==== Front Ann Saudi MedAnn Saudi MedAnnals of Saudi Medicine0256-49470975-4466King Faisal Specialist Hospital and Research Centre 1850018910.5144/0256-4947.2008.203asm-3-203Brief ReportPerformance of the Genotype MTBDR assay for molecular detection of multidrug-resistant strains of Mycobacterium tuberculosis Al-Mutairi Noura Ahmad Suhail Mokaddas Eiman From the Department of Microbiology, Kuwait University, KuwaitCorrespondence and reprints: Suhail Ahmad, Department of Microbiology, Kuwait University, Faculty of Medicine, PO Box 24923, Safat 13110, Kuwait, T: +965-5312-300-6503, F: +965-531-8454, suhail_ah@hsc.edu.kwMay-Jun 2008 28 3 203 206 01 3 2008 Copyright © 2008, Annals of Saudi Medicine2008This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License. ==== Body Tuberculosis (TB) is an infectious disease of global impact, killing nearly two million people every year.1 Efforts to control TB are hampered by expanding human immunodeficiency virus (HIV) infection and its association with active disease and increasing resistance of Mycobacterium tuberculosis strains to most effective (first-line) anti-TB drugs.1 New cases that are resistant to both rifampin (RMP) and isoniazid (INH) (defined as multidrug-resistant TB, MDR-TB) exemplify the problem of drug-resistant TB in a given country since fatality rates for MDR-TB are much higher.2 Rapid identification of MDR-TB strains is crucial for starting of effective chemotherapy and for initiation of infection control measures. The resistance of M tuberculosis strains to anti-TB drugs develops due to mutations in resistance-conferring genes and MDR-TB strains evolve due to sequential accumulation of these mutations.3 Mutations in the 81-base pair (bp) (hot-spot) region of the rpoB gene, encoding the β-subunit of RNA polymerase, occur in 90% to 95% of RMP-resistant strains.3–6 In contrast, mutations in several regions of multiple genes can cause INH resistance.3,7 However, mutations in the katG gene, encoding catalase-peroxidase, occur more frequently and 50% to 95% of INH-resistant strains worldwide contain mutations at the katG codon 315 (katG315).3,7–9 Based on this knowledge, a DNA strip (Genotype MTBDR) assay has been developed for simultaneous detection of resistance of M tuberculosis to RMP and INH.10–12 This PCR-based assay involves hybridization with oligonucleotide probes to detect either wild-type sequences or specific mutations. In this study, Genotype MTBDR assay was evaluated by using 35 MDR and 20 pansusceptible M tuberculosis strains isolated in Kuwait. The results were compared to conventional drug susceptibility testing (DST) performed on each isolate. METHODS Thirty-five MDR (MDR1 to MDR35) and 20 pansusceptible (S1 to S20) M tuberculosis strains isolated in Kuwait were used. The rpoB mutations in some MDRTB strains were previously detected by another strip (INNO-LiPA Rif. TB, Innogenetics, Ghent, Belgium) assay.13 Isolation, identification and DST of M tuberculosis isolates were performed as described previously.14,15 The isolates were defined as MDR-TB strains when bacterial growth occurred in the presence of 2 mg/L RMP and 0.1 mg/L INH, added separately. Resistance to ethambutol (EMB, 2.5 mg/L) and streptomycin (SM, 2 mg/L) was also determined (Table 1). The M tuberculosis reference strain H37Rv was used as a control in the Genotype MTBDR assay (Hain Life Sciences, Nehren, Germany). Genomic DNA from M tuberculosis H37Rv and BACTEC cultures of MDR and pansusceptible M tuberculosis isolates was prepared as described previously.16 The Genotype MTBDR assay was performed as recommended by the manufacturer. Briefly, multiplex PCR was performed with extracted DNA and the biotinylated amplicons were hybridized with oligonucleotides immobilized on nitrocellulose strips. Hybridized amplicons were detected by the addition of streptavidin-alkaline phosphatase conjugate followed by color development with a chromogenic substrate. Assay results were interpreted according to manufacturer’s instructions and were confirmed by DNA sequencing. The sequencing of the rpoB hot-spot region and the katG315 DNA region was performed as described previously.9,13 The sensitivity of detecting RMP-resistant, INH-resistant and MDR-TB strains by the Genotype MTBDR assay was calculated using phenotypic DST as the gold standard. RESULTS The multiplex PCR yielded expected amplicons from all 35 MDR and 20 pansusceptible M tuberculosis strains (data not shown). The amplicons were hybridized to probes (organized on membrane strips as shown in Figure 1, lane reference) under controlled conditions. The UC and Tub probes as well as rpoB and katG probes were positive for all pansusceptible and MDR M tuberculosis strains, as expected. All pansusceptible strains (banding patterns for S1 and S5 are shown in Figure 1, lanes 1 and 11) reacted with all five rpoB wild-type (WT1 to WT5) and katG WT probes but not with any rpoB and katG mutant probes, as expected. The 35 MDR-TB strains exhibited 11 different hybridization patterns (Table 1). RMP resistance was detected in 33 of 35 (94%) isolates by lack of reaction with an rpoB WT probe and with 28 of 33 (85%) isolates also reacting with an rpoB MUT (mutant) probe (banding patterns for selected isolates are shown in Figure 1, lanes 2–10). Two isolates reacted with all rpoB WT and none of the rpoB MUT probes (RMP susceptible pattern, Table 1). The majority (21 of 35, 60%) of MDR-TB strains reacted with rpoB MUT3 (S531L) with concomitant lack of hybridization with rpoB WT5 probe (Table 1). DNA sequencing of the rpoB gene confirmed the strip results for 34 of 35 MDR-TB strains. One isolate (MDR13) identified as RMP susceptible by the Genotype MTBDR assay contained an insertion mutation at rpoB codon 514 (insertion 514TTC). As for INH resistance, a mutation at katG315 was detected in 22 of 35 (63%) MDR-TB strains by absence of a signal with a katG WT probe (Table 1) with 21 of 22 isolates also reacting with a katG T1 (S315T) probe (as shown in Figure 1, lane 3, 5, 7, 8 or 10). DNA sequencing of the katG315 DNA region confirmed these results and also identified the AGC315AAC (S315N) mutation in one isolate (MDR31) that could not be specifically detected by katG mutant probes (Figure 1, lane 9). DISCUSSION The conventional DST of M tuberculosis isolates is time consuming. A simple strip assay (INNO-LiPA Rif. TB) was developed in late 1990s for rapid detection of M tuberculosis isolates resistant to RMP, a surrogate marker for MDR-TB.17 However, detection of RMP-resistant M tuberculosis isolates may not reflect infection with MDR-TB strains as monoresistance to RMP develops frequently during HIV infection and some other underlying conditions.2,18 The Genotype MTBDR strip assay combines probes targeting the rpoB hot-spot region with katG315 probes for simultaneous detection of the majority of M tuberculosis isolates resistant to both RMP and INH.10–12 Although the Genotype MTBDR assay may be directly applied to smear-positive respiratory specimens, it is not economical for countries with a high incidence of extrapulmonary TB and low rates of MDR-TB such as Kuwait.15 Application of the test on isolates from suspected patients is more practical. All of the 20 pansusceptible M tuberculosis strains were correctly identified as susceptible to RMP and INH. Resistance to RMP was correctly identified in 33 of 35 (94%) MDR-TB strains by the Genotype MTBDR assay (Table 1). The DNA sequencing data confirmed the results and specific mutations identified by the strip assay. The S531L mutation was most common (21 of 35, 60%). A high frequency of S531L mutation in the rpoB gene in MDR-TB strains has also been reported from several other countries.3,10,11 Two isolates (MDR13 and MDR30) were identified as RMP susceptible. DNA sequencing identified insertion of the 514TTC mutation in the rpoB gene in one isolate (MDR13). Another strip assay (INNO-LiPA Rif. TB) also failed to detect M tuberculosis isolates containing the insertion 514TTC mutation as RMP-resistant. 13,17 Thus, the rates of concordance of the Genotype MTBDR assay results for detecting RMP resistance with conventional DST and rpoB gene sequencing were 94% and 97%, respectively. One isolate (MDR30) contained a wild-type sequence in the rpoB hot-spot region. The molecular basis of resistance in this isolate either involves mutations in other regions of the rpoB gene or in other genes mediating resistance of M tuberculosis to RMP.3,5,13 The Genotype MTBDR assay identified resistance to both RMP and INH in 20 of 35 (57%) MDR-TB strains. This is mainly because the assay identified INH resistance in only 22 of 35 (63%) MDR-TB strains. Nearly all the isolates with the katG315 mutation (21 of 22, 95%) were detected by katG T1 (S315T) probe. One isolate (MDR31) was detected as INH-resistant by lack of reaction with the katG WT probe only. This isolate contained the AGC315AAC mutation at katG315 and no specific probe is present on the strip for detecting this mutation. The low (63%) sensitivity of the Genotype MTBDR assay for detection of INH resistance is a limitation of this assay. It is likely due to the fact that the assay targets only katG315 mutations while INH resistance in M tuberculosis strains could also involve mutations in other katG gene regions or in other loci.3,7 For example, mutations in the inhA regulatory region occur in 15% to 35% of INH-resistant M tuberculosis strains from some geographical locations.3,7,12 However, mutations in the inhA regulatory region cause low-level of resistance to INH and their detection may have little impact on INH therapy.7,12 Since mutations at katG315 in M tuberculosis strains cause a high-level of resistance to INH, their detection by the Genotype MTBDR assay is valuable in clinical settings and will have highest impact on INH therapy.2,7,12 In conclusion, the Genotype MTBDR assay reliably identifies frequently observed and clinically important mutations associated with resistance of M tuberculosis isolates to RMP and INH. Acknowledgments This study was supported by Research Administration grant YM 03/06 and College of Graduate Studies, Kuwait University. Figure 1 Representative Genotype MTBDR strip patterns obtained with pansusceptible and MDR M tuberculosis isolates. The positions of oligonucleotide probes on the strip, their specificity and the targeted genes as shown (from top to bottom) on the left side (Reference lane) are as follows: CC, conjugate control (for test of kit components); UC, amplification control for high G + C gram-positive bacteria; Tub, M tuberculosis complex-specific control; rpoB, control for rpoB amplification; WT1 to WT5, controls for presence of wild-type sequences in 81-bp hot-spot region of rpoB gene; MUT1, MUT2A, MUT2B and MUT3, probes for D516V (GAC516GTC), H526Y (CAC526TTC), H526D (CAC526GAC) and S531L (TCG531TTG) mutation, respectively, at the three most frequently mutated (516, 526 and 531) rpoB codons; katG, control for katG gene amplification; WT, control for presence of wild-type sequence at katG315; T1 and T2, probes for S315T (AGC315ACC) and S315T (AGC315ACA) mutation, respectively, at katG315. Near the bottom is a marker line (marked by horizontal M) for alignment of strips with the key (reference lane) provided with the kit. Representative patterns on strips from pansusceptible (S1, lane 1 and S2, lane 11) and MDR (MDR1, lane 2, rpoB D516V and katG S315; MDR3, lane 3, rpoB S531L and katG S315T; MDR4, lane 4, rpoB Q513K and katG S315; MDR6, lane 5, rpoB D516V and katG S315T; MDR10, lane 6, rpoB S531L and katG S315; MDR12, lane 7, rpoB H526D and katG S315T; MDR13, lane 8, rpoB insertion 514TTC and katG S315T; MDR31, lane 9, rpoB Q513K and katG S315N and MDR33, lane 10, rpoB H526R and katG S315T) M tuberculosis isolates are shown on the right. Table 1 Genotype MTBDR assay results for detecting RMP and INH resistance in 20 pansusceptible (S) and 35 multidrug-resistant (MDR) M. tuberculosis isolates and comparison with conventional drug susceptibility data. M. tuberculosis strains Susceptibility to RMP and INH Other resistance Genotype MTBDR assay patterns for rpoB hot-spot region katG315 H37Rv Sensitive None WT (susceptible) WT (susceptible) S1-S20 Sensitive None WT (susceptible) WT (susceptible) MDR1,2 Resistant EMB ΔWT2,MUT1,D516V WT MDR3,5,14,27 Resistant EMB, SM ΔWT5,MUT3,S531L ΔWT,T1,S315T MDR4 Resistant EMB ΔWT1 WT MDR6,26,35 Resistant EMB, SM ΔWT2,MUT1,D516V ΔWT,T1,S315T MDR7-9,19,20,32 Resistant None ΔWT5,MUT3,S531L ΔWT,T1,S315T MDR10,24,29 Resistant None ΔWT5,MUT3,S531L WT MDR11 Resistant SM ΔWT5,MUT3,S531L ΔWT,T1,S315T MDR12 Resistant EMB, SM ΔWT4,MUT2B,H526D ΔWT,T1,S315T MDR13 Resistant EMB, SM WT ΔWT,T1,S315T MDR15,23,25 Resistant EMB, SM ΔWT5,MUT3,S531L WT MDR16 Resistant EMB ΔWT4,MUT2B,H526D WT MDR17 Resistant SM ΔWT4 ΔWT,T1,S315T MDR18 Resistant SM ΔWT5,MUT3,S531L WT MDR21,22 Resistant None ΔWT5,MUT3,S531L WT MDR28 Resistant None ΔWT1 ΔWT,T1,S315T MDR30 Resistant EMB WT ΔWT,T1,S315T MDR31 Resistant EMB ΔWT1 ΔWT MDR33 Resistant None ΔWT4 ΔWT,T1,S315T MDR34 Resistant EMB ΔWT5,MUT3,S531L ΔWT,T1,S315T RMP, rifampin; INH, isoniazid; EMB, ethambutol; SM, streptomycin; WT, wild-type; ΔWT, absence of hybridization with a wild-type probe ==== Refs REFERENCES 1 Harries AD Dye C Tuberculosis Ann Trop Med Parasitol 2006 100 415 31 16899146 2 Zignol M Hosseini MS Wright A Weezenbeek CL Nunn P Watt CJ Global incidence of multidrug-resistant tuberculosis J Infect Dis 2006 194 479 85 16845631 3 Ramaswamy S Musser JM Molecular genetic basis of antimicrobial agent resistance in Mycobacterium tuberculosis : 1998 update Tuberc Lung Dis 1998 79 3 29 4 Telenti A Imboden P Marchesi F Lowrie D Cole S Colston MJ Detection of rifampicin-resistance mutations in Mycobacterium tuberculosis Lancet 1993 341 647 50 8095569 5 Heep M Brandstatter B Rieger U Lehn N Richter E Rusch-Gerdes 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 10 11136757 6 Ahmad S Mokaddas E Fares E Characterization of rpoB mutations in rifampin-resistant clinical Mycobacterium tuberculosis isolates from Kuwait and Dubai Diagn Microbiol Infect Dis 2002 44 245 52 12493171 7 Slayden RA Barry CE III The genetics and biochemistry of isoniazid resistance in Mycobacterium tuberculosis Microbes Infect 2000 2 659 69 10884617 8 Abal AT Ahmad S Mokaddas E Variations in the occurrence of S315T mutation within the katG gene in isoniazid-resistant clinical Mycobacterium tuberculosis isolates from Kuwait Microb Drug Resist 2002 8 99 105 12118524 9 Ahmad S Mokaddas E Contribution of AGC to ACC and other mutations at codon 315 of the katG gene in isoniazid-resistant Mycobacterium tuberculosis isolates from the Middle East Int J Antimicrob Agents 2004 23 473 9 15120726 10 Hillemann D Weizenegger M Kubica T Richter E Niemann S Use of the Genotype MTBDR assay for rapid detection of rifampin and isoniazid resistance in Mycobacterium tuberculosis complex isolates J Clin Microbiol 2005 43 3699 703 16081898 11 Miotto P Piana F Penati V Canducci F Migliori GB Cirillo DM Use of Genotype MTBDR assay for molecular detection of rifampin and isoniazid resistance in Mycobacterium tuberculosis clinical strains isolated in Italy J Clin Microbiol 2006 44 2485 91 16825369 12 Brossier F Veziris N Truffot-Pernot C Jarlier V Sougakoff W Performance of the Genotype MTBDR line probe assay for detection of resistance to rifampin and isoniazid in strains of Mycobacterium tuberculosis with low- and high-level resistance J Clin Microbiol 2006 44 3659 64 17021094 13 Ahmad S Mokaddas E The occurrence of rare rpoB mutations in rifampicin-resistant Mycobacterium tuberculosis isolates from Kuwait Int J Antimicrob Agents 2005 26 205 12 16099622 14 Mokaddas E Ahmad S Development and evaluation of a multiplex PCR for rapid detection and differentiation of Mycobacterium tuberculosis complex members from non-tuberculous mycobacteria Jap J Infect Dis 2007 60 140 4 17515652 15 Mokaddas E Ahmad S Samir I Secular trends in susceptibility patterns of Mycobacterium tuberculosis isolates in Kuwait, 1996–2005 Int J Tuberc Lung Dis 2008 12 319 25 18284839 16 Ahmad S Jaber A-A Mokaddas E Frequency of embB codon 306 mutations in ethambutolsusceptible and -resistant clinical Mycobacterium tuberculosis isolates in Kuwait Tuberculosis 2007 87 123 9 17289435 17 Cooksey RC Morlock GP Glickman S Crawford JT Evaluation of a line probe assay kit for characterization of rpoB mutations in rifampinresistant Mycobacterium tuberculosis isolates from New York City J Clin Microbiol 1997 35 1281 3 9114427 18 Mokaddas E Ahmad S Abal AT Al-Shami AS Molecular fingerprinting reveals familial transmission of rifampin-resistant tuberculosis in Kuwait Ann Saudi Med 2005 25 150 3 15977695