 GGT97AGT Gly97Ser 0.01497006 0.98502994 6 GGT97GAT Gly97Asp 0.01497006 0.98502994 4 GGT97GCT Gly97Ala 0.01497006 0.98502994 1 TAC99TAA Tyr99stop 0.01497006 0.98502994 2 ACC100CCC Thr100Pro 0.01497006 0.98502994 2 ACC100GCC Thr100Ala 0.01497006 0.98502994 1 GTG130GCG Val130Ala 0.01497006 0.98502994 1 GTG130GGG Val130Gly 0.01497006 0.98502994 1 Ins391¡ G FSC 131 (ins) 0.01497006 0.98502994 3 Ins391¡ GG FSC 131 (ins) 0.01497006 0.98502994 2 Ins392¡ G FSC 131 (ins) 0.01497006 0.98502994 2 Ins392¡ GG FSC 131 (ins) 0.01497006 0.98502994 4 Ins393¡ G FSC 131 (ins) 0.01497006 0.98502994 2 Ins393¡ GG FSC 131 (ins) 0.01497006 0.98502994 1 Ins394¡ ATGTGGTCG FSC 131 (ins) 0.01497006 0.98502994 1 TGC131GGTGC FSC 131 (ins) 0.01497006 0.98502994 1 GGT132AGT Gly132Ser 0.01497006 0.98502994 1 GGT132GAT Gly132Asp 0.01497006 0.98502994 1 GGT132GCT Gly132Ala 0.01497006 0.98502994 1 GGT132TGT Gly132Cys 0.01497006 0.98502994 2 ATT133ACT Ile133Thr 0.01497006 0.98502994 17 Del398¡ T FSC 133 (del) 0.01497006 0,98502994 1 GCC134GTC Ala134Val 0.01497006 0.98502994 2 ACC135AAC Thr135Asn 0.01497006 0.98502994 3 ACC135CCC Thr135Pro 0.01497006 0.98502994 4 GAT136TAT Asp136Tyr 0.01497006 0.98502994 3 Ins408¡ A FSC 136 (ins) 0.01497006 0.98502994 4 CAT137CCT His137Pro 0.01497006 0.98502994 1 CAT137CGT His137Arg 0.01497006 0.98502994 1 CAT137GAT His137Asp 0.01497006 0.98502994 1 TGT138CGT Cys138Arg 0.01497006 0.98502994 3 TGT138TGG Cys138Trp 0.01497006 0.98502994 1 Del417¡ G FSC 139 (del) 0.01497006 0.98502994 1 GTG139CTG Val139Leu 0.01497006 0.98502994 3 GTG139GGG Val139Gly 0.01497006 0.98502994 5 CGC140CCC Arg140Pro 0.01497006 0.98502994 1 CAG141CCG Gln141Pro 0.01497006 0.98502994 11 CAG141TAG Gln141stop 0.01497006 0.98502994 1 Ins423¡ CAGACGGCGCCAG FSC 141 (ins) 0.01497006 0.98502994 1 ACG142AAG Thr142Lys 0.01497006 0.98502994 1 ACG142ATG Thr142Met 0.01497006 0.98502994 3 ACG142GCG Thr142Ala 0.01497006 0.98502994 3 GCC143GGC Ala143Gly 0.01497006 0.98502994 1 CTG172CCG Leu172Pro 0.01497006 0.98502994 9 Del514¡ C FSC 172 (del) 0.01497006 0.98502994 1 Ins516¡ CG FSC 172 (ins) 0.01497006 0.98502994 1 ATG175ACG Met175Thr 0.01497006 0.98502994 1 ATG175ATA Met175Ile 0.01497006 0.98502994 10 ATG175GTG Met175Val 0.01497006 0.98502994 6 A-11C, nucleotide change A to C in position 11; Del-5¡ G, deletion of nucleotide G in position 5; ATC6AAC, ATC at codon 7 changed to AAC; WT  GTC7GGC, double pattern wild-type  GTC at codon 7 changed to GGC; Ins37¡ GACT, GACT inserted at codon 37. Promoter 11, nucleotidic mutation affecting the promoter region at position 11; del, deletion; FSC, frameshift codon; ins, insertion. p.S, probability associated with the susceptible phenotype. p.R, probability associated with the resistant phenotype. 6 mbio.asm.org September/October 2014 Volume 5 Issue 5 e01819-14 Molecular Detection of PZA Resistance Mutations conferring PZA at high confidence. Nine genetic tients. Phenotypic tests, however, have a long turnaround time, variants (32 strains, category E) were found in both PZA and are expensive, and are considered poorly reliable. As a conse- s r r PZA isolates, but with a proportion higher than 70% in PZA quence, the design of a molecular test for predicting PZA is a strains. These mutations were mainly associated with an increase priority. The development of a rapid molecular PZA DST is ham- in free energy and/or structural constraints and were most fre- pered by the diverse nature of resistance-associated mutations quently associated with PZA (93.5% of cases). We confirmed a mainly scattered over the full length of the pncA gene, and by the reduced but still present PZase activity for some of these variants fact that the impact of individual mutations has not been system- as a development of faint color during the enzymatic assay. atically investigated (4, 12). Therefore, we performed a large-scale Whereas Leu172Pro was found to be associated with homoplasy, study linking pncA sequence diversity with phenotypic, structural for other substitutions, the number of cases was too low to con- biology and population biology data to develop the first encyclo- r s sider convergent evolution in different lineages. pedia of pncA sequence variations linked to either a PZA or PZA Mutations with an unclear role in conferring PZA . Five ge- phenotype. This is likely to pave the way for application of new r r netic variants (21 cases, category D) were found in both PZA and genome-based sequencing technologies for predicting PZA ,al- s r PZA isolates but at a proportion less than 70% in PZA strains. lowing for personalized treatment algorithms. Two genetic variants (15 cases) showed borderline behavior in Strikingly, nearly 85% of the genetic variants identified in the terms of structure/free energy variation and enzyme activity. Ho- pncA gene were associated with phenotypic resistance to PZA and moplasy was found for the Val139Ala mutation, thus suggesting a were classified as “high-confidence” PZA resistance mutations. All putative role in phenotypic resistance or at least in increasing the in-frame and frameshift indel mutations within the coding region MIC. Pro62Arg, Asp63Ala, and Ser65Pro substitutions (6 cases) were included in this group. We found that nearly 90% of ob- represent another group of mutations belonging to this ambigu- served mutations are associated with protein structural destabili- ous category. Further characterization of these mutations is zation that causes loss of enzymatic activity. Equally importantly, needed to better understand their correlation with the PZA phe- we described 27 mutations most likely not involved in PZA resis- notype. tance that should be “filtered out” in future molecular tests and Mutations not involved in phenotypic resistance. Twenty- labeled as not “clinically relevant” (Fig. 4). Only five mutations seven genetic variants were not associated with PZA according to cannot be classified by our approach and remain without clear our classification. Eighteen mutations (163 cases, category C) association with a resistance or susceptible phenotype. These mu- s r were most frequently associated with PZA (91.4% of cases). It tations need further validation for association with PZA and/or should be noted that the Val21Ala mutation was also found asso- with a specific genetic background by an allelic exchange proce- ciated with other mutations in category A responsible for PZA / dure as performed for other drugs (19). PZase negativity. Interestingly, all these mutations were found to Discrepancies between molecular and phenotypic DST are be associated with single lineages; thus, no homoplasy was ob- confusing for clinicians managing patients; 180 isolates investi- served. Further characterization of these mutations is needed to gated here showed discrepant results between phenotypic and ge- better understand their role (if any) in PZA susceptibility. The notypic tests (WT pncA gene sequence and resistance by Bactec remaining genetic variants (27 cases, category B) did not affect the MGIT 960 DST). It has been reported that the Bactec MGIT 960 amino acid sequence of the PZase enzyme. We observed two silent mycobacterial detection system may overestimate resistance even mutations: TCC65TCT (Ser65Ser), GCG38GCC (Ala38Ala). The in the best laboratory settings (due to changes in the medium pH Ser65Ser silent mutation was found associated with the Delhi/CAS and/or variability in the inoculum size). Alternatively, a different lineage. In some cases, sequencing of the upstream region of pncA mechanism of resistance, such as mutations in rpsA, could also be allowed the identification of a deletion at position 125 or an hypothesized for a few cases, although these were not clearly con- insertion at nucleotide 3; however, isolates harboring these ge- firmed in clinical isolates (data not shown) (20–23). In Fig. S1 in netic variants were found associated with both phenotypic resis- the supplemental material, we modeled the impact of these “dis- tance and susceptibility. According to these data, and supported crepant cases” in different hypothetical diagnostic scenarios to by the lack of homoplasy for these mutations, the indels detected provide worst and best performances of pncA sequencing-based do not represent a marker for PZA . assay as follows. If all 180 cases were truly susceptible, the diag- A total of 1,062 clinical isolates (1,062/1,950 [55.4%]) showed nostic accuracy of a molecular test for PZA based on sequence a WT sequence for the PZase enzyme (included in category B), would be 98.77% (95% confidence interval [95% CI], 98.18 to and the sequence was associated with PZA in more than 80% of 99.17%) (Fig. S1C) in the range of the rifampin and isoniazid test cases. Enzymatic assay results were not available for all: 17 isolates results (11). If the 180 cases were truly PZA strains (wrongly (out of 138 tested; 12.3%) gave a negative PZase enzymatic activ- predicted as PZA by pncA gene sequence), the diagnostic accuracy ity, indicating that a WT PZase does not exclude phenotypic re- of pncA sequencing in detecting PZA would be 89.54% (95% CI, sistance a priori. 89.21 to 90.82%), in the range of isoniazid resistance (Fig. S1B) (11). DISCUSSION Based on our findings, any future molecular test for PZA resis- PZA DST is crucial for successful management of patients with tance should be able not only to detect the absence of the wild-type susceptible and drug-resistant TB, especially with MDR TB. Fur- sequence but also to identify the specific SNPs. We found, indeed, thermore, future shorter regimens for both drug-resistant and a relevant number (10%) of mutations previously not reported as drug-susceptible TB will include PZA as a key drug for achieving associated with drug resistance (DR) and the degree of variability both sterilization and prevention of the development of drug re- in terms of indel mutations. In addition, we found mutations not sistance to new drugs (17, 18). Thus, reliable PZA data for clinical associated with DR, including the previously reported lineage- isolates are crucial for guiding the clinical management of pa- specific genetic variants (e.g., TCC65TCT in Delhi/CAS) (14). Ac- September/October 2014 Volume 5 Issue 5 e01819-14 mbio.asm.org 7 Miotto et al. cordingly, only an assay with the capacity to provide in-depth PZase assay. PZase activity was evaluated as described by Singh et al. (30). Briefly, a Middlebrook 7H9 (BD, Franklin Lakes, NJ, USA) 1.5% sequence information could comply with the minimal require- agarose containing PZA (Sigma-Aldrich Corporation, Saint Louis, MO, ments for a new molecular PZA test. Fully automated, low-cost g/ml was prepared. Melted PZA USA) at a final concentration of 400 medium-density arrays and user-friendly whole-gene/whole- agar was distributed in glass tubes by using an agarose base to obtain a genome sequencing-based approaches will become a reality in the semitransparent medium allowing the detection of a faint pink band very near future and will be the most suitable assays to fulfill this against a white background. A heavy loopful of actively growing culture task. In particular, new next-generation sequencing (NGS)-based was carefully inoculated on the surface of the PZA agar medium and diagnostics could represent innovative tools to reduce false PZA incubated at 37°C for 4 days. One milliliter of ferrous ammonium sulfate cases and to improve safe and fast detection of drug resistances by (1%) was added to each tube after incubation and observed for 4 h for the molecular DST (24). Our work has generated the minimum sets of appearance of a pink band (positive) in the subsurface agar. PZA-resistant mutations that should be included in any molecular test for PZA isolates of M. bovis (negative by the PZase test) were used as negative controls, and the PZA-susceptible strain M. tuberculosis H37Rv was used and provide a start point for a pncA genetic variation encyclopedia as a positive control. All isolates showing discrepant results (namely, pncA needed for the valid interpretation of data generated by massive mutant and PZase positive or WT pncA and PZase negative) were retested sequencing approaches. at 4 and 10 days (31). An additional aspect that is highlighted by our study is the great PZase structure. For each amino acid substitution, we performed an advantage in sharing large data sets generated by several groups. in silico analysis of the free energy variation associated with the specific The establishment of a common database combining data from mutation taking into account an acidic environmental pH (6.0), very close clinical isolates collected in a large number of settings was crucial to the one required for PZA activity. The crystal structure of the PZase to improve our understanding the role of pncA gene mutations in enzyme determined to 2.2-Å resolution (PDB code 3PL1) (32) was used in determining the PZA susceptibility phenotype of M. tuberculosis. conjunction with the program FoldX (33). Mean free energy variation was The high number of samples providing sufficient reiteration of calculated for triplicates of predicted structures, and based on statistical analysis, a free energy variation greater than 2 kJ/mol was considered to less frequent mutations together with the inclusion of different destabilize the enzyme. Frameshifts and mutations affecting the promoter parameters (phenotype, genotype, enzymatic activity, structure, region were not considered. Free energy variation was then integrated and free energy analyses) in a decision tree allowed us to define with a visual structural analysis in order to identify substitutions tolerated specific operational categories of mutations relevant from a clin- by the free energy term but detrimental for the specific activity of the ical point of view. This enabled us to build a user-friendly diag- enzyme. nostic algorithm through the classification of specific SNPs in a Statistical analysis. For understanding the significance of each muta- shared database collecting more-complex information. These tion, we predicted the DST by fitting a conditional inference tree model large shared databases of mutations involved in drug resistance considering results of sequencing, activity, and the combination of struc- could contribute to a better understanding of molecular mecha- ture and energy analyses as predictors. In the model, we applied recursive nisms of resistance, improved molecular diagnostics, new diag- partitioning based on conditional permutation tests. Furthermore, at each step, P values were adjusted for multiplicity by the procedure of nostic algorithms, and better public health control of drug- Benjamini and Yekutiely (34). The majority of recursive partitioning al- sensitive and drug-resistant TB. gorithms introduced since 1963 (35), such as CHAID and CART, yield trees with too many branches and can also fail to pursue branches which MATERIALS AND METHODS can add significantly to the overall fit. This leads to potential drawbacks: Strain selection. Strains were made available by six TB National/Supra- overfitting and a selection bias toward covariates with many possible splits national Reference and partner laboratories within the TB-PANNET or missing values (36, 37). Consortium to provide wide coverage for most of the lineages observed This approach is able to address missing data, since it uses surrogate for the M. tuberculosis complex. Strains were tested for PZA susceptibility splits to determine the daughter node where the observations with miss- and included in the study regardless of testing for other antitubercular ing values in the primary split variable are sent (for further details, see drugs. PZA drug susceptibility testing (DST) was performed by using a references 38 and 39). As output of the model, given an isolate’s profile, a Bactec MGIT 960 mycobacterial detection system and MGIT 960 PZA kits conditional probability of being PZA resistant is given. As a general rule, (BD, Franklin Lakes, NJ, USA) according to the manufacturer’s instruc- adjusted P values of less than 0.05 were considered significant. In the tions. A total of 1,950 clinical isolates were incorporated in the database. model, we applied recursive partitioning based on conditional permuta- Whenever available, genotyping information (spoligotyping and/or my- tion tests. In fact, when splitting, the use of the conditional distribution of cobacterial interspersed repetitive-unitvariable-number tandem-repeat the statistics ensures an unbiased selection of the covariates. This statisti- [MIRU-VNTR] typing [25]) were collected. The MIRU-VNTRplus web cal approach prevented overfitting and overgrown trees, and no further tool (26, 27) was used to define lineage information (similarity search pruning or cross-validation was needed. settings for identification: 0.17; distance measure for MIRU-VNTR: cate- Further details on the rationale used for the analysis is available in the gorical, weighting 1; distance measure for spoligotyping: categorical, supplemental material. weighting 1). SUPPLEMENTAL MATERIAL pncA gene sequencing. DNA was extracted as described elsewhere (28). The pncA gene (Rv2043c, NCBI gene identifier [ID] 888260), includ- Supplemental material for this article may be found at http://mbio.asm.org/ ing the proximal promoter region, was amplified. On a subset of samples, lookup/suppl/doi:10.1128/mBio.01819-14/-/DCSupplemental. the distal promoter region (100 bp upstream of the start codon) was also Table S1, XLSX file, 0.2 MB. included in the amplified region according to the protocol described in Table S2, XLSX file, 0.04 MB. Figure S1, DOCX file, 0.3 MB. reference 29. Amplicons were sequenced with an automated DNA se- Text S1, DOCX file, 0.01 MB. quencer. The pncA gene sequence of isolates from Samara, Russian Fed- eration, was determined from whole-genome sequencing data as previ- ACKNOWLEDGMENTS ously described (15). Mutations in the pncA gene were identified by comparison with the wild-type M. tuberculosis H37Rv pncA gene se- We thank Federica Piana (S. Croce and Carle Hospital, Cuneo, Italy) for quence. providing additional M. tuberculosis complex isolates. We thank the fol- 8 mbio.asm.org September/October 2014 Volume 5 Issue 5 e01819-14 Molecular Detection of PZA Resistance lowing for helpful discussions: Vladyslav Nikolayevskyy, Yanina Bal- Kontsevaya I, Corander J, Bryant J, Parkhill J, Nejentsev S, Horstmann RD, Brown T, Drobniewski F. 2014. Evolution and transmission of abanova, Irina Kontsevaya, and Olga Ignatyeva. drug-resistant tuberculosis in a Russian population. Nat. Genet. 46: This work was supported by the European Community’s Seventh 279 –286. http://dx.doi.org/10.1038/ng.2878. Framework Programme (EU FP7/2007–2013) under grant agreement 16. Comas I, Coscolla M, Luo T, Borrell S, Holt KE, Kato-Maeda M, http://www.tbpannet.org) and the TB-PANNET FP7-223681 to D.M.C. 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