|Year : 2015 | Volume
| Issue : 5 | Page : 46-52
Early detection of multi-drug resistance and common mutations in Mycobacterium tuberculosis isolates from Delhi using GenoType MTBDRplus assay
R Singhal1, VP Myneedu1, J Arora1, N Singh1, M Bhalla1, A Verma1, R Sarin2
1 Department of Microbiology, National Institute of Tuberculosis and Respiratory Diseases, New Delhi, India
2 Department of TB and Chest, National Institute of Tuberculosis and Respiratory Diseases, New Delhi, India
|Date of Submission||28-Dec-2013|
|Date of Acceptance||19-Mar-2014|
|Date of Web Publication||6-Feb-2015|
V P Myneedu
Department of Microbiology, National Institute of Tuberculosis and Respiratory Diseases, New Delhi
Source of Support: None, Conflict of Interest: None
Purpose: There is scarcity of prevalence data of multi-drug-resistant tuberculosis (MDR-TB) data and common mutations responsible in North India. This study aimed to detect MDR-TB among MDR-TB suspects from Delhi and mutation patterns using GenoType MTBDRplus assay. Materials and Methods: All MDR suspects in five districts of New Delhi were referred to the laboratory from 1 st October 2011 to 31 st December 2012 as per criterion defined by Programmatic Management of Drug Resistant Tuberculosis (PMDT). GenoType MTBDRplus assay was performed on 2182 samples or cultures and mutations in the rpoB gene for rifampicin (RIF) and katG and inhA genes for isoniazid (INH) were analyzed. Results: A total of 366 (16.8%) MDR-TB cases were diagnosed. MDR rate was found to be 32%, 16.6% and 10.2% during criterion A, B and C respectively. The most common mutation detected for RIF was S531L (59.0%) and for INH was S315T1 (88.3%). Mutations S531L and S315T1 occurred significantly higher in MDR strains as compared to RIF mono-resistant and INH mono-resistant strains, respectively. Average laboratory turn-around time (TAT) for dispatch of result to districts for test conducted on samples was 4.4 days. Conclusion: GenoType MTBDRplus is a useful assay for rapid detection of MDR-TB. The common mutations for RIF and INH were similar to those seen in other regions. However, mutations determining MDR strains and mono-resistant strains differed significantly for both RIF and INH.
Keywords: Criterion, line probe assay, MDR suspect, mutation
|How to cite this article:|
Singhal R, Myneedu V P, Arora J, Singh N, Bhalla M, Verma A, Sarin R. Early detection of multi-drug resistance and common mutations in Mycobacterium tuberculosis isolates from Delhi using GenoType MTBDRplus assay. Indian J Med Microbiol 2015;33, Suppl S1:46-52
|How to cite this URL:|
Singhal R, Myneedu V P, Arora J, Singh N, Bhalla M, Verma A, Sarin R. Early detection of multi-drug resistance and common mutations in Mycobacterium tuberculosis isolates from Delhi using GenoType MTBDRplus assay. Indian J Med Microbiol [serial online] 2015 [cited 2020 Jan 26];33, Suppl S1:46-52. Available from: http://www.ijmm.org/text.asp?2015/33/5/46/150879
| ~ Introduction|| |
Multi-drug-resistant tuberculosis (MDR-TB), defined as the resistance to at-least rifampicin (RIF) and isoniazid (INH), poses grave challenge because of prolonged, limited and expensive treatment options with 10-30% of cases resulting in failure of treatment and death.  Timely diagnosis and treatment of infectious cases is crucial in curtailing the spread of infection in the community. Hence, it is imperative to perform drug susceptibility testing (DST) for appropriate management of drug-resistant cases.
Conventional DST such as 1% proportion method has been the gold standard but takes up to 4-6 weeks after the growth of Mycobacterium tuberculosis (M. tuberculosis). Liquid cultures are sensitive and faster.  Complexity and high cost of liquid culture systems, however, pose overwhelming challenge for their widespread implementation.
Commercial assays based on reverse hybridization of amplicons to immobilized membrane-based probes, covering wild type sequences of rpoB, katG and inhA and their mutations have been developed for rapid detection of drug resistance.  Sensitivity and specificity of GenoType MTBDRplus assay for RIF and INH resistance are found to be comparable to conventional phenotypic DST.  RIF resistance is caused by altered beta-subunit of DNA dependent RNA polymerase, caused by missense mutation commonly found in 81-bp hot-spot region of the rpoB gene.  Resistance to INH is most frequently associated with a specific mutation S315T in katG gene and/or C15T, A16G, T8A and T8A in the inhA gene. 
Tuberculosis and MDR-TB control in India is integrated under the Revised National Tuberculosis Control Programme (RNTCP) for timely detection of DR-TB and its management. Hence, all MDR-TB suspects are considered for DST using culture methods or line probe assay (LPA). 
The present study was conducted in the National Reference Laboratory (NRL), proficiency tested for M. tuberculosis DST by Supranational Laboratory (Antewerp Belgium). There is limited data of MDR-TB among MDR-suspects using LPA and its genetic determinants in this region. The mutations responsible for resistance could differ as per region and thus provide an insight into the epidemiology of the disease. Therefore, the present study was conducted to determine the MDR and mutations in rpoB, katG and inhA gene using GenoType MTBDRplus assay.
| ~ Materials and Methods|| |
A total of 2891 sputum samples from MDR-TB suspect patients were received from five districts of Delhi under RNTCP from 1 st October 2011 to 31 st December 2012. The TB patients were suspected as MDR-TB as per criterion A, B and C implemented in New Delhi [Table 1].  The study is approved by institute's ethical committee.
|Table 1: Patients considered as MDR‑TB suspects as per programmatic management of drug resistant tuberculosis; PMDT criterion|
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All specimens were screened for presence of acid fast bacilli (AFB) by Ziehl-Neelsen (ZN) staining.  The samples were processed by N-acetyl-L cysteine-Sodium hydroxide (NALC-NaOH) method of digestion and decontamination.  The pellet obtained was suspended in 2 ml of phosphate buffer saline (PBS) at pH 6.8. All smear-positive processed sputum samples (≥1 + for version 1.0) were directly processed for GenoType MTBDRplus assay as per the national guidelines. 
All smear-negative processed samples and scanty positive samples (version 1.0) were inoculated in MGIT 960 tubes for culture as per national guidelines.  Tubes with positive alerts were identified for presence of M. tuberculosis by smear microscopy for serpentine cording and rapid immunochromatographic test for detection of MPT64 TB Ag (SD BIOLINE). Cultures positive for M. tuberculosis were subjected to LPA.
GenoType MTBDRplus assay
The Genotype MTBDRplus assay version 1.0 or version 2.0 (Hain Life Sciences, Nehran, Germany) was carried out on the processed sample as per manufacturer's instructions.  DNA extraction, master-mix preparation, DNA amplification and hybridization were done after thorough cleaning in dedicated rooms. 
For DNA extraction, 500 μl of NaLC-NaOH processed samples and 1 ml of M. tuberculosis cultures from MGIT tubes were taken. The processed samples or cultures were pelleted in 100 μl sterile molecular grade water. In version 1.0, bacterial pellets were heat inactivated at 95°C for 20 minutes and sonicated; in version 2.0, bacterial pellets were subjected to chemical lysis using the Genolyse kit. The supernatant containing DNA was transferred into the fresh tube and stored at -20°C.
Master-mix was prepared using reagents provided in the kit and addition of TAQ polymerase as supplied in the two versions of the kit. DNA solution was added in volumes of 5 μl to PCR tubes and amplified. Detection of PCR products was done using hybridization by LPA methodology by GenoType MTBDRplus. 
The result of DNA strips was interpreted with the help of reporting card as resistant or sensitive for RIF or INH or invalid based on the kit insert. In addition, presence of mutation bands and absence of sensitive bands were also recorded.  Testing of LPA on any invalid results were repeated using processed sample deposits, stored at −20°C. M. tuberculosis H37Rv (ATCC 27294) was run as positive control and sterile molecular grade water was run as negative control, in each run for quality control.
The reports were communicated electronically to the District TB Officers (DTO) of the respective districts within 24 hours of report generation. Turn-around time for each sample was calculated.
Data were presented as frequency tables, and mean was calculated wherever required. Fisher's exact test for statistical significance (defined as the probability that an effect is not due to by chance alone) was calculated wherever applicable using Graphpad computer software. P < 0.05 was considered significant.
| ~ Results|| |
Out of 2891 samples from MDR suspects received, 2043 were males and 848 were females (ratio of 2.4:0). Maximum patients were found in the age groups of 16-35 (1507; 52.1%). A total of 181 and 161 patients were found to be aged more than 60 years and less than or equal to 15 years, respectively. Smear-positive and smear-negative patients were found to be 2120/2891 (73.3%) and 771/2891 (26.7%), respectively. The district-wise distribution of patients is detailed in [Table 2].
|Table 2: District‑wise distribution of AFB smear positive and negative samples as per revised national tuberculosis control programme guidelines|
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A total of 2182 GenoType MTBDRplus tests were conducted. Of these, 1961 were done directly on samples and 221 on positive cultures. Valid LPA results were obtained for 2038 out of 2182 samples (93.4%) on which LPA was performed. Of 2038, 366 (17.9%) tests were found to be resistant to RIF and INH, 93/2038 (4.6%) as mono-RIF-resistant, 148/2038 (7.3%) as mono-INH-resistant and 1431/2038 (70.2%) as sensitive to both RIF and INH [Table 3]. The MDR-TB rate detected among MDR suspects was found to be 39/122 (32%), 108/652 (16.6%) and 219/2117 (10.2%) during criteria A, B and C, respectively. This difference of MDR-TB rate diagnosed in criteria A, B and C was significant (P < 0.0001).
Correlation of valid GenoType MTBDRplus with smear microscopy result
Overall, 144/2038 (6.6%) of tests were found to be invalid. The invalid rate increased as 4/572 (0.7%), 10/371 (2.7%), 123/907 (13.6%) and 7/40 (17.5%) as AFB concentration decreased as 3+, 2+, 1+ and scanty. This correlation was statistically highly significant. (P < 0.0001).
Turn-around time (TAT)
An average TAT of 5.5 days, 4.2 days, 5.3 days, 4.3 days and 3.3 days was achieved in IV th Q 2011, I st Q 2012, II nd Q 2012, III rd Q 2012 and IV th quarter 2012, respectively, for LPA performed directly on samples with overall average of 4.4 days. TAT for phenotypic DST by liquid culture was 27.2 days.
Mutation pattern of RIF
Among 459 RIF-resistant strains, missing wild type with known mutations were found in 339 (73.9%) strains [Table 4]. Commonest specific mutation was S531L in rpoB (267/459, 58.2%) followed by D516V (37/459, 8%), H526Y (23/459, 5%) and H526D (6/459, 1.3%). In six strains, more than one mutation was found. The mutation S531L was found in 231/366 (63.1%) and 36/93 (38.7%) of MDR strains and mono-RIF strains, respectively. This difference was highly statistically significant (P < 0.0001). In 120/459 (26.1%) of RIF-resistant strains, one or more wild type probes were missing with no presence of mutant probes. Such mutations were found more commonly in mono-RIF strains than MDR strains (P < 0.0001). These included missing wild type bands WT8 (53/120, 44.2%), WT7 (14/120, 11.7%), WT3 (4/120, 3.3%), WT2 (10/120, 8.3%), WT1 (4/120, 3.3%), WT6-WT7 (1/120, 0.8%), WT5-WT6 (2/120, 1.7%), WT3-W8 (1/120, 0.8%), W3-W4 (20/120, 16.7%), W2-W3 (5/120, 4.2%), WT6-WT7-WT8 (1/120, 0.8%), WT4-WT5-W8 (1/120, 0.8%), WT3-W4-W7 (1/120, 0.8%) and WT2-WT3-WT4 (3/120, 2.5%) [Table 4]. Mixed pattern to RIF with all wild type probes present along with one or more mutant bands was found in (22/459; 4.8%) cases, most common being S531L (8/459; 1.7%) and D516V mutation (7/459; 1.5%).
|Table 4: Mutations detected by genotype MTBDRplus assay in rifampicin– resistant Mycobacterium tuberculosis|
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Mutation pattern of INH
Among 514 INH-resistant strains detected, katG mutation occurred in 479 (93.2%). Known mutation S315T1 was found in 454/514 (88.3%) of INH-resistant strains or 454/479 (94.8%) of katG mutant strains. Missing wild type with absence of mutant probes among katG was found in 19/479 (4.0%) strains. Mutations in S315T1 and katG were significantly higher in MDR strains than INH mono-resistant strains (P value < 0.0001). InhA mutations were found in 69/514 (13.4%) of INH-resistant strains which included C15T (53/69, 76.8%), T8C (4/69, 5.8%), T8A (2/69, 2.9%), A16G (1/69, 1.5%) and (9/69, 13.0%) due to missing wild type with no mutation band. Mutations in C15T and inhA were significantly higher in INH mono-resistant than MDR strains (P < 0.0001). Both inhA and katG mutations were seen in 34/514 (6.6%) of isolates [Table 5]. Mixed wild-type and mutant pattern to INH was found in 26/514 (5.1%) cases.
|Table 5: Mutations detected by genotype MTBDR plus in isoniazid‑resistant Mycobacterium tuberculosis|
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| ~ Discussion|| |
A study of MDR-TB detection among MDR-TB suspects in North Indian RNTCP network using the GenoType MTBDRplus assay was conducted. The genotypic profiles of the rpoB, katG and inhA regions associated with resistance were also reviewed.
Males were found to be predominant in the present study with 70.7%. Maximum patients were found in young age group of 16-35 years, 1507 (52.1%) as seen earlier.  As young adult males are economically productive segment of society, high MDR-TB in this group has several socioeconomic implications.
Overall, MDR rate among MDR suspects tested was 17.9%. There was a significant reduction in MDR-TB rate from criteria A to B and then C. This could be because under PMDT, MDR-TB are suspected among the notified TB cases initially from highly selective group of patients with high probability of MDR-TB to gradually expand and provide universal access of MDR-TB diagnostics for most TB patients. 
Various Indian studies have reported MDR rates to be varying from 17.4% to 53% among re-treatment cases. , World-wide surveillance of MDR in re-treatment cases ranged from 9.4% to 36.5%, from 1994-2000 across the world.  Previous exposure to anti-tuberculosis agents is the most common cause of developing MDR.
GenoType MTBDRplus assay directly performed on samples gave an average TAT of 4.4 days, a significant reduction by 83.8% compared to phenotypic DST by liquid culture (27.2; unpublished information). This is one of the main advantages of the assay as reported earlier.  One lot of DNA extraction to hybridization procedure is completed in less than two working days; however, initial AFB screening, report typing and dispatch adds to TAT. The management of MDR-TB in our hospital has been accelerated as almost 95% of patients diagnosed as MDR-TB are started on category IV treatment within 1 week of communication of report.
The invalid results obtained for some samples are well within the acceptable limits.  The rate of invalid obtained correlated inversely to AFB microscopy grading as has also been reported earlier.  Therefore, the test may have limited use in pauci-bacillary extra-pulmonary cases.
The GenoType MTBDRplus provides comprehensive information about the mutations leading to resistance, which highly correlates with the sequencing results.  In the present study, the test identified RIF resistance by one of the four rpoB mutant probes in 73.9% isolates, much lesser than in a South African study (91%).  Mutation S531L was detected in 59.0% of RIF resistance cases, similar to other Indian studies; 59.8% and 84.6% cases. , Internationally, S531L mutation has been detected in rates varying from 47% to 70.5%. ,, In the present study, S531L mutation occurred significantly more in MDR strains in comparison to RIF mono-resistant strains (P value < 0.0001). This has been seen in a South African study and an Indian study. , In South African and Indian studies, S531L mutation accounted for 76.4% and 37.5% and 55% and 19% of MDR-TB and RIF mono-resistant strains, respectively, which is a significant difference. ,
The missing wild type probe without any mutant bands was found in 26.1% of RIF-resistant isolates as also reported in other studies in France (29%), New Delhi (11.1%) and Vietnam (33.3%). ,, As the GenoType MTBDRplus incorporates the probes for common mutations only, these isolates could have uncommon mutations. Interestingly, such mutations were found significantly more in mono-RIF-resistant strains. One limitation of the study is the lack of sequencing which could have identified such mutations.
The katG gene mutations account for commonest mechanism of resistance for INH. Specific mutation S315T1 in katG was found in 88.3% of INH-resistant isolates, similar to other studies. , However, variations have been reported in studies from France (62.5%) and South Africa (37.6%). , High prevalence of katG mutations contributes to most cases of INH resistance in high-burden countries and leads to high-level INH resistance. A significant difference in prevalence of S315T1 in MDR strains as compared to mono-INH-resistant strains was found (P < 0.0001) as also reported in South African and Indian studies. 
Mutation in the inhA gene accounts for low-level resistance. It was found to be 13.4%, similar to studies elsewhere 5.4-21.1%. ,,,,, The inhA mutations accounted for 30% and 9% of mono-INH and MDR strains, respectively. This difference was found to be highly significant. In the South African and Indian studies, difference in prevalence of inhA mutations in MDR and Mono-INH-resistant strains was not significant. , The South African study reported high prevalence of inhA mutations among all INH-resistant cases (41.7%).  The current assay offers additional detection of INH-resistant strains due to mutations in inhA gene, which was lacking in the earlier version (Genotype MTBDR assay).
Hetero-resistance, i.e. presence of all wild type probes along with presence of one or more mutant bands was found in 4.8% of RIF-resistant strains and 5.1% of INH-resistant strains. Tolani et al. from Mumbai have reported high number of hetero-resistant strains.  The detection of hetero-resistant strains would ensure the timely initiation of MDR-TB regimens in such patients. Patients harbouring such strains would have better treatment outcomes compared to pure MDR-TB patients.  A disadvantage of this genotypic test is a possibility of silent mutation, i.e. mutations that do not lead to amino acid change with, the strain being sensitive by phenotypic DST. In an earlier study from NRL, the test was compared with phenotypic DST. 
To conclude, LPA with GenoType MTBDRplus has revolutionized the MDR-TB diagnosis. Less staff is able to complete far more number of DSTs per day. In addition, TAT is substantially reduced promoting early detection and management of drug-resistant cases. The test provides additional information of the common mutations imparting resistance to RIF and INH, which helps in understanding the epidemiology of the disease. More studies need to be instituted to assess the performance of the test in smear negative patients at larger platforms.
| ~ Acknowledgements|| |
The kits, consumables and machines for GenoType MTBDR assay have been provided free of cost by FIND, India for management of MDR-TB patients under the PMDT programme. We acknowledge the technical help of Mr. Abdul, Mr. Grish and Mr. Arun.
| ~ References|| |
Centers for Disease Control and Prevention (CDC). Emergence of Mycobacterium tuberculosis
with extensive resistance to second line drugs--worldwide, 2000-2004. MMWR Morb Mortal Wkly Rep 2006;55:301-5.
Balabanova Y, Drobniewski F, Nikolayevskyy V, Kruuner A, Malomanova N, Simak T, et al
. An integrated approach to rapid diagnosis of tuberculosis and multi-drug resistance using liquid culture and rapid methods in Russia. PLoS One 2009;4:e7129.
Brossier F, Veziris N, Truffot-Pernot C, Jarlier V, Sougakoff W. Performance of the GenoType MTBDR line probe assay for detection of resistance to rifampicin and isoniazid in strains of Mycobacterium tuberculosis with low and high level resistance. J Clin Microbiol 2006;44:3659-64.
Ling DI, Zwerling AA, Pai M. GenoType MTBDR assays for the diagnosis of multi-drug resistant tuberculosis: A meta-analysis. Eur Respir J 2008;32:1165-74.
Telenti A, Honore N, Bernasconi C, March J, Ortega A, Heym B, et al
. Genotypic assessment of isoniazid and rifampin resistance in Mycobacterium tuberculosis: A blind study at reference laboratory level. J Clin Microbiol 1997;35:719-23.
Kent PT, Kubica GP. Public health mycobacteriology, a guide for level III lab. Department of Health and Human Services, Public health services, (Center for Disease Control, Atlanta GA USA); 1985. p. 207.
Sharma SK, Kaushik G, Jha B, George N, Arora SK, Gupta D. Prevalence of multidrug-resistant tuberculosis among newly diagnosed cases of sputum-positive pulmonary tuberculosis. Indian J Med Res 2011;133:308-11.
Ramachandran R, Nalini S, Chandershekhar V, Dave PV, Sanghvi AS, Wares F, et al.
Surveillance of drug resistant tuberculosis in the state of Gujrat, India. Int J Tuberc Lung Dis 2009;134:1154-60.
Parmasivan CN, Rehman F, Wares F, Sundar Mohan N, Sundar S, Devi S, et al
. First and second line drug resistance patterns among previously treated tuberculosis patients in India. Int J Tuberc Lung Dis 2010;14:243-6.
Zignol M, van Gemert W, Falzon D, Sismanidis C, Glaziou P, Floyda K, et al
. Surveillance of anti-tuberculosis drug resistance in the world: An updated analysis 2007-2010. Bull World Health Organ 2012;90:111-9D.
Barnard M, Albert H, Coetzee G, Brien R, Bosman ME. Rapid molecular screening for multi-drug resistant tuberculosis in a high-volume public health laboratory in South Africa. Int J Respir Crit Care Med 2008;177:787-92.
Makinen J, Marttila HJ, Marjamaki M, Viljanen MK, Soini H. Comparison of two commercially available DNA line probe assays for detection of multi-drug resistant Mycobacterium tuberculosis. J Clin Microbiol 2006;44:350-2.
Raveendran R, Wattal C, Oberoi JK, Goel N, Datta S, Prasad KJ. Utility of GenoType MTBDRplus assay in rapid diagnosis of multidrug resistant tuberculosis at a tertiary care centre in India. Indian J Med Microbiol 2012;30:58-63.
Singhal R, Arora J, Lal P, Bhalla M, Myneedu VP, Behera D. Comparison of line probe assay with liquid culture for rapid detection of multi-drug resistance in Mycobacterium tuberculosis. Indian J Med Res 2012;136:1044-7.
Huyen MN, Tiemersma EW, Lan NT, Cobelens FG, Dung NH, Sy DN, et al
. Validation of the GenoType MTBDRplus assay for diagnosis of multi-drug resistant tuberculosis in South Vietnam. BMC Infect Dis 2010;10:149.
Singh LS, Mazumder PB, Sharma GD. Analysis of mutational pattern in multidrug resistant tuberculosis (MDR TB) in a geographically isolated northeastern region of India. IOSR-J Pharm Biol Sci 2014;9:4-10.
Mokrousov I, Narvskaya O, Otten T, Limenschenko E, Steklova L, Vyshnevskiy B. High prevalence of katG Ser315Thr substitution among isoniazid-resistant Mycobacterium tuberculosis
clinical isolates from Northwestern Russia 1996-2001. Antimicrob Agents Chemother 2002;46:1417-24.
Anek-Vorapong R, Sinthuwattanawibool C, Podewils LJ, McCarthy K, Ngamlert K, Promsarin B, et al
. Validation of the GenoType MTBDRplus assay for detection of MDR-TB in a public health laboratory in Thailand. BMC Infect Dis 2010;10:123.
Tolani MP, D′souza DT, Mistry NF. Drug resistance mutations and heteroresistance detected using the GenoType MTBDRplus assay and their implication for treatment outcomes inpatients from Mumbai, India. BMC Infect Dis 2012;12:9.
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]