|Year : 2015 | Volume
| Issue : 4 | Page : 547-553
Detecting mutation pattern of drug-resistant Mycobacterium tuberculosis isolates in Himachal Pradesh using GenoType® MTBDRplus assay
C Thakur, V Kumar, AK Gupta
Intermediate Reference Laboratory, Government Tuberculosis Sanatorium, Dharampur, Solan, Himachal Pradesh, India
|Date of Submission||18-Nov-2014|
|Date of Acceptance||06-May-2015|
|Date of Web Publication||16-Oct-2015|
Intermediate Reference Laboratory, Government Tuberculosis Sanatorium, Dharampur, Solan, Himachal Pradesh
Source of Support: None, Conflict of Interest: None
Context: Tuberculosis (TB) is a major public health problem in India and a principal cause of death in adults, especially among the economically productive age group. India accounts for one-fifth of the global burden of TB. It is estimated that about 40% of Indian population is infected with TB bacillus. The GenoType® MTBDRplus molecular method allows rapid diagnosis of the clinical samples and detection of the most common mutations in the genes associated with rifampicin (R) and isoniazid (H) resistance. Aims: To study the drug resistance and mutational patterns in multidrug-resistant (MDR) suspects clinical strains using GenoType® MTBDRplus assay. Subjects and Methods: A total of 770 sputum samples of the MDR-TB suspects were included in this study, which were received at Intermediate Reference Laboratory, Government TB Sanatorium, Dharampur, Solan, Himachal Pradesh from the Designated Microscopy Centres of Himachal Pradesh for the culture and susceptibility testing. All the 521 Mycobacterium tuberculosis complex (MTBC) strains were subjected to GenoType® MTBDRplus (HAIN Lifescience) assay to detect molecular resistance pattern to first line anti-tubercular drugs (isoniazid and rifampicin). Results: Of 770 samples, 556 (72.20%) were from male and 214 (27.80%) were from female. Among the 521 MTBC strains, 19.76% were found to be MDR and mono-resistance to isoniazid and rifampicin was detected in 8.63% and 6.14% strains respectively. About 74.81%, 76.35% and 5.40% strains harboured known mutation in rpoB, katG and inhA genes respectively. Conclusions: In rpoB gene, the most common mutation is associated with S531 L region. The GenoType® MTBDRplus assay is a rapid test for the detection of the most common mutations in MDR-TB strains. In our study, unknown rpoB gene mutations were found in 25.18% strains that may further be detected by gene sequencing.
Keywords: Drug resistance, GenoType® MTBDRplus assay, mutation, tuberculosis
|How to cite this article:|
Thakur C, Kumar V, Gupta A K. Detecting mutation pattern of drug-resistant Mycobacterium tuberculosis isolates in Himachal Pradesh using GenoType® MTBDRplus assay. Indian J Med Microbiol 2015;33:547-53
|How to cite this URL:|
Thakur C, Kumar V, Gupta A K. Detecting mutation pattern of drug-resistant Mycobacterium tuberculosis isolates in Himachal Pradesh using GenoType® MTBDRplus assay. Indian J Med Microbiol [serial online] 2015 [cited 2019 May 19];33:547-53. Available from: http://www.ijmm.org/text.asp?2015/33/4/547/167336
| ~ Introduction|| |
Tuberculosis (TB) is a major public health problem in India and a leading cause of death in adults, especially among the economically productive age group. Historically, TB has been associated with significant morbidity and mortality and remains a major global health problem. India accounts for one-fifth of the global burden of TB. It is estimated that about 40% of Indian population is infected with TB bacillus. The prevalence and mortality due to TB in India were estimated to be 249 and 26 respectively per 100,000 population.
India and China alone accounted for 26% and 12% of global cases respectively. India is estimated to have the highest number of multidrug-resistant-TB (MDR-TB) cases among notified TB patients.
The emergence of anti-tubercular drug resistance is an increasing public health problem and TB control programmes in industrialised and developing countries alike. In 2012, there were an estimated 12 million TB cases globally, including 8.6 million new cases and 1.3 million fatal cases. According to the WHO, there were about 440,000 new cases of MDR-TB per year, accounting for 150,000 deaths and 25,000 cases of extensively drug-resistant worldwide by 2012. Currently, there is no countrywide drug resistance survey, and that is why the exact data about the prevalence and pattern of drug-resistant strains of Mycobacterium tuberculosis is unknown. Worldwide, 3.7% of new cases and 20% of previously treated cases were estimated to have MDR-TB. According to drug resistance surveillance of Gujarat and Maharashtra, the prevalence of MDR-TB is estimated to be about 3% in new cases and 12–17% in retreatment cases. The prevalence of MDR-TB among new and previously treated cases is increasing all over the world as well as in India.,
Thus, it is extremely important to develop quicker diagnostic methods to identify resistant cases and provide suitable chemotherapy for effective treatment to prevent further spread of drug-resistant TB. During the past few years, several molecular techniques have been developed, including conventional sequencing, pyrosequencing, real-time polymerase chain reaction (PCR) and reverse hybridisation assay with DNA probes. These molecular techniques have been proposed for detection of mutation frequency and patterns associated with drug resistance.,
Now, WHO recommends the use of molecular line probe assay (LPA) (GenoType ® MTBDRplus Hain Lifescience, GmbH) for the diagnosis of MDR-TB. The GenoType ® MTBDRplus assay molecular method allows rapid diagnosis of the clinical samples and detection of the most common mutations in the genes associated with rifampicin (R) and isoniazid (H) resistance. LPA uses multiplex PCR and reverses hybridisation to identify M. tuberculosis complex (MTBC) and is designed to detect the most important rpoB, katG and inhA gene mutations conferring R and high-level H resistance in the clinical isolates simultaneously. It can be performed directly from Ziehl-Neelsen (ZN) smear-positive sputum or from culture isolates and provide results in 1–2 days. A recent systematic review concluded that LPAs are highly sensitive and specific for detection of rifampicin resistance (≥97% and ≥99%) and isoniazid resistance (≥90% and ≥99%) on culture isolates and smear-positive sputum samples.,
The present study was planned to understand drug resistance and mutational patterns in sputum samples from MDR-TB suspect cases by GenoType ® MTBDRplus assay in Himachal state of India. Sputum from MDR-TB suspect patients who were smear or culture positive for M. tuberculosis were subjected to drug susceptibility testing (DST).
| ~ Subjects and Methods|| |
Patients included in the study were new or previously treated pulmonary TB cases suspected to be MDR-TB from all age groups and in whom DST against MTBC had been performed. Those infected with mycobacterial other than TB and with an unknown bacteriological profile were not included in the present study.
Sputum samples of the MDR-TB suspects were included in this study were received from the Designated Microscopy Centres (DMCs) of Himachal Pradesh for the culture and susceptibility testing during the period from April 2013 to October 2013.
All the sputum samples received were subjected to direct smear microscopy by ZN staining method and samples were treated with N-acetyl-L-cysteine-NAOH for decontamination. All the ZN negative samples were inoculated in Lowenstein Jensen (LJ) media for MTB culture. All the ZN smear positive and LJ culture positive samples were subjected to GenoType ® MTBDRplus (HAIN Lifescience) assay to detect molecular resistance pattern to first line anti-tubercular drugs (isoniazid and rifampicin) as per manufacturer's instructions. Data were analysed as per the age, sex, drug resistance, and their mutations patterns associated with MDR-TB strains using the GenoType ® MTBDRplus assay.
| ~ Results|| |
A total of 770 sputum samples suspected to be MDR-TB cases were received from the DMCs of Himachal Pradesh during the period between April, 2013 and October, 2013. The samples received were classified into two groups namely, group A consisting of patients undergoing category-I treatment (primary drug resistance) that is, new TB cases and group B with patients who were undergoing category-II treatment (acquired drug resistance) that is, retreatment cases as per Revised National TB Control Programme guidelines. Out of 770 samples, 170 (22.07%) samples were under group A and 600 (77.92%) were in group B. Of 770 samples, 556 (72.20%) were from male and 214 (27.80%) were from female [Table 1].
Occurrence of tuberculosis among different age groups
All the 770 MDR-TB suspect sputum samples were collected between the ages of 05–94 years. The data obtained from the samples collected revealed that the rate of TB infection is more in males than females. In males, the frequency of TB was found to be highest in the age group of 31–40 (21.04%) and in females it was in age group of 21–30 (35.04%). Overall, the highest frequency was found in the age group of 21–30 (24.02%) followed by in the age group of 31–40 (21.29%) [Table 2].
Drug susceptibility profile
Of 770 sputum samples, 529 (68.70%) were exhibited as ZN smear positive and 241 (31.29%) as ZN smear negative. Among these ZN smear negative specimens, 45 (18.67%) were found to be culture positive. DST was performed by LPA in 574 (74.54%) samples, including 529 ZN smear positive and 45 culture positive isolates. Of these, 521 (90.76%) were identified as MTBC positive and 53 (9.23%) as MTBC negative. These 53 (9.23%) MTBC negative samples were found to be non-TB Mycobacterium on the basis of their growth characteristics, pigmentation and by biochemical identification.
From the 521 MTBC positive strains, 103 (19.76%) were found to be under category-I treatment and 418 (80.23%) were under category-II treatment [Table 3]. There are 103 (19.76%) strains were found to be MDR that is, resistant to both drugs H as well as R and 341 (65.45%) were found sensitive for both drugs among the 521 samples. However, mono-resistance to isoniazid and rifampicin was detected in 45 (8.63%) and 32 (6.14%) strains respectively in both groups [Table 3].
|Table 3: Drug susceptibility profile of Mycobacterium tuberculosis isolates|
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Resistance to isoniazid and rifampicin in group A
In group A (under category-I treatment), 13 (12.62%) strains were exhibited MDR and 74 (71.89%) strains were sensitive to both drugs. However, 16 (15.53%) isolates were found mono-resistant, of which 9 (8.73%) were isoniazid and 7 (6.8%) strains were rifampicin resistant out of 103 samples [Table 3].
Resistance to isoniazid and rifampicin in group B
In group B (under category-II treatment), of 418 strains, 90 (21.53%) strains were found MDR and 267 (63.87%) strains were sensitive to both drugs. However, 36 (8.61%) strains from this group were showed mono resistant to isoniazid and 25 (5.98%) were mono-resistant to rifampicin [Table 3].
An analysis of the frequency and mutational patterns associated with MDR-TB as well as mono-resistant strains were performed using the GenoType ® MTBDRplus assay. Readable bands pattern of rpoB, katG, and inhA section in GenoType ® MTBDRplus assay results were obtained from the 521 (90.76%) MTBC strains [Table 4].
|Table 4: Pattern of gene mutations detected by GenoType® MTBDRplus assay in drug-resistant Mycobacterium tuberculosis strains|
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Among the 135 rifampicin resistant and 148 isoniazid resistant strains, 101 (74.81%), 113 (76.35%) and 8 (5.40%) strains harboured known mutation in rpoB, katG and inhA genes respectively. The frequency of rpoB mutation was 84 (62.22%) in S531 L, 6 (4.44%) in D516V, 7 (5.18%) in H526Y, 4 (2.96%) in H526D region and 34 (25.18%) were showed unknown mutations (absence of wild type and with no confirmed mutation). The katG gene mutations were 110 (74.32%) in S315T1 and 3 in S315T2 (2.06%) region that is most prominent and inhA gene mutation were 8 (5.40%) in C15T region. None of the confirmed mutation found in other region such as A16G, T8C and T8A of inhA gene. In inhA and katG region 15 (10.13%) and 12 (8.10%) isolates respectively showed other unknown mutations, with the absence of one or more wild-type.
| ~ Discussion|| |
Increasing trends of TB and MDR-TB rates in high-burden countries required development and implementation of rapid diagnostic techniques. Phenotypic DST is a time-consuming process because it requires culture, which may take 4–6 weeks or a longer time. In the present study, we detected the MDR-TB and frequency of different resistance mutational patterns among clinical strains from the MDR-TB suspects in the state of Himachal Pradesh. Sputum samples from the confirmed pulmonary MDR-TB suspect patients were processed for M. tuberculosis DST by GenoType ® MTBDRplus assay. The results of this study showed that the rate of TB infection were found to be more in males and the male to female ratio is 2.6:1, which is similar to other reports.,, Age-wise distribution of patients also revealed some interesting observations. Of 770 MTB positive patients, 63.23% of the patients were young and reproductive age group (20–50 years). This observation may have strong implications in TB control strategies.
Primary drug resistance in India and abroad (undergoing category-I treatment)
Primary drug resistance to isoniazid in the present study was found to be 21.35% that is higher than other reported studies in India such as New Delhi 18.5%, Jabalpur 17%, Raichur 18.7%, Tamil Nadu 15.4%, Wardha 15%, Jaipur 13.6% and Ranchi 2.86% in newly diagnosed cases. However, primary drug resistance to isoniazid in other countries was reported from Uganda 5.8%, Germany 7.1% and Australia 8.9%. In other studies, higher resistance was reported from Bangladesh with 54.5% and Saudi Arabia with 33.8%. On the other hand, primary drug resistance to rifampicin in the present study was observed to be 19.42% that is higher as compared to other studies in India; New Delhi 0.6%, Wardha 0.5%, Jabalpur 2% and Raichur 2.5%, Bengaluru 2.6%, Ranchi 3.51%, Tamil Nadu 4.4%, Lucknow 4.7% and Jaipur 6.8%. Internationally, resistance to rifampicin was reported from China 6.6%, Uganda 1.5%, Germany 1.6% and Australia 2.6%. Higher resistance was reported from Saudi Arabia 23.5% and Bangladesh 50%. The rate of primary MDR-TB in our study was 12.62% that is comparatively higher to several reports elsewhere in India, from Lucknow 4.7% and Jaipur 4.5%. A study from Mumbai revealed the highest proportion of MDR-TB in new cases with 24% MDR-TB rate were also reported from other countries such as China 5.7% (WHO, 2011), Bangladesh 40.9%, and Saudi Arabia 20.6%. In our study, higher rate of primary drug resistance was observed as compared to other studies in India, as patients included in this study were exclusively MDR-TB suspects.
Acquired drug resistance in India and abroad (undergoing category-II treatment)
Acquired drug resistance to isoniazid in the present study was found to be 30.14%, which is lower than Mumbai with 53.2%, Jaipur 39.7% from Indian studies. This is also lower than Ethiopia with 56.1%, Bangladesh 82.6% and China 38.8%, but higher than Germany with 15.4%, Sri Lanka 5.3% and Uganda 20% in addition to very similar rate of resistance reported from Australia with 29.2% from the studies of other countries. Similarly, acquired drug resistance to rifampicin in the present study was showed 27.51%, a very similar findings reported by Malhotra et al., from Jaipur 28.2%. Higher rate of resistance were reported by other studies in India from Mumbai with 74.4% and New Delhi 33.7%. It is also higher in other countries such as Ethiopia with 46.1%, China 29.7%, Uzbekistan 62.5% and Bangladesh 80%; however, lower rate of resistance reported from Uganda with 13.4%, Germany 7.7% and Sri Lanka 2.6%. Acquired MDR-TB in the present study was exhibited 21.53% as similar resistance was reported from Jaipur with 24.3% but higher than Bengaluru 12.8%. Comparatively higher resistance was reported at New Delhi with 33.7% and Mumbai 41%.
Mutational pattern in multidrug-resistant tuberculosis cases
VanRie et al. reported in 2001 that rifampicin resistance was highly associated with mutation in the 81 base pair region of the rpoB gene. In this study, we also found that the most common mutation is associated with rpoB 530–533 region, mostly S531 L mutation (62.2%). This mutation was more frequently found in MDR-TB strains (64.07%) than in rifampicin mono-resistant strains (56.25%), similar findings were reported in South Africa, Uganda. Recently, Maurya et al. also reported 62.3% mutation in this region. Other mutations were detected in the rpoB codon, such as D516V (4.44%), H526Y (5.18%) and H526D (2.96%). Maurya et al. reported higher frequency of mutation in that codon  isoniazid resistance was most commonly associated with katG S315T1 mutation, in many high TB burden countries, presumably related to ongoing transmission of these strains., In the present investigation, we were found to be 74.32% mutation in the codon S315T1, whereas 93.3% mutation was reported by Maurya et al. In our study regarding this mutation variable were seen 86.40% in MDR strains and 46.66% in INH mono-resistant strains. In previous study, vanRie et al. reported that the mutation in the katG was less frequent (37.6%). Studies from a number of countries have been reported variability in the association of isoniazid resistance with mutations in katG or inhA. Mutation in the 315 region of katG was 93.3%, present in all INH resistant isolates worldwide and predominantly reported from Germany, Russia and other countries., In this study, the confirmed mutation in inhA gene was found 5.40% only in the C15T region with 5.40% strains and no confirmed mutation was found in other regions such as A16G, T8C and T8A of inhA gene. In MDR-TB suspect cases of Himachal Pradesh, the primary MDR-TB was found in 12.62% and acquired MDR-TB was found in 21.53% cases. Rifampicin mono-resistance was found in 6.14% cases. In rpoB gene, the most common mutation is associated with S531 L region. The GenoType ® MTBDRplus assay is a rapid test for the detection of the most common mutations in MDR-TB strains. In our study, unknown rpoB gene mutations were found in 25.18% strains that may further be detected by gene sequencing.
One limitation of the study is that phenotypic drug resistance testing (liquid culture or solid culture) was not performed. So the performance of GenoType ® MTBDRplus was not assesses as compared to culture method.
In this study, occurrence of TB infection is more in males as compared to females. The rate of primary MDR-TB was 12.62% that is comparatively higher because of patients included in the study were exclusively MDR-TB suspects. The GenoType ®
MTBDRplus assay is a rapid test for the detection of the most common mutations in MDR-TB strains. In rpoB gene, the most common mutation is associated with S531 L region. In this study, unknown rpoB gene mutations were found in 25.18% strains that may further be detected by gene sequencing.
| ~ Acknowledgment|| |
The authors would like to thank the technical members of Intermediate Reference Laboratory, Government TB Sanatorium Dharampur, Solan, Himachal Pradesh, for their technical support during research work.
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[Table 1], [Table 2], [Table 3], [Table 4]