Rapid detection of drug resistance and mutational patterns of extensively drug-resistant strains by a novel GenoType® MTBDRsl assayAK Singh1, AK Maurya2, S Kant3, J Umrao1, RAS Kushwaha3, VL Nag1, TN Dhole1
1 Department of Microbiology, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow, Uttar Pradesh, India
2 Department of Microbiology, Sanjay Gandhi Postgraduate Institute of Medical Sciences; Department of Pulmonary Medicine, King George Medical University, Lucknow, Uttar Pradesh, India
3 Department of Pulmonary Medicine, King George Medical University, Lucknow, Uttar Pradesh, India
Correspondence Address: Source of Support: Indian Council of Medical Research, New Delhi, India,
(Extramural ICMR Project Sanction No. 5/8/5/4/2007.ECD.I), Conflict of Interest: None DOI: 10.4103/0022-3859.118034
Source of Support: Indian Council of Medical Research, New Delhi, India, (Extramural ICMR Project Sanction No. 5/8/5/4/2007.ECD.I), Conflict of Interest: None
Background: The emergence of extensively drug-resistant tuberculosis (XDR-TB) is a major concern in the India. The burden of XDR-TB is increasing due to inadequate monitoring, lack of proper diagnosis, and treatment. The GenoType ® Mycobacterium tuberculosis drug resistance second line (MTBDRsl) assay is a novel line probe assay used for the rapid detection of mutational patterns conferring resistance to XDR-TB. Aim: The aim of this study was to study the rapid detection of drug resistance and mutational patterns of the XDR-TB by a novel GenoType ® MTBDRsl assay. Materials and Methods: We evaluated 98 multidrug-resistant (MDR) M. tuberculosis isolates for second line drugs susceptibility testing by 1% proportion method (BacT/ALERT 3D system) and GenoType ® MTBDRsl assay for rapid detection of conferring drug resistance to XDR-TB. Results: A total of seven (17.4%) were identified as XDR-TB by using standard phenotypic method. The concordance between phenotypic and GenoType ® MTBDRsl assay was 91.7-100% for different antibiotics. The sensitivity and specificity of the MTBDRsl assay were 100% and 100% for aminoglycosides; 100% and 100% for fluoroquinolones; 91.7% and 100% for ethambutol. The most frequent mutations and patterns were gyrA MUT1 (A90V) in seven (41.2%) and gyrA + WT1-3 + MUT1 in four (23.5%); rrs MUT1 (A1401G) in 11 (64.7%), and rrs WT1-2 + MUT1 in eight (47.1%); and embB MUT1B (M306V) in 11 (64.7%) strains. Conclusions: These data suggest that the GenoType ® MTBDRsl assay is rapid, novel test for detection of resistance to second line anti-tubercular drugs. This assay provides additional information about the frequency and mutational patterns responsible for XDR-TB resistance.
Keywords: Extensively drug-resistant, Mycobacterium tuberculosis complex, multi drug-resistant
Tuberculosis (TB) remains a major threat to public health worldwide.  In 2009, out of the estimated global annual incidence of 9.4 million TB cases, 2 million were estimated to have occurred in India, thus contributing to a fifth of the global burden of TB. An important aspect of Mycobacterium tuberculosis is the recent rise to multi drug-resistant (MDR) and extremely drug-resistant (XDR) TB cases.  MDR-TB (defined as in vitro resistance to isoniazid and rifampicin, and XDR-TB defined as M. tuberculosis resistant to isoniazid, rifampicin, any fluoroquinolones (FLQ) and at least one of three injectable second-line drugs) are now being widely reported. ,,, Timely identification of XDR-TB is essential for management and prevention of spread in the community. , The number of XDR-TB cases proportionally depends upon the number of increased MDR-TB cases, since 69 countries have reported XDR-TB as per the World Health Organization (WHO) report in 2011. It is an estimated that 25,000 confirmed XDR-TB cases are emerging every year.  Various studies from India have been shown the prevalence of XDR-TB ranging from 2.4% to 15.3%. , Polymerase chain reaction (PCR)-based techniques provide new possibilities for the rapid diagnosis of first- and second-line drug resistance; however, not all mycobacterial laboratories have access to deoxyribose nucleic acid (DNA) sequencing facilities. DNA strip assays have been developed for the detection of rifampin (INNO-LiPA Rif. TB; Innogenetics, Ghent, Belgium) or rifampin and isoniazid resistance of M. tuberculosis in a single assay (GenoType ® Mycobacterium tuberculosis drug resistance plus (MTBDRplus) assay; Hain Lifescience, Nehren, Germany) and are now commercially available. , The GenoType ® MTBDRsl assay is a specific test for the detection of resistance to FLQ, aminoglycosides (AMG), and ethambutol (EMB) in M. tuberculosis complex. This assay has been evaluated for M. tuberculosis both from cultures and clinical specimens. ,,,, The DNA strip assays are based on PCR or multiplex PCR in combination with reverse hybridization. The existence of a resistant strain is signalled either by the omission of a wild-type band or the appearance of bands representing specific mutations. The main target of FLQ in M. tuberculosis is the DNA gyrase gene encoded by gyrA and gyrB, which is essential for DNA supercoiling. The genetic mechanism of FLQ resistance is mainly due to alterations in the DNA gyrase gene, especially mutation(s) in a short sequence called the quinolone-resistance-determining region of the gyrA.  Broad-based knowledge about mutation(s) that causes resistance to EMB and some second-line drugs has been available recently. Resistance to FLQs, AMG, and EMB in M. tuberculosis is most frequently attributed to mutations in the gyrA, rrs, and embB genes, respectively. First investigations have shown that by targeting mutations in codons 90, 91, and 94 in the gyrA gene, approximately 70-90% of all FLQ-resistant strains can be correctly detected. ,, Previous reports have linked mutations A1401G, C1402T, and G1484T in the rrs gene to amikacin (AMK), capreomycin (CPM), and kanamycin (KAN) resistance, respectively, ,, each of them being responsible for a specific resistance patterns. Furthermore, mutations at embB codon 306 are found in 30-68% of EMB-resistant clinical strains. ,, As per our knowledge, this is first study to know the rapid drug resistance and mutational patterns conferring resistance to XDR-TB strains among MDR-TB using the novel GenoType ® MTBDRsl assay for second line drugs in Northern India.
Clinical specimens and data collection
The study was performed prospectively in a blinded manner. This study was conducted after approval by the local research ethics committee. Written, informed consent was taken from all participants. Patients who were attending the outpatient department (OPD) and indoor patients department (IPD) of various wards/units of two tertiary care centers and referred cases from other peripheral healthcare centers in Northern India were screened for XDR-TB from January 2011 to August 2012 (primarily treatment failures and complicated TB cases).
Criteria for inclusion
Patients included in the study were either treatment naïve or previously treated pulmonary TB (PTB) and extra-pulmonary TB (EPTB) cases patients of any age, in whom the diagnosis of TB was confirmed by culture and in whom drug susceptibility testing (DST) against M. tuberculosis complex strains had been performed. Patients infected with non-tuberculous mycobacteria (NTM), bacillary patients with an unknown bacteriological profile, and those unwilling to consent were excluded.
Mycobacterium tuberculosis complex strains
A total of 98 consecutive MDR-TB strains, that were already typed and identified were recruited for DST. The laboratory receives clinical specimens from the surrounding peripheral health centers and hospitals in and around Lucknow, which is a geographical area with a high incidence of drug-resistant TB. All strains were freshly sub-cultured in Middlebrook 7H12 broth (BacT/ALERT MP vial) before performing DST.
Phenotypic DST of 2 nd line antitubercular drugs and EMB by 1% proportion method using BacT/ALERT 3D system.
MDR-TB confirmed strains were selected for second line antitubercular drugs and EMB sensitivity test by standard 1% proportion method using BacT/ALERT 3D system for AMK, KAN, FLQ/ciprofloxacin (CIP), and EMB procured from Sigma, USA. The BacT/ALERT MP bottle containing the growth of M. tuberculosis complex (≤36 hours, subcultured and then diluted 1:1 in sterile distilled water (SDW) were taken as test broth for DST. This formed the direct growth control (DGC) with Mc Farland 2 turbidity. This was put into all drug containing and control BacT/ALERT MP bottles. A 100-fold dilution of the DGC (0.1 ml of DGC + 9.9 ml of SDW) was prepared and 0.5 ml was added to another BacT/ALERT MP bottle and this was the 1% growth control (1% GC). These bottles were incubated in the BacT/ALERT 3D system at 35°C for 12 days and monitored every 10 min to detect growth. An isolate was considered as resistant if the bottles containing the drug flagged positive at the same time or before the 1% GC. An isolate was considered as susceptible if the bottle containing the drug remained negative during the test period or flagged positive after the 1% GC. If the DGC did not flag positive in 12 days, the test was invalidated and had to be repeated. ,,,,
XDR-TB detection and mutational analysis by the GenoType ® MTBDRsl assay. All MDR-TB strains were tested for second line anti-tubercular drugs panel tested by line probe assay using GenoType ® MTBDRsl assay as per manufacturer's instructions (Hain Lifesciences GmbH, Nehren Germany).  For all samples derived from culture suspensions, the strip assays were performed as recommended by the manufacturer. Briefly, for amplification, 35 μl of a primer-nucleotide mixture (provided with the kit), amplification buffer containing 2.5 mM MgCl 2 , 1.25 U Hot Start Taq DNA polymerase (Qiagen, Hilden, Germany), and 5 μl of the preparation of mycobacterial DNA in a final volume of 50 μl were used. The amplification protocol consisted of 15 min of denaturing at 95°C; 10 cycles comprising 30 s at 95°C and 120 s at 58°C; an additional 20 cycles comprising 25 s at 95°C, 40 s at 53°C, and 40 s at 70°C; and a final extension at 70°C for 8 min. Hybridization and detection were performed in an automated washing and shaking device (Profiblot; Tekan, Maennedorf, Switzerland). Steps taken to avoid amplicon contamination were manual pipetting of the amplicon, use of separate wells and tubes for each strip, and extensive rinsing after each use. The program was started after mixing 20 μl of the amplification products with 20 μl of denaturing reagent (provided with the kit) for 5 min in separate troughs of a plastic well. Automatically, 1 ml of pre-warmed hybridization buffer was added, followed by a stop to put the membrane strips into each trough. The hybridization procedure was performed at 45°C for 0.5 h, followed by two washing steps. For colorimetric detection of hybridized amplicons, streptavidin conjugated with alkaline phosphatase and substrate buffer was added. After a final washing, strips were air dried and fixed on paper. To control cross contamination, a no-template control was included in each run. For the sputum specimens, an altered amplification protocol was applied which consisted of 15 min of denaturing at 95°C; 10 cycles comprising 30s at 95°C and 120 s at 58°C; an additional 30 cycles comprising 25 s at 95°C, 40 s at 53°C, and 40 s at 70°C; and a final extension at 70°C for 8 min. Hybridization and detection were done as described above. The MTBDRsl assay strip contains 22 probes, including two amplification and hybridization controls to verify the test procedures. The control (conjugate, amplification, and M. tuberculosis complex-specific controls), targeted gene (gyrA, rrs, and embB), wild-type, and mutation bands are shown from up to down, as follows: CC, conjugate control; AC, amplification control; TUB as M. tuberculosis complex-specific control; gyrA, control for gyrA amplification; gyrA WT1 to WT3, gyrA wild-type probes located at codons 85 to 97; gyrA MUT1 to MUT3D, gyrA mutant probes in codons A90V, S91P, D94A, D94N/Y, D94G, and D94H, respectively; rrs, control for rrs amplification; rrs WT1 and WT2, rrs WT probes located at nucleotides 1401/1402 and 1484, respectively; rrs MUT1 and MUT2, rrs mutant probes for A1401G and G1484T, respectively; embB, control for embB amplification; embB WT1, embB WT probe located at codon 306; embB MUT1A and MUT1B, embB mutant probes in codons M306I and M306V, respectively [Figure 1]. 
The sensitivity and specificity of the GenoType ® MTBDRsl assay results were compared to the conventional BacT/ALERT 1% proportion DST results for XDR-TB. An analysis of frequency and mutational patterns associated with XDR-TB strains were performed using Statistical Package for the Social Sciences 15.0 (SPSS, Chicago, IL, USA) for Windows. The sensitivity and specificity of the molecular methods were analyzed using cross-tabulation after arranging the results of the GenoType ® MTBDRsl assay and gold standard 1% proportion method using BacT/ALERT 3D system. A P < 0.05 was considered statistically significant.
A total of 98 confirmed MDR-TB were tested by both phenotypic and genotypic methods for the detection of XDR-TB strains. Only 17/98 MDR-TB (17.5%) strains were identified as XDR-TB strains. The GenoType ® MTBDRsl assay produced interpretable results for all of the XDR-TB isolates and the MTBC-specific control band appeared accurately. By using this assay, all the 17 XDR-TB strains were correctly identified as XDR-TB strains with interpretable results. Of the 17 XDR-TB patients, 16 (94.2%) were from pulmonary specimens and one was (5.8%) from extra-pulmonary TB specimens (P < 0.05). The turnaround times for DST ranged from 6 to 21 days for the BacT/ALERT 3D system and from 2 to 3 days for the GenoType ® MTBDRsl assay. Performance of GenoType ® MTBDRsl assay was calculated by comparison with conventional DST results over a total of 17 XDR-TB isolates tested [Table 1]. The different mutational patterns of FLQ, AMG/AMK-KAN and EMB conferring resistance to XDR-TB strains (n = 17) were shown in [Table 2].
The sensitivity and specificity of the GenoType ® MTBDRsl assay for AMK/KAN resistance were respectively 100% (95% CI: 80.33-100%) and 100% (95% CI: 95.50-100%), respectively [Table 1]. Of the 17 XDR-TB isolates tested, 15 (88.2%) were resistant to AMK, and 17 (100%) were resistant to KAN by 1% proportional method. But all 17 XDR-TB strains were resistant to AMG/AMK-KAN by GenoType® MTBDRsl assay. The predominant mutations of the GenoType ® MTBDRsl assay identified as conferring AMG/AMK-KAN (rrs gene) was rrs MUT1 (A1401G) in 11 (64.7%) strains. In a total of eight strains the most common (47.1%) mutational pattern was rrs WT1-2 + MUT1, followed by rrs WT1-2 + MUT2 (23.5%) seen in rrs gene [Table 3].
The sensitivity and specificity of the GenoType ® MTBDRsl assay for CIP/FLQ resistance were 100% (95% CI: 80.33-100%) and 100% (95% CI: 95.5-100%), respectively [Table 1]. Of the 17 XDR-TB isolates tested, 17 (100%) were resistance to CIP/FLQ by 1% proportional method and further confirmed by GenoType ® MTBDRsl assay (100% concordance). The predominant mutations detected by the GenoType ® MTBDRsl assay as for FLQ resistance were gyrA MUT1 (A90V) (41.2%) and gyrA MUT3C (D94G) (35.3%) [Table 3]. No mutations in the gyrA MUT2 (S91P) and gyrA MUT3D (D94H) were found in our study. We found common mutational patterns in gyrA gene, four (23.5%) gyrA ΔWT + MUT1, three (17.6%) gyrA ΔWT + MUT3C, and two (9.1%) gyrA ΔWT + MUT3B strains [Table 3].
In 17 XDR-TB strains tested for EMB resistance by both phenotypic and genotypic method, only 11/17 (64.7%) strains were resistant to EMB by the GenoType ® MTBDRsl assay. The sensitivity and specificity of the GenoType ® MTBDRsl assay for EMB resistance were 91.7% (95% CI: 61.46-98.61%) and 100% (95% CI: 95.76-100%), respectively [Table 1]. The predominant mutations of the GenoType ® MTBDRsl assay identified as conferring EMB (embB gene) resistance were 11 (64.7%) embBMUT1B (M306V) [Table 3]. Among EMB resistant isolates, 11/17 all isolates showed (64.7%) embB ΔWT + MUT1B the common mutational pattern seen in embB gene conferring resistance to EMB [Table 3].
The increasing burden of XDR-TB is of serious concern in developing countries, especially in India, because of limited facilities for culture, identification and DST of M. tuberculosis complex especially for second line anti-tubercular drugs. This significantly hinders TB control. Phenotypic drug sensitivity methods are cumbersome and difficult to interpret especially for second line drugs. There is thus an urgent need for laboratories to find a rapid and efficient method for diagnosis of XDR-TB. The GenoType ® MTBDRsl assays are rapid and technically simple to perform and do not require sophisticated equipment. This assay has been studied in other countries with variable results. Different studies have reported false negative results due to unique genetic mutations associated with resistance to second line anti-TB drugs. ,,,,
In the present study, we have evaluated the role and feasibility of the GenoType ® MTBDRsl assay to detect mutations conferring resistance to EMB, second line drugs like FLQ and cyclic peptides/AMG. Considering the phenotypic 1% proportion DST method as the gold standard, the concordance for detection of AMK/KAN, CIP/FLQ, and EMB by the GenoType ® MTBDRsl assay was 100%, 100%, and 91.7%, respectively. A study by Ingen et al., 2010 showed the excellent concordance of phenotypic method with the GenoType ® MTBDRsl assay for AMK and kapreomycin (CPM) was 100% and 97% for these second line drugs. 
Previous studies demonstrated the accuracy of the GenoType ® MTBDRsl assay to be 75.6-90.6% for detecting FLQ, 77-100% for detecting KAN, 80-86.7% for detecting CPM resistance, 84.8-100% for detecting AMK, and 57-69.2% for detecting EMB resistance. ,, In our study, we have reported the sensitivity of 100%, 100%, and 91.7% for AMG/AMK-KAN, FLQ, and EMB, respectively, by using the GenoType ® MTBDRsl assay. The much shorter turn-around times for the GenoType ® MTBDRsl assay (mean, 2 versus 11 days) is a major advantage over that of the phenotypic 1% proportion method using BacT/ALERT 3D system. Fast and robust laboratory results are of paramount importance to guide the choice of drugs in XDR-TB treatment.
The sensitivity and specificity of the GenoType ® MTBDRsl assay for AMK/KAN resistance detection was 100% in this study. However, only 15 MTBC strains were resistant to AMK; and remaining two strains were sensitive by using 1% proportion method. But by using the GenoType ® MTBDRsl assay either presence of mutational probe (s) or absence of any wild type probe (s) conferring resistance to AMG was 100% as per standard definition of XDR-TB. A previous study demonstrated the accuracy of the GenoType ® MTBDRsl assay to be 80-100% for detecting AMK/CPM resistance as compared with our study (100%). ,, But 17 strains were identified as KAN resistant by using both methods (100% concordance). Conversely, two strains susceptible to AMK by phenotypic method but resistant to KAN were correctly identified as resistant by using the GenoType ® MTBDRsl assay. Although other reasons cannot be excluded, an explanation might be that the high-level of resistance to AMK, with preserved susceptibility to KAN as already explained by Alangaden et al.,  and using 1% proportional method as compared to the GenoType ® MTBDRsl assay.
Previously studies reported less frequent occurrence of gyrB mutations than gyrA mutations.  In this study, we have reported 100% concordance between phenotypic and genotypic methods for FLQ. The phenotypic method identified 17 MTBC strains as resistant to FLQ; of this 15/17 (88.2%) had mutations in the any one or combinations of many genes in the GenoType ® MTBDRsl assay and remaining 2/17 (11.8%) showed ΔWT genes. This result was supported by a study in which author demonstrated mutation in 70.3% as compared with absence of WT (29.7%) (ΔWT ) gene.  Among resistance genes conferring resistance to FLQ the most common resistant/mutant gene was gyrA MUT1 in 7 (A90V) (41.2%) MTBC strains [Table 3]. But other study reported that gyrA MUT3C (D94G) was most common i.e., 14/33 (42.4%) followed by gyrA MUT3D (D94H) 10/33 (30.3%).  Among most common mutational patterns observed were gyrA + WT1-3 + MUT1 in four (23.5%) followed by gyrA + WT1-2 + MUT3C in three (17.6%) strains in our study.
In 17 XDR-TB strains tested for EMB resistance, only 11/17 (64.7%) strains were resistant to EMB by the GenoType ® MTBDRsl assay. But the phenotypic method detected 12/17 (70.6%) as resistant to EMB. Discrepant result was obtained for one isolates EMB in this study. That means the sensitivity of this assay for detection of EMB resistance was low (91.7%) as compared with AMG and FLQ in this study. Previous other studies were reported sensitivities from 42% to 69% by using this assay. ,,,,,, It proved that probably in one discrepant strain other mutation (s) (other than embB region) or some other mechanism of drug resistance exists and may have been responsible for phenotypic drug resistance. For EMB resistant isolates, 11/17 (64.7%) showed embB ΔWT + MUT1B the common mutational pattern seen in embB gene. It is well known that, EMB shows a problematic result during DST and yields less reproducible results. , The present study together with different previous reports highlights the fact that the molecular basis of EMB resistance in M. tuberculosis complex is still insufficiently understood to allow detection of EMB resistance by using this assay. Among resistant strains to EMB, only mutations were noted in the embB MUT1B gene (100%) and none of these strains showed resistance in other genes like embB MUT1A. One limitation of this study was that no sequencing was done for one discrepant strain to EMB for detection of other known mutation (s) responsible for EMB resistance.
The performance of the GenoType ® MTBDRsl assay in this study was good with reproducible results. The different molecular methods only detect mutations in most common genes conferring resistance to particular antibiotic screened for, while phenotypic methods detect resistance independent of the underlying mechanism. Furthermore, not all mutations conferring resistance to second line ATT drugs are known till now, especially for AMK, KAN, and EMB. Although, a standard guideline exists for phenotypic DST against second line ATT and is often not as excellent in accuracy with poor clinical predictive values. 
In conclusion, the GenoType ® MTBDRsl assay showed good sensitivity (91.7-100%) for different second line anti-tubercular drugs and EMB in this study. This assay has short turnaround times for the rapid detection of second line drugs and EMB resistance. Our study provides additional information about frequency and mutational patterns that are common in XDR-TB strains in the Northern India. Further studies are essential in high-burden countries to determine the optimal algorithm for implementation of this assay by the Revised National TB Control Program in settings of high XDR-TB prevalence. The rapid detection of XDR-TB cases will allow early initiation of appropriate targeted therapy and infection control to prevent further transmission of XDR-TB strains in the community.
This work was supported by grant from Indian Council of Medical Research, New Delhi (Extramural ICMR Project Sanction No. 5/8/5/4/2007-ECD-I). Authors would like to thank the Technical Member of Mycobacteriology Laboratory, Department of Microbiology, Sanjay Gandhi Postgraduate Institute of Medical Science, Lucknow, India for their technical support during research work.
[Table 1], [Table 2], [Table 3]