Correlation between Genotypic and Phenotypic Testing for Resistance to Rifampin in Mycobacterium tuberculosis Clinical Isolates in Haiti: Investigation of Cases with Discrepant Susceptibility Results
The World Health Organization has recommended use of molecular-based tests MTBDRplus and GeneXpert MTB/RIF to diagnose multidrug-resistant tuberculosis in developing and high-burden countries. Both tests are based on detection of mutations in the Rifampin (RIF) Resistance-Determining Region of DNA-dependent RNA Polymerase gene (rpoB). Such mutations are found in 95–98% of Mycobacterium tuberculosis strains determined to be RIF-resistant by the “gold standard” culture-based drug susceptibility testing (DST). We report the phenotypic and genotypic characterization of 153 consecutive clinical Mycobacterium tuberculosis strains diagnosed as RIF-resistant by molecular tests in our laboratory in Port-au-Prince, Haiti. 133 isolates (86.9%) were resistant to both RIF and Isoniazid and 4 isolates (2.6%) were RIF mono-resistant in MGIT SIRE liquid culture-based DST. However the remaining 16 isolates (10.5%) tested RIF-sensitive by the assay. Five strains with discordant genotypic and phenotypic susceptibility results had RIF minimal inhibitory concentration (MIC) close to the cut-off value of 1 μg/ml used in phenotypic susceptibility assays and were confirmed as resistant by DST on solid media. Nine strains had sub-critical RIF MICs ranging from 0.063 to 0.5 μg/ml. Finally two strains were pan-susceptible and harbored a silent rpoB mutation. Our data indicate that not only detection of the presence but also identification of the nature of rpoB mutation is needed to accurately diagnose resistance to RIF in Mycobacterium tuberculosis. Observed clinical significance of low-level resistance to RIF supports the re-evaluation of the present critical concentration of the drug used in culture-based DST assays.
References
[1]
Molecular line probe assays for rapid screening of patients at risk of multidrug-resistant tuberculosis (MDR-TB): Policy Statement. Geneva, World Health Organization, 27 June 2008; Available: http://www.who.int/tb/features_archive/p？olicy_statement.pdf. Accessed7 February 2014.
[2]
Rapid implementation of the Xpert MTB/RIF diagnostic test. WHO/HTM/TB/2011.2. Geneva, World Health Organization, 2011. Available: http://whqlibdoc.who.int/publications/20？11/9789241501569_eng.pdf. Accessed 7 February 2014.
[3]
Ramaswamy S, Musser JM (1998) Molecular genetic basis of antimicrobial agent resistance in Mycobacterium tuberculosis: 1998 update. Tuber Lung Dis. 79(1): 3–29. doi: 10.1054/tuld.1998.0002
[4]
Telenti A, Imboden P, Marchesi F, Lowrie D, Cole S, et al. (1993) Detection of rifampicin-resistance mutations in Mycobacterium tuberculosis. Lancet. 341: 647–650. doi: 10.1016/0140-6736(93)90417-f
[5]
Guidelines for surveillance of drug resistance in tuberculosis. Geneva, World Health Organization, 1994, (document WHO/TB/94.178). Available: http://who.int/tb/publications/1994/en/i？ndex.html. Accessed7 February 2014.
[6]
Traore H, Fissette K, Bastian I, Devleeschouwer M, Portaels F (2000) Detection of rifampicin resistance in Mycobacterium tuberculosis isolates from diverse countries by a commercial line probe assay as an initial indicator of multidrug resistance. Int J Tuberc Lung Dis. 4(5): 481–4.
[7]
World Health Organization. Global tuberculosis control: 2011. Geneva: WHO; Available from: http://www.who.int/tb/publications/globa？l_report/2011/gtbr11_ full.pdf. Accessed7 February 2014.
[8]
Ocheretina O, Morose W, Gauthier M, Joseph P, D'Meza R, et al. (2012) Multidrug- resistant tuberculosis in Port-au-Prince,. Haiti. Rev. Panam. Salud Publica. 31: 221–224. doi: 10.1590/s1020-49892012000300006
[9]
Boehme CC, Nabeta P, Hillemann D, Nicol MP, Shenai S, et al. (2010) Rapid molecular detection of tuberculosis and rifampin resistance. N Engl J Med. 363(11): 1005–15. doi: 10.1056/nejmoa0907847
[10]
Lawn SD, Brooks SV, Kranzer K, Nicol MP, Whitelaw A, et al. (2011) Screening for HIV-associated tuberculosis and rifampicin resistance before antiretroviral therapy using the Xpert MTB/RIF assay: a prospective study. PLoS Med. 8(7): e1001067. doi: 10.1371/journal.pmed.1001067
[11]
Ocheretina O, Merveille YM, Mabou MM, Escuyer VE, Dunbar SA, et al. (2013) Use of Luminex MagPlex Magnetic Microspheres for High-Throughput Spoligotyping of Mycobacterium tuberculosis Isolates in Port-au-Prince, Haiti. J Clin Microbiol. 51(7): 2232–7. doi: 10.1128/jcm.00268-13
[12]
Kamerbeek J, Schouls L, Kolk A, van Agterveld M, van Soolingen D, et al. (1997) Simultaneous detection and strain differentiation of Mycobacterium tuberculosis for diagnosis and epidemiology. J Clin Microbiol. 35(4): 907–914.
[13]
Kent PT, Kubica GP. (1985) Public health mycobacteriology: a guide for the level III laboratory. U.S. Department of Health and Human Services, Centers for Disease Control and Prevention, Atlanta, USA.
[14]
National Committee on Clinical Laboratory Standards. Susceptibility testing of Mycobacteria, Nocardiae, and other aerobic Actinomycetes. Approved standard. NCCLS document M24-A. Wayne, Pennsylvania: NCCLS; 2003.
[15]
Franzblau SG, Witzig RS, McLaughlin JC, Torres P, Madico G, et al. (1998) Rapid, low-technology MIC determination with clinical Mycobacterium tuberculosis isolates by using the microplate Alamar Blue assay. J Clin Microbiol. 36(2): 362–6.
[16]
Van Deun A, Barrera L, Bastian I, Fattorini L, Hoffmann H, et al. (2009) Mycobacterium tuberculosis strains with highly discordant rifampin susceptibility test results. J Clin Microbiol. 47(11): 3501–6. doi: 10.1128/jcm.01209-09
[17]
Boehme CC, Nicol MP, Nabeta P, Michael JS, Gotuzzo E, et al. (2011) Feasibility, diagnostic accuracy, and effectiveness of decentralized use of the Xpert MTB/RIF test for diagnosis of tuberculosis and multidrug resistance: a multicentre implementation study. Lancet. 377(9776): 1495–505. doi: 10.1016/s0140-6736(11)60438-8
[18]
Huang WL, Chen HY, Kuo YM, Jou R (2009) Performance assessment of the GenoType MTBDRplus test and DNA sequencing in detection of multidrug-resistant Mycobacterium tuberculosis. J Clin Microbiol. 47(8): 2520–4. doi: 10.1128/jcm.02499-08
[19]
Theron G, Peter J, van Zyl-Smit R, Mishra H, Streicher E, et al. (2011) Evaluation of the Xpert MTB/RIF assay for the diagnosis of pulmonary tuberculosis in a high HIV prevalence setting. Am J Respir Crit Care Med. 184(1): 132–40. doi: 10.1164/rccm.201101-0056oc
[20]
Marlowe EM, Novak-Weekley SM, Cumpio J, Sharp SE, Momeny MA, et al. (2011) Evaluation of the Cepheid Xpert MTB/RIF assay for direct detection of Mycobacterium tuberculosis complex in respiratory specimens. J Clin Microbiol. 49(4): 1621–3. doi: 10.1128/jcm.02214-10
[21]
Van Rie A, Mellet K, John MA, Scott L, Page-Shipp L, et al. (2012) False-positive rifampicin resistance on Xpert？ MTB/RIF: case report and clinical implications. Int J Tuberc Lung Dis. 16(2): 206–8. doi: 10.5588/ijtld.11.0395
[22]
Yang B, Koga H, Ohno H, Ogawa K, Fukuda M, et al. (1998) Relationship between antimycobacterial activities of rifampicin, rifabutin and KRM-1648 and rpoB mutations of Mycobacterium tuberculosis. J Antimicrob Chemother. 42(5): 621–8. doi: 10.1093/jac/42.5.621
[23]
Campbell EA, Pavlova O, Zenkin N, Leon F, Irschik H, et al. (2005) Structural, functional, and genetic analysis of sorangicin inhibition of bacterial RNA polymerase. EMBO J. 24(4): 674–82. doi: 10.1038/sj.emboj.7600499
[24]
Angra PK, Taylor TH, Iademarco MF, Metchock B, Astles JR, et al. (2012) Performance of Tuberculosis Drug Susceptibility Testing in the United States Laboratories from 1994-2008. J Clin Microbiol. 50(4): 1233–9. doi: 10.1128/jcm.06479-11
[25]
Tenover FC, Crawford JT, Huebner RE, Geiter LJ, Horsburgh CR Jr, et al. (1993) The resurgence of tuberculosis: is your laboratory ready? J Clin Microbiol. 31(4): 767–70.
[26]
Williamson DA, Roberts SA, Bower JE, Vaughan R, Newton S, et al. (2011) Clinical failures associated with rpoB mutations in phenotypically occult multidrug-resistant Mycobacterium tuberculosis. Int J Tuberc Lung Dis. 16(2): 216–20. doi: 10.5588/ijtld.11.0178
[27]
Comas I, Borrell S, Roetzer A, Rose G, Malla B, et al. (2011) Whole-genome sequencing of rifampicin-resistant Mycobacterium tuberculosis strains identifies compensatory mutations in RNA polymerase genes. Nat Genet. 44(1): 106–10. doi: 10.1038/ng.1038
[28]
Kim BJ, Kim SY, Park BH, Lyu MA, Park IK, et al. (1997) Mutations in the rpoB gene of Mycobacterium tuberculosis that interfere with PCR-single-strand conformation polymorphism analysis for rifampin susceptibility testing. J Clin Microbiol. 35(2): 492–4.
[29]
Yuan X, Zhang T, Kawakami K, Zhu J, Li H, et al. (2012) Molecular characterization of multidrug- and extensively drug-resistant Mycobacterium tuberculosis strains in Jiangxi, China. J Clin Microbiol. 50(7): 2404–13. doi: 10.1128/jcm.06860-11
[30]
Mani C, Selvakumar N, Narayanan S, Narayanan PR (2001) Mutations in the rpoB gene of multidrug-resistant Mycobacterium tuberculosis clinical isolates from India. J Clin Microbiol. 39(8): 2987–90. doi: 10.1128/jcm.39.8.2987-2990.2001
[31]
Williamson DA, Basu I, Bower J, Freeman JT, Henderson G, et al. (2011) An evaluation of the Xpert MTB/RIF assay and detection of false-positive rifampicin resistance in Mycobacterium tuberculosis. Diagn Microbiol Infect Dis. 74(2): 207–9. doi: 10.1016/j.diagmicrobio.2012.06.013
[32]
Kapur V, Li LL, Iordanescu S, Hamrick MR, Wanger A, et al. (1994) Characterization by automated DNA sequencing of mutations in the gene (rpoB) encoding the RNA polymerase beta subunit in rifampin-resistant Mycobacterium tuberculosis strains from New York City and Texas. J Clin Microbiol. 32(4): 1095–8.
[33]
Alonso M, Palacios JJ, Herranz M, Penedo A, Menéndez A, et al. (2011) Isolation of Mycobacterium tuberculosis strains with a silent mutation in rpoB leading to potential misassignment of resistance category. J Clin Microbiol. 49(7): 2688–90. doi: 10.1128/jcm.00659-11
[34]
Moure R, Martín R, Alcaide F (2011) Silent mutation in rpoB detected from clinical samples with rifampin-susceptible Mycobacterium tuberculosis. J Clin Microbiol. 49(10): 3722. doi: 10.1128/jcm.05314-11
[35]
Scott LE, McCarthy K, Gous N, Nduna M, Van Rie A, et al. (2011) Comparison of Xpert MTB/RIF with other nucleic acid technologies for diagnosing pulmonary tuberculosis in a high HIV prevalence setting: a prospective study. PLoS Med. 8(7): e1001061. doi: 10.1371/journal.pmed.1001061
[36]
Ling DI, Zwerling AA, Pai M (2008) GenoType MTBDR assays for the diagnosis of multidrug-resistant tuberculosis: a meta-analysis. Eur. Respir. J. 32: 1165–1174. doi: 10.1183/09031936.00061808
Dalhoff A, Ambrose PG, Mouton JW (2009) A long journey from minimum inhibitory concentration testing to clinically predictive breakpoints: deterministic and probabilistic approaches in deriving breakpoints. Infection. 37(4): 296–305. doi: 10.1007/s15010-009-7108-9
[39]
Gumbo T (2010) New susceptibility breakpoints for first-line antituberculosis drugs based on antimicrobial pharmacokinetic/pharmacodynamic science and population pharmacokinetic variability. Antimicrob Agents Chemother. 54(4): 1484–91. doi: 10.1128/aac.01474-09
