- Research article
- Open Access
Pyrazinamide resistance and mutations in pncA among isolates of Mycobacterium tuberculosis from Khyber Pakhtunkhwa, Pakistan
BMC Infectious Diseases volume 19, Article number: 116 (2019)
Pyrazinamide (PZA) is an important component of first-line drugs because of its distinctive capability to kill subpopulations of persistent Mycobacterium tuberculosis (MTB). The prodrug (PZA) is converted to its active form, pyrazinoic acid (POA) by MTB pncA-encoded pyrazinamidase (PZase). Mutation in pncA is the most common and primary cause of PZA resistance. The aim of the present study was to explore the molecular characterization of PZA resistance in a Pashtun-dominated region of Khyber Pakhtunkhwa, Pakistan.
We performed drug susceptibility testing (DST) on 753 culture-positive isolates collected from the Provincial Tuberculosis Control Program Khyber Pakhtunkhwa using the BACTEC MGIT 960 PZA method. In addition, the pncA gene was sequenced in PZA-resistant isolates, and PZA susceptibility testing results were used to determine the sensitivity and specificity of pncA gene mutations.
A total of 69 isolates were PZA resistant (14.8%). Mutations were investigated in 69 resistant, 26 susceptible and one H37Rv isolates by sequencing. Thirty-six different mutations were identified in PZA-resistant isolates, with fifteen mutations, including 194_203delCCTCGTCGTG and 317_318delTC, that have not been reported in TBDRM and GMTV Databases and previous studies. Mutations Lys96Thr and Ser179Gly were found in the maximum number of isolates (n = 4 each). We did not detect mutations in sensitive isolates, except for the synonymous mutation 195C > T (Ser65Ser). The sensitivity and specificity of the pncA sequencing method were 79.31% (95% CI, 69.29 to 87.25%) and 86.67% (95% CI, 69.28 to 96.24%).
Mutations in the pncA gene in circulating isolates of geographically distinct regions, especially in high-burden countries, should be investigated for better control and management of drug-resistant TB. Molecular methods for the investigation of PZA resistance are better than DST.
Tuberculosis (TB) is a common life-threatening infectious disease caused by Mycobacterium tuberculosis (MTB) . The standard new therapy includes a six-month treatment of four recommended first-line drugs, i.e., isoniazid, rifampin, pyrazinamide and ethambutol . However, the misuse of these antibiotics has led to the emergence of multidrug-resistant (MDR) strains of MTB . According to the WHO report in 2016, Pakistan is ranked among the top five countries, with a prevalence of 56% of global TB and among the high-burden drug-resistant countries . The incidence of drug resistance (MDR/RR-TB) is twenty-six thousand (16–36) with 14 (8.5–19) per hundred thousand individuals in a population. This situation leaves a high challenge to the TB control program in the country.
Khyber Pakhtunkhwa (KPK), a region in Pashtun, is the third largest province of Pakistan, with an area of 74,521 km2 and a population of approximately 30,523,371 individuals. Tuberculosis remains a major public health problem and one of the neglected health areas in the past. Recently, a TB control program has been launched at Hayatabad Medical Complex Peshawar that regularly monitors the incidence of TB as well as drug susceptibility testing (DST) in the population.
Pyrazinamide (PZA), a key first-line antibiotic used for the short-course treatment of 6 months, kills dormant tubercle bacilli at an acidic pH, whereas other drugs fail to work during the early severe stages of chemotherapy . Due to some technical and buffering issues of drug susceptibility testing (DST) for PZA, the results of phenotypic resistance are not always reliable . The MGIT 960 system is the most reliable method to perform PZA-DST [7, 8] and is currently the only available phenotypic test method to explore PZA susceptibility. MTB isolates are cultured in the presence of PZA under acidic conditions as required for conversion into pyrazinoic acid (POA) , the active form of pyrazinamide in vivo, activated by MTB PZase (PncA). These issues have prompted efforts towards molecular methods of PZA resistance .
In Khyber Pakhtunkhwa, there are no molecular studies to explore pncA mutations in the KPK Pashtun-dominant region. Here, we aimed to compare phenotypic resistance to PZA to genotype and identify mutations in pncA among PZA-resistant isolates circulating in this epidemiologically distinct area in a Pashtun-dominant population, which may be useful in tracing the transmission in patients.
Material and methods
The present investigation was approved by the Institutional Ethics Committee of CUST Islamabad and Provincial Tuberculosis Reference Laboratory (PTRL) KPK under reference number PTP/PTRL-402/16. Prior to the study, informed consent was obtained from each TB patient, however, the results were not linked back to individual patients.
All samples were processed at the BSL-III facility of PTRL, Hayatabad Medical Complex (HMC). The lab receives TB cases from the entire province, which is facilitated by the MGIT 960 system of drug susceptibility testing. The data for TB patients were collected from their guardians or caretakers.
Sample processing, isolation and mycobacterial culture
The samples were processed using the N-acetyl-L-cysteine–sodium hydroxide (NALC–NaOH) concentration method  in a Falcon tube containing an equal volume of the NaOH/N-acetyl-L-cysteine (NALC), which was subsequently vortexed and incubated at room temperature for 15 min for decontamination and digestion. Next, 50 ml of phosphate buffer was transferred to each tube, followed by centrifugation at 3000 rpm for 15 min. The supernatant was transferred to a fresh tube containing 5% phenol, while the pellet was mixed with phosphate buffer and cultured on Lowenstein–Jensen medium (LJ) in MGIT tubes containing 7H9 media.
Drug susceptibility testing (DST)
Drug susceptibility testing of PZA was performed through the automated BACTEC MGIT 960 system (BD Diagnostic Systems, NJ, USA) . Mycobacterium tuberculosis H37Rv and Mycobacterium bovis were used as susceptible and resistant controls, respectively. A sample was marked as PZA resistant if growth was found at 100 μg/ml of the PZA critical concentration. DST for resistant isolates was repeated for confirmation of drug resistance. The PZA-resistant samples were further subjected to DST with isoniazid (INH), rifampin (RIF), ethambutol (EMB), amikacin (AMK), streptomycin (SM), capreomycin (CAP), ofloxacin (OFX) and kanamycin (KM) through the BACTEC MGIT 960 system, with critical concentrations of drugs as per the policy guidelines of the WHO (WHO 2014) . The resistant samples were further manually assessed to confirm the growth of MTB against the critical drug concentration.
DNA extraction, PCR amplification and sequencing
Genomic DNA from PZA-resistant isolates were extracted by sonication [16, 17]. One microliter of fresh culture was transferred from a Mycobacterium Growth Indicator Tube (MGIT) to a microcentrifuge tube and boiled at 86 °C for 30 min using an Echotherm™ IC22 Digital, Chilling/Heating Dry Bath followed by 15 min sonication using a sonicator (ELMASONIC S30). All the samples were centrifuged for 5 min at 10,000 rpm. The supernatant containing DNA was stored at − 20 °C. The fragments containing pncA were amplified using previously reported primers (pncA-F = 5GCGTCATGGACCCTATATC-3 and pncA-R = 5 AACAGTTCATCCCGGTTC-3=) . Each 50-μl PCR contained 0.1 μl of each DNTs, 0.8 μl of Taq (New England Biolabs, UK)), 5 μl of PCR buffer, 3 μl of MgCl2, 1 μl of each forward and reverse primer, 34.8 μl of molecular grade water and 4 μl of genomic DNA. The PCR conditions were 5 min at 94 °C for denaturation, followed by 30 cycles of 30 s at 94 °C, 30 s at 56 °C, and 72 °C for 1 min, with an extension step at 72 °C for 5 min, as previously described. The PCR product was analyzed by 6 Applied Biosystems 3730xl (Macrogen Korea).
The sequence data obtained were loaded into Mutation Surveyor V5.0.1 software . The data were analyzed and compared with the PncA (Rv2043c) gene of RefSeq database of NCBI (NC_000962), while the patient data were entered by using Epi-Data entry version 3.1 software and analyzed through Epi-Data analysis software.
A total of 4518 samples were collected from TB subjects from all districts of KPK. Among these individuals, 753 subjects were culture positive, with ages ranging from 8 to 76 years (median age = 34.34). A majority of the cases were never treated (diagnostics) (44/69). All patients were KPK residents with Pushto as the main language (Table 1).
Drug susceptibility pattern
By using the BACTEC MGIT 960 system, 69 (14.8%) isolates were classified as PZA resistant. All PZA resistant isolates, 26 PZA-sensitive isolates and one H37Rv isolate were sequenced to analyze the mutations in the 561 bp region of pncA. Multidrug drug resistance (MDR) and extreme drug resistance (XDR) were detected in 52/69 (75.35%) and 6/69 (8.69%) isolates, respectively.
Risk factors such as age, gender, history, reason, disease type and resistance type are presented in Table 1. The presence of a high number of MDR isolates (52/69) in PZA-resistant isolates shows the major risk factor in transmission and treatment failure.
Mutation in PZA-resistant and PZA-susceptible isolates
Mutations were investigated in both resistance and susceptible isolates in the coding region (561 bp) of pncA (Table 2). Among the 69 PZA-resistant isolates 51 (74%), thirty-six different mutations with fifteen novel mutations, including 194_203delCCTCGTCGTG and 317_318delTC, were detected; but these variations were not found in TBDRM and GMTV Databases [20, 21]. The most common mutations detected at positions 287 and 423 were Lys96Thr (n = 4) and Ser179Gly (n = 4), respectively. A majority of these variations were substitution mutations, except for three deletions, 194_203 del CCTCGTCGTG (n = 1), 317_318delTC (n = 1), 530 del C (n = 3). A common synonymous mutation at position 195 C > T (Ser65Ser) was observed in both resistant (n = 22) and susceptible isolates (n = 16).
Comparing phenotypic PZA-resistant with DNA sequencing
Among sensitive isolates we did not detect any nonsynonymous mutations in the coding region of pncA. To estimate the performance of DST compared with the pncA sequencing result, the genotypic data and phenotypic data for all 69 resistance isolates were evaluated. Considering phenotype as a reference, among the 69 resistant isolates, 51 (74%) isolates showed mutations, with sensitivity of 79.31% (95% CI, 69.29 to 87.25%) and specificity of 86.67% (95% CI, 69.28 to 96.24%).
PZA is a distinctive antituberculosis drug that plays a key role in shortening TB treatment. PZA kills nonreplicating persistent MTB and is prescribed in both susceptible and MDR-TB treatment. After conversion into its active form POA by pyrazinamidase (PZase), PZA remains active at low pH during acidic stress . However, in a large number of cases, MTB patients develop resistance against PZA that led to the survival of persistent bacteria. Conventional methods of PZA DST increase the level of false resistance that may result from media buffering issues and large inoculum sizes, where the acidic environment is required for drug action but inhibits the growth of MTB [6, 22, 23]. Under such conditions, the most reliable method in the present scenario is the molecular detection of PZA-resistance, which involves sequencing the pncA gene to assess mutations in the 561 bp coding region and upstream regulatory region. In the present study, more than half of the tested PZA-resistant isolates were also MDR-TB, 52/69 (75.35%) isolates, consistent with the results of previous studies [12, 24]. Previous studies [25, 26] have also shown a correlation between mutations in the pncA gene and phenotypic PZA-resistance. Based on these findings, we report mutations in 51 (74%) resistant isolates that harbor 36 mutations in the coding region of pncA, with sensitivity and specificity of pncA sequencing of 79.31% (95% CI, 69.29 to 87.25%) and 86.67% (95% CI, 69.28 to 96.24%), respectively. Streicher et al. and Whitfield reported better sensitivity and specificity for pncA sequencing compared with that of MGIT 960 DST of 90.9, 100%  and 95.0 (95% CI 92.1–98.0), 99.1 (95% CI 98.4–99.9) . Miotto et al. (2014) identified 280 mutations in 1950 clinical strains , which were categorized into four groups, 1) very high confidence resistance mutations, 2) high-confidence resistance mutations, 3) mutations with an unclear role and 4) mutations not associated with phenotypic resistance based on confidence level. We detected 12 mutations with very high confidence resistance, while the rest of mutations detected have been found in Miotto unclear category (Table 2). The mutations 211C > T, 212A > G, 226A > C, 286A > C and 422A > C in the present study (Table 2) were previously shown as very high confidence resistance mutations [9, 12, 29]. Molecular biomarkers that could specifically target the first two categories should be developed .
Tan et al. (2014) reported that each geographical region has a diverse type of variations in pncA. Isolates from Southern China exhibited a scattered type of mutations in 561 bp region, which remains a complex target in the development of diagnostic biomarkers in identification of all resistance conferring mutations . Some strains, which were PZA resistant by conventional DST, lack any mutations in PncA and its regulatory gene, suggesting other targets of drug and issues concerning DST.
The residues Cys138, Asp8, Lys96 and Asp49, His51, His57, and His71 are present in the active and metal binding sites [30, 31] of the pncA-encoded enzyme pyrazinamidase (PZase). We identified mutations dispersed throughout the pncA gene (35A > C---538G > T) nearby the area of metal binding and active site amino acids (46–76 and 133–146). We detected mutations that are important for enzyme catalysis and metal binding (Table 2). However, we did not detect any mutations in the 18 PZA-resistant MTB isolates, suggesting the involvement of other genes RpsA and PanD (aspartate decarboxylase) [32, 33]. A potential new target of PZA, the clpC1 (Unfoldase) gene, which encodes a family of ATPases, was identified in PZA-resistance isolates in addition to the previously identified genes pncA, rpsA and panD . However, the role of these genes (rpsA, panD and clpC1) in PZA resistance is small compared to that of PncA. In a more recent study four new efflux proteins Rv0191, Rv1667c, Rv3756c and Rv3008 were implicated in PZA/POA resistance . These findings suggest a new mechanism for PZA resistance in MTB. Further investigations are needed to identify the quantitative role of all these targets and mechanisms in PZA-resistant MTB for better management of drug-resistant TB.
In conclusion, considering phenotype as a reference, among the 69 PZA resistant isolates, 51 (74%) showed mutations with sensitivity of 79.31% (95% CI, 69.29 to 87.25%) and specificity of 86.67% (95% CI, 69.28 to 96.24%). The mutations 33C > A, 53C > A, 194_203 Del CCTCGTCGTG, 205C > A, 317–18 Del TC, 331G > T, 376G > A, 419G > A, 430G > A, 449G > C, 508G > C, 519G > A, 522G > A, 530DEL C and 535A > G were not found in the GMTV and TBDRM databases and neither in previous studies suggesting the need of some more studies from distinct geographical regions must be carried out for some novel mutations confined to that specific areas. Majority of mutations were of high confidence intervals and uncharacterized category in resistance. Molecular methods to investigate PZA resistance by screening mutations in pncA gene in distinct epidemiological regions offer a much more rapid alternative compared to that of conventional bacteriology. Mutations in pncA gene are highly linked with resistance to PZA and scattered in the entire coding region of pncA. Further, we found an association between PZA resistance and resistance to other important first line drugs, INH and RIF, which is a major hurdle in the treatment management of MDR TB. This high frequency of pncA mutations from geographically distinct regions recommends the WHO guidelines to empirically use the pyrazinamide in drug-resistant TB should be considered. Further studies with large sample size may strengthen these findings to identify the mutations in PZA-resistant isolates specific to certain geographical areas for the better treatment and development of geographically specific biomarkers.
Drug susceptibility testing
Genome-wide Mycobacterium tuberculosis variation
Provincial tuberculosis control program
Tuberculosis Drug Resistance Mutation Database
Extreme drug resistance
Dutta NK, Mehra S, Didier PJ, Roy CJ, Doyle LA, Alvarez X, et al. Genetic requirements for the survival of tubercle bacilli in primates. J Infect Dis. 2010;201:1743–52.
WHO. Drug-resistant TB: surveillance and response: supplement to global tuberculosis report 2014. 2014;
Cavusoglu C, Durmaz R, Bilgic A, Gunal S. Genotyping of rifampin-resistant Mycobacterium tuberculosis isolates from western Turkey. Ann Saudi Med. 2004;24:102–5.
Organization WH, others. Global tuberculosis report 2016. 2016; Available from: http://apps.who.int/iris/bitstream/10665/250441/1/9789241565394-eng.pdf
Heifets L, Higgins M, Simon B. Pyrazinamide is not active against Mycobacterium tuberculosis residing in cultured human monocyte-derived macrophages [unresolved issues]. Int J Tuberc Lung Dis. 2000;4:491–5.
Hoffner S, Ängeby K, Sturegård E, Jönsson B, Johansson A, Sellin M, et al. Proficiency of drug susceptibility testing of Mycobacterium tuberculosis against pyrazinamide: the Swedish experience. Int J Tuberc Lung Dis. 2013;17:1486–90.
Simons SO, van der Laan T, Mulder A, van Ingen J, Rigouts L, Dekhuijzen PNR, et al. Rapid diagnosis of pyrazinamide-resistant multidrug-resistant tuberculosis using a molecular-based diagnostic algorithm. Clin Microbiol Infect. 2014;20:1015–20.
Zhang Y, Mitchison D, Int J. The curious characteristics of pyrazinamide: a review. Tuberc. 2003;7(1):6–21.
Miotto P, Cabibbe AM, Feuerriegel S, Casali N, Drobniewski F, Rodionova Y, et al. Mycobacterium tuberculosis pyrazinamide resistance determinants: a multicenter study. MBio. 2014;5:e01819–4.
Boshoff HI, Mizrahi V, Barry CE. Effects of pyrazinamide on fatty acid synthesis by whole mycobacterial cells and purified fatty acid synthase I. J Bacteriol. 2002;184:2167–72.
Scorpio A, Zhang Y. Mutations in pncA, a gene encoding pyrazinamidase/nicotinamidase, cause resistance to the antituberculous drug pyrazinamide in tubercle bacillus. Nat Med. 1996;2:662–7.
Sengstake S, Bergval IL, Schuitema AR, de Beer JL, Phelan J, de Zwaan R, et al. Pyrazinamide resistance-conferring mutations in pncA and the transmission of multidrug resistant TB in Georgia. BMC Infect Dis [Internet]. 2017;17. Available from: https://0-www-ncbi-nlm-nih-gov.brum.beds.ac.uk/pmc/articles/PMC5506614/
Kubica GP, Dye WE, Cohn ML, Middlebrook G. Sputum digestion and decontamination with N-acetyl-L-cysteine-sodium hydroxide for culture of mycobacteria. Am Rev Respir Dis. 1963;87:775–9.
Siddiqi SH, Gerdes SR. Foundation for Innovative new Diagnostics (FIND) MGITTM procedure manual for BACTEC™ MGIT 960™ TB system. Switzerland; 2006.
WHO | Policy guidance on drug-susceptibility testing (DST) of second-line antituberculosis drugs. WHO. World Health Organization; 2014;
Buck GE, O’Hara LC, Summersgill JT. Rapid, simple method for treating clinical specimens containing Mycobacterium tuberculosis to remove DNA for polymerase chain reaction. J Clin Microbiol. 1992;30:1331–4.
Kirschner P, Springer B, Vogel U, Meier A, Wrede A, Kiekenbeck M, et al. Genotypic identification of mycobacteria by nucleic acid sequence determination: report of a 2-year experience in a clinical laboratory. J Clin Microbiol. 1993;31:2882–9.
Xia Q, Zhao L, Li F, Fan Y, Chen Y, Wu B, et al. Phenotypic and genotypic characterization of pyrazinamide resistance among multidrug-resistant Mycobacterium tuberculosis isolates in Zhejiang, China. Antimicrob Agents Chemother. 2015;59:1690–5.
Dong C, Yu B. Mutation surveyor: an in silico tool for sequencing analysis. Methods Mol Biol Clifton NJ. 2011;760:223–37.
Chernyaeva EN, Shulgina MV, Rotkevich MS, Dobrynin PV, Simonov SA, Shitikov EA, et al. Genome-wide Mycobacterium tuberculosis variation (GMTV) database: a new tool for integrating sequence variations and epidemiology. BMC Genomics. 2014;15:308.
Sandgren A, Strong M, Muthukrishnan P, Weiner BK, Church GM, Murray MB. Tuberculosis Drug Resistance Mutation Database. PLoS Med [Internet]. 2009;6. Available from: https://journals.plos.org/plosmedicine/article?id=10.1371/journal.pmed.1000002
Chang K-C, Leung C-C, Yew W-W, Leung EC-C, Leung W-M, Tam C-M, et al. Pyrazinamide may improve fluoroquinolone-based treatment of multidrug-resistant tuberculosis. Antimicrob Agents Chemother. 2012;56(11):5465–75 https://0-doi-org.brum.beds.ac.uk/10.1128/AAC.01300-12.
Piersimoni C, Mustazzolu A, Giannoni F, Bornigia S, Gherardi G, Fattorini L. Prevention of false resistance results obtained in testing the susceptibility of Mycobacterium tuberculosis to pyrazinamide with the Bactec MGIT 960 system using a reduced inoculum. J Clin Microbiol Am Soc Microbiol. 2013;51:291–4.
Pang Y, Zhu D, Zheng H, Shen J, Hu Y, Liu J, et al. Prevalence and molecular characterization of pyrazinamide resistance among multidrug-resistant Mycobacterium tuberculosis isolates from Southern China. BMC Infect Dis [Internet]. 2017;17. Available from: https://0-www-ncbi-nlm-nih-gov.brum.beds.ac.uk/pmc/articles/PMC5674869/.
Alexander DC, Ma JH, Guthrie JL, Blair J, Chedore P, Jamieson FB. Gene sequencing for routine verification of pyrazinamide resistance in Mycobacterium tuberculosis: a role for pncA but not rpsA. J Clin Microbiol. 2012;50:3726–8.
Tan Y, Hu Z, Zhang T, Cai X, Kuang H, Liu Y, et al. Role of pncA and rpsA gene sequencing in detection of pyrazinamide resistance in Mycobacterium tuberculosis isolates from southern China. J Clin Microbiol. 2014;52:291–7.
Streicher EM, Maharaj K, York T, Van Heerden C, Barnard M, Diacon A, et al. Rapid sequencing of the Mycobacterium tuberculosis pncA gene for detection of pyrazinamide susceptibility. J Clin Microbiol. 2014;52:4056–7.
Whitfield MG, Streicher EM, Dolby T, Simpson JA, Sampson SL, Van Helden PD, et al. Prevalence of pyrazinamide resistance across the spectrum of drug resistant phenotypes of Mycobacterium tuberculosis. Tuberc Edinb. 2016/07/28. 2016;99:128–130.
Xu P, Wu J, Yang C, Luo T, Shen X, Zhang Y, et al. Prevalence and transmission of pyrazinamide resistant Mycobacterium tuberculosis in China. Tuberc Edinb Scotl. 2016;98:56–61.
Petrella S, Gelus-Ziental N, Maudry A, Laurans C, Boudjelloul R, Sougakoff W. Crystal structure of the pyrazinamidase of Mycobacterium tuberculosis: insights into natural and acquired resistance to pyrazinamide. PLoS One. 2011;6:e15785.
Zhang Y, Yew WW. Mechanisms of drug resistance in Mycobacterium tuberculosis [state of the art series. Drug-resistant tuberculosis. Edited by CY. Chiang. Number 1 in the series]. Int J Tuberc Lung Dis. 2009;13:1320–30.
Gu Y, Yu X, Jiang G, Wang X, Ma Y, Li Y, et al. Pyrazinamide resistance among multidrug-resistant tuberculosis clinical isolates in a national referral center of China and its correlations with pncA, rpsA, and panD gene mutations. Diagn Microbiol Infect Dis Elsevier Inc. 2016;84:207–11.
Zhang S, Chen J, Shi W, Liu W, Zhang W, Zhang Y. Mutations in panD encoding aspartate decarboxylase are associated with pyrazinamide resistance in Mycobacterium tuberculosis. Emerg Microbes Infect. 2013;2:e34.
Zhang S, Chen J, Shi W, Cui P, Zhang J, Cho S, et al. Mutation in clpC1 encoding an ATP-dependent ATPase involved in protein degradation is associated with pyrazinamide resistance in Mycobacterium tuberculosis. Emerg Microbes Infect. 2017;6:e8.
Zhang Y, Zhang J, Cui P, Zhang W, Zhang Y. Identification of novel efflux proteins Rv0191, Rv3756c, Rv3008 and Rv1667c involved in pyrazinamide resistance in Mycobacterium tuberculosis. Antimicrob Agents Chemother. 2017;AAC.00940–17.
Rahman A, Ferdous SS, Ahmed S, Rahman SMM, Uddin MKM, Pholwat S, et al. Pyrazinamide susceptibility and pncA mutation profiles of Mycobacterium tuberculosis among multidrug-resistant tuberculosis patients in Bangladesh. Antimicrob Agents Chemother. 2017;61:e00511–7.
Cui Z, Wang J, Lu J, Huang X, Zheng R, Hu Z. Evaluation of methods for testing the susceptibility of clinical Mycobacterium tuberculosis isolates to pyrazinamide. J Clin Microbiol. 2013;51:1374–80.
Kim HJ, Kwak HK, Lee J, Yun YJ, Lee JS, Lee MS, et al. Patterns of pncA mutations in drug-resistant Mycobacterium tuberculosis isolated from patients in South Korea. Int J Tuberc Lung Dis Off J Int Union Tuberc Lung Dis. 2012;16:98–103.
Therese KL, Gayathri R, Balasubramanian S, Natrajan S, Madhavan HN. Phenotypic and genotypic characteristics of drug resistance in Mycobacterium tuberculosis isolates from pediatric population of Chennai, India. Indian J Med Microbiol. 2012;30:411.
The present study was ethically and technically supported by Dr. Sahar Fazal and Dr. Aamer Nadeem, Associate Professors of Bioinformatics and Bioscience, and Dr. Nayyer Masood, Professor, and Muhammad Tanvir Afzal, Associate Professor, Faculty of Computing, Capital University of Science and Technology Islamabad Capital University of Science and Technology Islamabad.
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The present investigation was approved by the Institutional Ethics Committee of CUST Islamabad and Provincial Tuberculosis Reference Laboratory (PTRL) KPK under reference number PTP/PTRL-402/16. Prior to the study, all the participants provided a written informed consent. However, the results were not linked back to individual patients.
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khan, M.T., Malik, S.I., Ali, S. et al. Pyrazinamide resistance and mutations in pncA among isolates of Mycobacterium tuberculosis from Khyber Pakhtunkhwa, Pakistan. BMC Infect Dis 19, 116 (2019). https://0-doi-org.brum.beds.ac.uk/10.1186/s12879-019-3764-2