Utility of the REBA MTB-Rifa® assay for rapid detection of rifampicin resistant Mycobacterium tuberculosis

Eunjin Cho, Isdore Chola Shamputa, Hyun-Kyung Kwak, Jiim Lee, Myungsun Lee, Soohee Hwang, Doosoo Jeon, Cheon Tae Kim, Sangnae Cho, Laura E Via, Clifton E Barry 3rd, Jong Seok Lee, Eunjin Cho, Isdore Chola Shamputa, Hyun-Kyung Kwak, Jiim Lee, Myungsun Lee, Soohee Hwang, Doosoo Jeon, Cheon Tae Kim, Sangnae Cho, Laura E Via, Clifton E Barry 3rd, Jong Seok Lee

Abstract

Background: Drug-resistant tuberculosis (TB), including resistance to both rifampicin (RIF) and isoniazid (INH) referred to as multidrug-resistant tuberculosis (MDR-TB), has become an increasing global threat in recent years. Effective management of patients infected with MDR-TB strains requires identifying such patients by performing conventional drug-susceptibility testing (DST) on bacteria isolated from sputum, a process that can take up to 2 months. This delay in diagnosis can result in worsening and continued transmission of MDR-TB. Molecular methods that rely upon nucleic acid amplification of specific alleles known to be associated with resistance to specific drugs have been helpful in shortening the time to detect drug resistant TB.

Methods: We investigated the utility of the REBA MTB-Rifa®, a commercially available line probe assay (LPA) for detecting rifampicin (RIF) resistance in the RIF resistance-determining region (RRDR) of the rpoB gene. Altogether, 492 Mycobacterium tuberculosis (M. tuberculosis) clinical isolates and additional 228 smear- and culture-positive sputum samples with confirmed M. tuberculosis were collected from subjects with suspected MDR-TB in South Korea. The results were compared with conventional phenotypic DST and sequencing of the rpoB gene.

Results: A total of 215 of the 492 isolates were resistant to RIF by conventional DST, and of which 92.1% (198/215) were MDR-TB strains. The REBA MTB-Rifa® assay identified RIF resistance in 98.1% (211/215) of these isolates but failed to identify resistance in four phenotypically RIF resistant isolates. These four isolates lacked mutations in the RRDR but three were confirmed to be MDR-TB strains by sequencing. The sensitivity and specificity of this test for clinical isolates was thus 98.1% (211/215) and 100% (277/277), respectively. When applied directly to 228 smear positive sputum samples, the sensitivity and the specificity of REBA MTB-Rifa® assay was 100% (96/96, 132/132), respectively.

Conclusions: These findings support the use of the REBA MTB-Rifa® assay for rapid detection of RIF resistance on clinical isolates and smear positive sputum samples. The results also suggest that RIF resistance is a good surrogate marker of MDR-TB in South Korea and the need to add more probes to other LPAs which can cover newly identified mutations relevant to RIF resistance.

Trial registration: ClinicalTrials.gov NCT00341601.

Figures

Figure 1
Figure 1
Schema of the study design. *, Number of samples; RIF, rifampicin; RRDR, rifampicin resistance determining region.
Figure 2
Figure 2
Representation of the RIF binding site region of RpoB and the 10 sites in which mutations were observed in this study. The green dotted lines depict hydrogen bonds anchoring the RIF molecule to RpoB. The numbering corresponds to the numbering of the E. coli protein but the structure corresponds to the RpoB from Thermus aquaticus deposited in PubMed database as 1I6V [32].

References

    1. World Health Organization. The global plan to stop TB 2011–2015: fast facts. Available at: 2010.
    1. Ling DI, Zwerling AA, Pai M. Rapid diagnosis of drug-resistant TB using line probe assays: from evidence to policy. Expert Rev Respir Med. 2008;2:583–588. doi: 10.1586/17476348.2.5.583.
    1. World Health Organization. Molecular line probe assays for rapid screening of patients at risk of multidrug resistant tuberculosis (MDR-TB) Available at: 2008.
    1. Bwanga R, Hoffner S, Haile M, Joloba ML. Direct susceptibility testing for multi drug resistant tuberculosis: a meta-analysis. BMC Infect Dis. 2009;20:67.
    1. Boehme CC, Nabeta P, Hillemann D, Nicol MP, Shenai S, Krapp F, Allen J, Tahirli R, Blakemore R, Rustomjee R, Milovic A, Jones M, O’Brien SM, Persing DH, Ruesch-Gerdes S, Gotuzzo E, Rodrigues C, Alland D, Perkins MD. Rapid molecular detection of tuberculosis and rifampin resistance. N Engl J Med. 2010;363:1005–1015. doi: 10.1056/NEJMoa0907847.
    1. Blakemore R, Story E, Helb D, Kop J, Banada P, Owens MR, Chakravorty S, Jones M, Alland D. Evaluation of the analytical performance of the GeneXpert MTB/RIF assay. J Clin Microbiol. 2010;48:2495–2501. doi: 10.1128/JCM.00128-10.
    1. Hillemann D, Rusch-Gerdes S, Richter E. Evaluation of the GenoType MTBDRplus assay for rifampin and isoniazid susceptibility testing of mycobacterium tuberculosis strains and clinical specimens. J Clin Microbiol. 2007;45:2635–2640. doi: 10.1128/JCM.00521-07.
    1. Ramaswamy S, Musser JM. Molecular genetic basis of antimicrobial agent resistance in mycobacterium tuberculosis: 1998 update. Tuber Lung Dis. 1998;79:3–29. doi: 10.1054/tuld.1998.0002.
    1. Akpaka PE, Baboolal S, Clarke D, Francis L, Rastogi N. Evaluation of methods for rapid detection of resistance to isoniazid and rifampin in mycobacterium tuberculosis isolates collected in the Caribbean. J Clin Microbiol. 2008;46:3426–3428. doi: 10.1128/JCM.01455-08.
    1. Alcaide F, Coll P. Advance in rapid diagnosis of tuberculosis disease and anti-tuberculous drug resistance. Enferm Infecc Microbiol Clin. 2011;29:34–40.
    1. Simon AW, Wilson SM, Yates MD, Drobniewski FA. Comparison of three molecular assays for rapid detection of rifampin resistance in mycobacterium tuberculosis. J Clin Microbiol. 1998;36:1969–1973.
    1. Traore H, Fissette K, Bastian I, Devleeschouwer M, Portaels F. 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. 2000;4:481–484.
    1. Lawn SD, Mwaba P, Bates M, Piatek A, Alexander H, Marais BJ, Cuevas LE, McHugh TD, Zijenah L, Kapata N, Abubakar I, McNerney R, Hoelscher M, Memish ZA, Migliori GB, Kim P, Maeurer M, Schito M, Zumla A. Advances in tuberculosis diagnostics: the Xpert MTB/RIF assay and future prospects for a point-of-care test. Lancet Infect Dis. 2013;13:349–361. doi: 10.1016/S1473-3099(13)70008-2.
    1. Jeon CY, Hwang SH, Min JH, Prevots DR, Goldfeder LC, Lee H, Eum SY, Jeon DS, Kang HS, Kim JH, Kim BJ, Kim DY, Holland SM, Park SK, Cho SN, Barry CE 3rd, Via LE. Extensively drug-resistant tuberculosis in South Korea: risk factors and treatment outcomes among patients at a tertiary referral hospital. Clin Infect Dis. 2008;46:42–49. doi: 10.1086/524017.
    1. World Health Organization. WHO report 2012: global tuberculosis control WHO/HTM.TB/2011.16. Geneva, Switzerland: WHO; 2011.
    1. Wayne LG. Simple pyrazinamidase and urease tests for routine identification of mycobacteria. Am Rev Respir Dis. 1974;109:147–151.
    1. Franzblau SG, Witzig RS, McLaughlin JC, Torres P, Madico G, Hernandez A, Degnan MT, Cook MB, Quenzer VK, Ferguson RM, Gilman RH. Rapid, Low-technology MIC determination with clinical mycobacterium tuberculosis isolates by using the microplate Alamar blue assay. J Clin Microbiol. 1998;36(2):362–366.
    1. Bang H, Park S, Hwang J, Jin H, Cho E, Kim DY, Song T, Shamputa IC, Via LE, Barry CE 3rd, Cho SN, Lee H. Improved rapid molecular diagnosis of multidrug-resistant tuberculosis using a new reverse hybridization assay, REBA MTB-MDR. J Med Microbiol. 2011;60:1447–1454. doi: 10.1099/jmm.0.032292-0.
    1. Heep M, Rieger U, Beck D, Lehn N. Mutations in the beginning of the rpoB gene can induce resistance to rifamycins in both helicobacter pylori and mycobacterium tuberculosis. Antimicrob Agents Chemother. 2000;44:1075–1077. doi: 10.1128/AAC.44.4.1075-1077.2000.
    1. Heep M, Brandstatter B, Rieger U, Lehn N, Richter E, Rusch-Gerdes S, Niemann S. Frequency of rpoB mutations inside and outside the cluster I region in rifampin-resistant clinical mycobacterium tuberculosis isolates. J Clin Microbiol. 2001;39:107–110. doi: 10.1128/JCM.39.1.107-110.2001.
    1. Cavusoglu C, Turhan A, Akinci P, Soyler I. Evaluation of the Genotype MTBDR assay for rapid detection of rifampin and isoniazid resistance in Mycobacterium tuberculosis isolates. J Clin Microbiol. 2006;44:2338–2342. doi: 10.1128/JCM.00425-06.
    1. Huang WL, Chen HY, Kuo YM, Jou R. Performance assessment of the GenoType MTBDRplus test and DNA sequencing in detection of multidrug-resistant mycobacterium tuberculosis. J Clin Microbiol. 2009;47:2520–2524. doi: 10.1128/JCM.02499-08.
    1. Telenti A, Imboden P, Marchesi F, Schmidheini T, Bodmer T. Direct, automated detection of rifampin-resistant mycobacterium tuberculosis by polymerase chain reaction and single-strand conformation polymorphism analysis. Antimicrob Agents Chemother. 1993;37:2054–2058. doi: 10.1128/AAC.37.10.2054.
    1. Hirano K, Abe C, Takahashi M. Mutations in the rpoB gene of rifampin-resistant mycobacterium tuberculosis strains isolated mostly in Asian countries and their rapid detection by line probe assay. J Clin Microbiol. 1999;37:2663–2666.
    1. Ozkutuk N, Gazi H, Surucuoglu S, Gunduz A, Ozbakkaloglu B. Characterization of rpoB mutations by line probe assay in rifampicin-resistant Mycobacterium tuberculosis clinical isolates from the aegean region in Turkey. Jpn J Infect Dis. 2007;60:211–213.
    1. Hillemann D, Kubica T, Rusch-Gerdes S, Niemann S. Disequilibrium in distribution of resistance mutations among mycobacterium tuberculosis Beijing and non-Beijing strains isolated from patients in Germany. Antimicrob Agents Chemother. 2005;49:1229–1231. doi: 10.1128/AAC.49.3.1229-1231.2005.
    1. Qian L, Abe C, Lin TP, Yu MC, Cho SN, Wang S, Douglas JT. RpoB genotypes of mycobacterium tuberculosis Beijing family isolates from east Asian countries. J Clin Microbiol. 2002;40:1091–1094. doi: 10.1128/JCM.40.3.1091-1094.2002.
    1. Paluch-Oles J, Koziol-Montewka M, Magrys A. Mutations in the rpoB gene of rifampin-resistant mycobacterium tuberculosis isolates from eastern Poland. New Microbiol. 2009;32:147–152.
    1. Dooley KE, Lahlou O, Ghali I, Knudsen J, Elmessaoudi MD, Cherkaoui I, Aouad RE. Risk factors for tuberculosis treatment failure, default, or relapse and outcomes of retreatment in Morocco. BMC Public Health. 2011;11:140–147. doi: 10.1186/1471-2458-11-140.
    1. Nettleman MD. Multidrug-resistant tuberculosis: news from the front. JAMA. 2005;293:2788–2790. doi: 10.1001/jama.293.22.2788.
    1. Temple B, Ayakaka I, Ogwang S, Nabanjja H, Kayes S, Nakubulwa S, Worodria W, Levin J, Joloba M, Okwera A, Eisenach KD, McNerney R, Elliott AM, Smith PG, Mugerwa RD, Ellner JJ, Jones-Lopez EC. Rate and amplification of drug resistance among previously-treated patients with tuberculosis in Kampala, Uganda. Clin Infect Dis. 2008;47:1126–1134. doi: 10.1086/592252.
    1. Campbell EA, Korzheva N, Mustaev A, Murakami K, Nair S, Goldfarb A, Darst SA. Structural mechanism for rifampicin inhibition of bacterial RNA polymerase. Cell. 2001;104:901–912. doi: 10.1016/S0092-8674(01)00286-0.

Source: PubMed

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