Background: Pyrazinamide (PZA) is one of the most important drugs for tuberculosis (TB) treatment, however, its susceptibility is not routinely tested. High-resolution melting (HRM) curve analysis has been widely used for many applications. In this study, HRM assay was developed and evaluated for the detection of PZA resistance in Mycobacteriumtuberculosis clinical isolates. Methods: Ninety five M. tuberculosis clinical isolates with different susceptibility patterns to anti-TB drugs were used to evaluate this assay. Isolates were phenotypically (Bactec MGIT 960) and genotypically (HRM and pncA gene sequencing) analysed for PZA resistance. Results: Bactec MGIT 960 analysis revealed that 29 of the 95 M. tuberculosis isolates were PZA resistant. In comparison to the Bactec MGIT 960, HRM showed a sensitivity of 47.7% and specificity of 74.6%, and the overall agreement between the two methods was 68.4%. Based on DNA sequencing, a correlation of 0.67 (significant at p-value < 0.05) between phenotypic resistance to PZA and pncA mutations was observed. PZA resistance was strongly associated with multi-drug resistant (MDR)-TB as it was shown in 79.3% of the MDR isolates included in the study. Conclusion: HRM is simple and useful for screening clinical M. tuberculosis isolates for PZA resistance, however, further modifications to improve its performance are required.
References
[1]
Ahmady, A., Poolad, T., Rafee, P., Tousheh, M., Kahbazi, M. and Arjomandzadegan, M. (2013) Study of Pyrazinamidase Structural Changes in Pyrazinamide Resistant and Susceptible Isolates of Mycobacterium tuberculosis. Tuberk Toraks, 61, 110-114.
http://dx.doi.org/10.5578/tt.3888
[2]
Yee, D.P., Menzies, D. and Brassard, P. (2012) Clinical Outcomes of Pyrazinamide-Mono-resistant Mycobacterium tuberculosis in Quebec. International Journal of Tuberculosis and Lung Disease, 16, 604-609.
[3]
Raynaud, C., Lanéelle, M., Senaratne, R.H., Draper, P., Lanéelle, G. and Daffé, M. (1999) Mechanisms of Pyrazinamide Resistance in Mycobacteria: Importance of Lack of Uptake in Addition to Lack of Pyrazinamidase Activity. Microbiology, 145, 1359-1367.
http://dx.doi.org/10.1099/13500872-145-6-1359
[4]
Sheen, P., Lozano, K., Gilman, R.H., Valencia, H.J., Loli, S., Fuentes, P., et al. (2013) pncA Gene Expression and Prediction Factors on Pyrazinamide Resistance in Mycobacterium tuberculosis. Tuberculosis, 93, 515-522. http://dx.doi.org/10.1016/j.tube.2013.03.005
[5]
Zimic, M., Loli, S., Gilman, R.H., Gutierrez, A., Fuentes, P., Cotrina, M., et al. (2012) A New Approach for Pyrazinamide Susceptibility Testing in Mycobacterium tuberculosis. Microbial Drug Resistance, 4, 372-375. http://dx.doi.org/10.1089/mdr.2011.0207
[6]
Ajbani, K., Nikam, C., Kazi, M., Gray, C., Boehme, C., Balan, K., et al. (2012) Evaluation of Genotype MTBDRsl Assay to Detect Drug Resistance Associated with Fluoroquinolones, Aminoglycosides and Ethambutol on Clinical Sediments. PLoS One, 7, e49433.
http://dx.doi.org/10.1371/journal.pone.0049433
[7]
Jureen, P., Werngren, J. and Hoffner, S.E. (2004) Evaluation of the Line Probe Assay (LiPA) for Rapid Detection of Rifampicin Resistance in Mycobacterium tuberculosis. Tuberculosis, 84, 311-316. http://dx.doi.org/10.1016/j.tube.2003.12.001
[8]
Morlock, G.P., Crawford, J.T., Butler, W.R., Brim, S.E., Sikes, D., Mazurek, G.H., et al. (2000) Phenotypic Characterization of pncA Mutants of Mycobacterium tuberculosis. Antimicrobial Agents and Chemotherapy, 44, 2291-2295.
http://dx.doi.org/10.1128/AAC.44.9.2291-2295.2000
[9]
Sreevatsan, S., Pan, X., Zhang, Y., Kreiswirth, B.N. and Musser, J.M. (1997) Mutations Associated with Pyrazinamide Resistance in pncA of Mycobacterium tuberculosis Complex Organisms. Antimicrobial Agents and Chemotherapy, 41, 636-640.
[10]
Scorpio, A., Lindholm-Levy, P., Heifets, L., Gilman, R., Siddiqi, S., Cynamon, M., et al. (1997) Characterization of pncA Mutations in Pyrazinamide-Resistant Mycobacterium tuberculosis. Antimicrobial Agents and Chemotherapy, 41, 540-543.
[11]
Slany, M., Vanerkova, M., Nemcova, E., Zaloudikova, B., Ruzicka, F. and Freiberger, T. (2010) Differentiation of Staphylococcus spp. by High-Resolution Melting Analysis. Canadian Journal of Microbiology, 56, 1040-1049. http://dx.doi.org/10.1139/W10-091
[12]
Merchant-Patel, S., Blackall, P.J., Templeton, J., Price, E.P., Tong, S.Y., Huygens, F., et al. (2010) Campylobacter jejuni and Campylobacter coli Genotyping by High-Resolution Melting Analysis of a flaA Fragment. Applied and Environmental Microbiology, 76, 493-499. http://dx.doi.org/10.1128/AEM.01164-09
[13]
Choi, G.E., Lee, S.M., Yi, J., Hwang, S.H., Kim, H.H., Lee, E.Y., et al. (2010) High-Resolution Melting Curve Analysis for Rapid Detection of Rifampin and Isoniazid Resistance in Mycobacterium tuberculosis Clinical Isolates. Journal of Clinical Microbiology, 48, 3893-3898. http://dx.doi.org/10.1128/JCM.00396-10
[14]
Scorpio, A. and Zhang, Y. (1996) Mutations in pncA, a Gene Encoding Pyrazinamidase/ Nicotinamidase, Cause Resistance to the Antituberculous Drug Pyrazinamide in Tubercle Bacillus. Nature Medicine, 2, 662-667. http://dx.doi.org/10.1038/nm0696-662
[15]
Jonmalung, J., Prammananan, T., Leechawengwongs, M. and Chaiprasert, A. (2010) Surveillance of Pyrazinamide Susceptibility among Multidrug-Resistant Mycobacterium tuberculosis Isolates from Siriraj Hospital, Thailand. BMC Microbiology, 10, 223.
http://dx.doi.org/10.1186/1471-2180-10-223
[16]
Minime-Lingoupou, F., Pierre-Audigier, C., Kassa-Kelembho, E., Barilone, N., Zandanga, G., Rauzier, J., et al. (2010) Rapid Identification of Multidrug-Resistant Tuberculosis Isolates in Treatment Failure or Relapse Patients in Bangui, Central African Republic. International Journal of Tuberculosis and Lung Disease, 14, 782-785.
[17]
Louw, G.E., Warren, R.M., Donald, P.R., Murray, M.B., Bosman, M., Van Helden, P.D., et al. (2006) Frequency and Implications of Pyrazinamide Resistance in Managing Previously Treated Tuberculosis Patients. International Journal of Tuberculosis and Lung Disease, 10, 802-807.
[18]
Ando, H., Mitarai, S., Kondo, Y., Suetake, T., Sekiguchi, J.I., Kato, S., et al. (2010) Pyrazinamide Resistance in Multidrug-Resistant Mycobacterium tuberculosis Isolates in Japan. Clinical Microbiology and Infection, 16, 1164-1168.
http://dx.doi.org/10.1111/j.1469-0691.2009.03078.x
[19]
Chiu, Y.C., Huang, S.F., Yu, K.W., Lee, Y.C., Feng, J.Y. and Su, W.J. (2011) Characteristics of pncA Mutations in Multidrug-Resistant Tuberculosis in Taiwan. BMC Infectious Diseases, 11, 240.
[20]
Rao, N.A., Irfan, M., Soomro, M.M. and Mehfooz, Z. (2010) Drug Resistance Pattern in Multidrug Resistance Pulmonary Tuberculosis Patients. Journal of the College of Physicians and Surgeons Pakistan, 20, 262-265.
[21]
Kim, H.J., Kwak, H.K., Lee, J., Yun, Y.J., Lee, J.S., Lee, M.S., et al. (2012) Patterns of pncA Mutations in Drug-Resistant Mycobacterium tuberculosis Isolated from Patients in South Korea. International Journal of Tuberculosis and Lung Disease, 16, 98-103.
http://dx.doi.org/10.5588/ijtld.10.0739
[22]
Shenai, S., Rodrigues, C., Sadani, M., Sukhadia, N. and Mehta, A. (2009) Comparison of Phenotypic and Genotypic Methods for Pyrazinamide Susceptibility Testing. Indian Journal of Tuberculosis, 56, 82-90.
[23]
Zhang, Y., Chiu Chang, K., Leung, C., Wai Yew, W., Gicquel, B., Fallows, D., et al. (2012) “ZS-MDR-TB” versus “ZR-MDR-TB”: Improving Treatment of MDR-TB by Identifying Pyrazinamide Susceptibility. Emerging Microbes & Infections, 1, e5.
http://dx.doi.org/10.1038/emi.2012.18
[24]
Nagai, Y., Iwade, Y., Hayakawa, E., Nakano, M., Sakai, T., Mitarai, S., et al. (2013) High Resolution Melting Curve Assay for Rapid Detection of Drug-Resistant Mycobacterium tuberculosis. Journal of Infection and Chemotherapy, 19, 1116-1125.
http://dx.doi.org/10.1007/s10156-013-0636-3
[25]
Hong, C.Y., Wang, F. and Liu, X.L. (2013) Detection of pncA Mutation Associated with Pyrazinamide Resistance in Mycobacterium tuberculosis by High-Resolution Melting Curve Analysis. Chinese Journal of Tuberculosis and Respiratory Diseases, 36, 198-201.
[26]
Pholwat, S., Stroup, S., Gratz, J., Trangan, V., Foongladda, S., Kumburu, H., et al. (2014) Pyrazinamide Susceptibility Testing of Mycobacterium tuberculosis by High Resolution Melt Analysis. Tuberculosis, 94, 20-25. http://dx.doi.org/10.1016/j.tube.2013.10.006
[27]
Bishop, K.S., Blumberg, L., Trollip, A.P., Smith, A.N., Roux, L., York, D.F., et al. (2001) Characterisation of the pncA Gene in Mycobacterium tuberculosis Isolates from Gauteng, South Africa. International Journal of Tuberculosis and Lung Disease, 5, 952-957.
[28]
Huang, T.S., Lee, S.S., Tu, H.Z., Huang, W.K., Chen, Y.S., Huang, C.K., et al. (2003) Correlation between Pyrazinamide Activity and pncA Mutations in Mycobacterium tuberculosis Isolates in Taiwan. Antimicrobial Agents and Chemotherapy, 47, 3672-3673.
http://dx.doi.org/10.1128/AAC.47.11.3672-3673.2003
[29]
Miyagi, C., Yamane, N., Yogesh, B., Ano, H. and Takashima, T. (2004) Genetic and Phenotypic Characterization of Pyrazinamide-Resistant Mycobacterium tuberculosis Complex Isolates in Japan. Diagnostic Microbiology and Infectious Disease, 48, 111-116.
http://dx.doi.org/10.1016/j.diagmicrobio.2003.09.013
[30]
Rodrigues Vde, F., Telles, M.A., Ribeiro, M.O., Cafrune, P.I., Rossetti, M.L. and Zaha, A. (2005) Characterization of pncA Mutations in Pyrazinamide-Resistant Mycobacterium tuberculosis in Brazil. Antimicrobial Agents and Chemotherapy, 49, 444-446.
http://dx.doi.org/10.1128/AAC.49.1.444-446.2005
[31]
Chan, R.C.Y., Hui, M., Chan, E.W.C., Au, T.K., Chin, M.L., Yip, C.K., et al. (2007) Genetic and Phenotypic Characterization of Drug-Resistant Mycobacterium tuberculosis Isolates in Hong Kong. Journal of Antimicrobial Chemotherapy, 59, 866-873.
http://dx.doi.org/10.1093/jac/dkm054
[32]
Pandey, S., Newton, S., Upton, A., Roberts, S. and Drinkovic, D. (2009) Characterisation of pncA Mutations in Clinical Mycobacterium tuberculosis Isolates in New Zealand. Pathology, 41, 582-584. http://dx.doi.org/10.1080/00313020903071587
[33]
Hou, L., Osei-Hyiaman, D., Zhang, Z., Wang, B., Yang, A. and Kano, K. (2000) Molecular Characterization of pncA Gene Mutations in Mycobacterium tuberculosis Clinical Isolates from China. Epidemiology and Infection, 124, 227-232.
http://dx.doi.org/10.1017/S0950268899003635
[34]
Mphahlele, M., Syre, H., Valvatne, H., Stavrum, R., Mannsåker, T., Muthivhi, T., et al. (2008) Pyrazinamide Resistance among South African Multidrug-Resistant Mycobacterium tuberculosis Isolates. Journal of Clinical Microbiology, 46, 3459-3464.
http://dx.doi.org/10.1128/JCM.00973-08
[35]
Hirano, K., Takahashi, M., Kazumi, Y., Fukasawa, Y. and Abe, C. (1997) Mutation in pncA Is a Major Mechanism of Pyrazinamide Resistance in Mycobacterium tuberculosis. Tubercle and Lung Disease, 78, 117-122. http://dx.doi.org/10.1016/S0962-8479(98)80004-X
[36]
Lee, K.W., Lee, J.M. and Jung, K.S. (2001) Characterization of pncA Mutations of Pyrazinamide-Resistant Mycobacterium tuberculosis in Korea. Journal of Korean Medical Science, 16, 537-543. http://dx.doi.org/10.3346/jkms.2001.16.5.537
[37]
Doustdar, F., Khosravi, A.D. and Farnia, P. (2009) Mycobacterium tuberculosis Genotypic Diversity in Pyrazinamide-Resistant Isolates of Iran. Microbial Drug Resistance, 15, 251-256. http://dx.doi.org/10.1089/mdr.2009.0066
[38]
Hillemann, D., Rusch-Gerdes, S. and Richter, E. (2007) Evaluation of the GenoType MTBDRplus Assay for Rifampin and Isoniazid Susceptibility Testing of Mycobacterium tuberculosis Strains and Clinical Specimens. Journal of Clinical Microbiology, 45, 2635-2640. http://dx.doi.org/10.1128/JCM.00521-07
[39]
Shi, W., Zhang, X., Jiang, X., Yuan, H., Lee, J.S., Barry, C.E., et al. (2011) Pyrazinamide Inhibits Trans-Translation in Mycobacterium tuberculosis. Science, 333, 1630-1632.
http://dx.doi.org/10.1126/science.1208813
[40]
Tan, Y., Hu, Z., Zhang, T., Cai, X., Kuang, H., Liu, Y., et al. (2014) Role of pncA and rpsA Gene Sequencing in Detection of Pyrazinamide Resistance in Mycobacterium tuberculosis Isolates from Southern China. Journal of Clinical Microbiology, 52, 291-297.
http://dx.doi.org/10.1128/JCM.01903-13
[41]
Bhuju, S., Fonseca, L.D., Marsico, A.G., de Oliveira Vieira, G.B., Sobral, L.F., Stehr, M., et al. (2013) Mycobacterium tuberculosis Isolates from Rio de Janeiro Reveal Unusually Low Correlation between Pyrazinamide Resistance and Mutations in the pncA Gene. Infection Genetics and Evolution, 19, 1-6. http://dx.doi.org/10.1016/j.meegid.2013.06.008
[42]
Watcharasamphankul, W., Houpt, E.R. and Foongladda, S. (2013) Rapid Detection of Pyrazinamide Resistant Mycobacterium tuberculosis by High Resolution Melting Curve Analysis. Journal of the Medical Association of Thailand, 96, 1218-1223.
[43]
Zhang, Y., Permar, S. and Sun, Z. (2002) Conditions That May Affect the Results of Susceptibility Testing of Mycobacterium tuberculosis to Pyrazinamide. Journal of Medical Microbiology, 51, 42-49. http://dx.doi.org/10.1099/0022-1317-51-1-42
