|Year : 2014 | Volume
| Issue : 3 | Page : 270-276
Association of MDR-TB isolates with clinical characteristics of patients from Northern region of India
A Gupta1, MR Nagaraja2, P Kumari3, G Singh4, R Raman3, SK Singh5, S Anupurb1
1 Department of Microbiology, Institute of Medical Sciences, Uttar Pradesh, India
2 Department of Endocrinology and Metabolism, Institute of Medical Sciences, Uttar Pradesh; Department of Biochemistry, Chikka Muniyappa Reddy Institute of Management Studies, Bangalore, Karnataka, India
3 Department of Zoology and Centre for Genetic Disorder, Banaras Hindu University, Varanasi, Uttar Pradesh, India
4 Division of Biostatistics, Institute of Medical Sciences, Uttar Pradesh, India
5 Department of Endocrinology and Metabolism, Institute of Medical Sciences, Uttar Pradesh, India
|Date of Submission||10-Apr-2013|
|Date of Acceptance||17-Dec-2013|
|Date of Web Publication||10-Jul-2014|
Department of Microbiology, Institute of Medical Sciences, Uttar Pradesh
Source of Support: None, Conflict of Interest: None
Purpose: We sought to determine the characteristics and relative frequency of transmission of MDR-TB in North India and their association with the clinical and epidemiological characteristics of TB-patients. Materials and Methods: To achieve the objectives PCR-SSCP, MAS-PCR and direct DNA sequencing were used against 101 Mycobacterium tuberculosis isolates. Results: Multidrug-resistant-TB isolates were found to be significantly higher (P = 0.000) in previously treated patients in comparison to newly diagnosed patients. Further, significant differences (P = 0.003) were observed between different age groups (Mean ± SD, 28.6 ± 11.77) of the TB patients and multidrug resistance. Most frequent mutations were observed at codons 531 and 315 of rpoB and katG genes, respectively, in MDR-TB isolates. Conclusion: Routine surveillance of resistance to anti-TB drugs will improve timely recognition of MDR-TB cases and help prevent further transmission in Northern India.
Keywords: Deoxyribonucleic Acid sequencing, Human Immunodeficiency Virus, Multidrug-resistant-tuberculosis, Polymerase Chain Reaction-single-strand conformation polymorphism, tuberculosis
|How to cite this article:|
Gupta A, Nagaraja M R, Kumari P, Singh G, Raman R, Singh S K, Anupurb S. Association of MDR-TB isolates with clinical characteristics of patients from Northern region of India. Indian J Med Microbiol 2014;32:270-6
|How to cite this URL:|
Gupta A, Nagaraja M R, Kumari P, Singh G, Raman R, Singh S K, Anupurb S. Association of MDR-TB isolates with clinical characteristics of patients from Northern region of India. Indian J Med Microbiol [serial online] 2014 [cited 2020 Apr 10];32:270-6. Available from: http://www.ijmm.org/text.asp?2014/32/3/270/136561
| ~ Introduction|| |
Multidrug-resistant (MDR) tuberculosis (TB), defined as resistance to both isoniazid (INH) and rifampicin (RIF), is a worldwide problem and is one of the greatest challenge facing public health particularly in resource-poor settings where adequate diagnosis and treatment are often unavailable.
MDR-TB reduces responses to standard short-course chemotherapy with first-line anti-TB drugs, leads to higher mortality and treatment failure rates, and increases the period of transmissibility of the disease  as a result of which super-resistant strains have emerged. India ranks first out of the 22 countries with the highest burden of the disease, but in spite of this, information on the magnitude of MDR-TB in the country is largely unavailable. 
The advent of HIV/AIDS in the 1980s resulted in an increase in transmission of TB associated with outbreaks of MDR-TB.  The dual epidemics of HIV infection and MDR-TB threaten global TB control, especially in developing countries including India. In the early 1990s, drug resistance surveillance was resumed in developed countries, but the true incidence remained unclear in the developing world. 
Treatment of MDR-TB is difficult even in resource rich settings. Patients are generally treated for a minimum of 18-24 months with second-line TB drugs that have significant adverse effects.  Most studies of MDR-TB treatment have come from countries where HIV co-infection is uncommon. ,, Very little is known about outcomes of MDR-TB treatment in HIV-prevalent settings such as India.
Prompt detection of anti-TB drug resistance is essential for controlling the development and spread of MDR-TB, as it facilitates appropriate and timely delivery of anti-TB therapy, reducing overall cost of treatment and transmission of resistant cases. Several methods such as direct DNA sequencing, , single-strand conformation polymorphism (SSCP)-PCR analysis, , restriction fragment length polymorphism-PCR,  multiplex-allele-specific (MAS)-PCR), , Genotype MTBDRplus  and many more, are designed to observe specific mutations responsible for drug resistance in Mycobacterium tuberculosis. Among these many techniques, automated direct DNA sequencing of PCR products has been considered as the gold standard.
We have used PCR-SSCP, MAS-PCR and direct DNA sequencing to understand the frequency and type of mutations in genes associated with multidrug resistance in M. tuberculosis isolates in Northern India and their association with the clinical and epidemiological characteristics of patients.
| ~ Materials and Methods|| |
Our University hospital has a vast catchment area, being the only tertiary care hospital in North-eastern Uttar Pradesh (UP) providing medical cover to over 15 crore of population of Eastern UP, Western Bihar, adjoining areas of Madhya Pradesh and Nepal.
Sputum specimens were collected during the period of January 2008 to January 2010, from clinically suspected pulmonary TB patients attending various OPDs of University hospital. Our hospital, a tertiary care centre with a referral bias towards non-responding cases, is located in Northern India.
M. tuberculosis isolates/conventional drug susceptibility testing
One-hundred and one M. tuberculosis isolates were isolated, identified and subjected to indirect DST according to the gold standard proportion method (PM). , H37Rv (ATCC 27294) and a known MDR strain were used as negative and positive controls, respectively.
For the purpose three rapid HIV test kits based on different antigens/principles were used according to National AIDS Control Organization (NACO) guidelines.
Multiplex-allele-specific PCR assay for detection of RIF and INH resistance determinants
Bacterial DNA was extracted by using the protocol of Van Embden et al., with slight modifications.
A two-step MAS-PCR assay was performed to detect mutations at rpoB codons (516, 526 and 531) and katG codon 315. ,
PCR-SSCP for rpoB mutation
A 193-bp fragment of the rpoB gene (GenBank accession number L27898, nucleotides 2288 to 2480) partial sequence including the rifampicin-resistant determining region (RRDR) (81-bp region within rpoB gene that encodes 507 to 533) was amplified by using the primers  given in [Table 1]. Concentrations of the PCR reaction mixture and PCR conditions were according to the protocol of Sheng et al., with minor modifications.
|Table 1: Primers sequence for amplification of rpoB and katG gene segments |
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PCR-SSCP for katG mutation
For each PCR reaction, to amplify part of katG (209 bp) gene, a standard 20 μl reaction mixture was used. Each reaction mix included two primers [Table 1]. 50 pmol in 5 μl, respectively, 0.6 μl of 200 μmol/l dNTP mix, 3 μl of Mg2+-containing 10 × PCR reaction buffer, 1 Unit (0.33 μl) Taq Polymerase Mix and 16.07 μl PCR-grade water. One microliter DNA solution containing 25 ng DNA template was used. The thermocycling parameters included an initial denaturing at 94°C for 5 min, 42 cycles of 94°C for 1 min, 66°C for 1 min and 72°C for 1 min and final extension at 72°C for 10 min.
Screening of PCR-SSCP products
The PCR products were analysed by PCR-SSCP by using the method of Bobadilla-del-Valle et al.,  with some modifications.
DNA sequencing of katG and rpoB gene segments
Samples showing shift in SSCP gel was eluted and purified from agarose gel using a HiYield TM Gel/PCR DNA Extraction Kit (Real Biotech Co., Taipei County, Taiwan) for DNA sequencing.
Automated sequencing was performed using the BigDye Terminator kit v3.1 (Applied Biosystems, USA) protocol. DNA sequence analysis and comparisons of rpoB and katG were carried out with Chromas V.2.3 and FinchTV softwares and web-based programmes. The rpoB and katG sequences from M. tuberculosis H37Rv strain, GenBank accession reference numbers L27898 and X68081, respectively, were used to construct the alignments.
We used the Chi-square test of proportions to identify significant differences between two or more groups of patients. A P < 0.05 was considered statistically significant. Odd ratios (ORs) and 95% confidence intervals (CI) were calculated to measure the association between patient characteristics and the outcome of interest. All analyses were performed using SPSS statistical software (version 16.0.0) (SPSS Inc., Chicago, IL, USA).
The study was approved by the ethical committee of the Institution.
| ~ Results|| |
M. tuberculosis isolates/conventional drug susceptibility
A total of 101 M. tuberculosis isolates, from pulmonary TB patients, were included in the present study. Forty nine (48.51%) were detected susceptible to all four first line drugs i.e., INH and RIF, STR and EMB while 52 (51.49%) were detected multidrug resistant with or without resistance to any other tested anti-tubercular drugs. Further, new and retreated cases were 52 (51.49%) and 49 (48.51%), respectively.
PCR for rpoB and katG genes
The results of PCR detection revealed that the expected band of 193 and 209 bp containing rpoB and katG gene segments of interest were obtained from all 101 clinical TB isolates.
Results of PCR-SSCP
All tested susceptible isolates (H37Rv strain and 49 clinical isolates) displayed identical SSCP patterns [Figure 1]a. Lane 1; [Figure 1]b. Lane 1-2]. Among 52 MDR-TB isolates, 37 (71.15%) and 39 (75%) RIF and INH-resistant mutants, respectively, showed abnormal SSCP patterns [Figure 1]a. Lane 2-4; [Figure 1]b. Lane 3-4]. Further, 25 (48.08%) isolates were detected as MDR.
|Figure 1: (a) SSCP analysis of standard sensitive and RIF-resistant strains. Lane 1: H37Rv standard sensitive; lane 2, 3, 4, RIF-resistant strains (b) SSCP analysis of katG-sensitive and resistant strains. Lane 1: H37Rv standard sensitive; lane 2 katG sensitive; lanes 3, 4 katG resistant strains|
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Results of DNA sequencing
Of 52 MDR-TB isolates rpoB and katG gene sequence analysis was done for 40 isolates whereas resistance pattern of the remaining 12 isolates had been previously analyzed by MAS-PCR assay. Further, four pan drug-susceptible TB isolates were also analyzed by DNA sequencing.
Sequencing of the 193-bp central region of the rpoB gene revealed point mutations at 4 different codons in 36/40 isolates. Most mutations were found at codons 531 (n = 27) and 526 (n = 6). In addition, 2 point mutations were detected at codon 511 and one isolate was found to have mutation at codon 521 [Table 2]. Analysis of MAS-PCR assays showed the presence of mutations in 531 (8/12), 526 (2/12) and 516 (2/12) codons respectively [Table 3]. All these mutations produced five changes in amino acid/nucleotide content [Table 2] and [Table 3]. In total, of 52 MDR-TB isolates 48 had mutation. Of 48 isolates, mutations in 531, 526, 516, 511 and 521 were found among 35 (67.31%), 8 (15.38), 2 (3.85%), 2 (3.85%) and 1 (1.92%) isolates, respectively. While, 4 of 52 MDR isolates had no mutations at this region of the rpoB gene [Table 2] and [Table 3]. No mutations were found in the core region of the rpoB gene in the 4 RIF-susceptible isolates.
|Table 2: Drug - resistance profile and mutations found in rpoB and katG genes in MDR-TB isolates as determined by DNA sequencing (n=40) |
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|Table 3: Drug - resistance profile and mutations found in rpoB and katG genes in MDR-TB isolates as determined by MAS-PCR (n=12) |
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Partial katG gene sequencing revealed mutations at codon 315 in 36/40 MDR-TB isolates. Further, all 12 MDR-TB isolates, analysed by MAS-PCR, had mutations at codon315 of katG gene segment. In total, of 52 MDR-TB isolates 48 (92.31%) had katG315 substitution. While, four of 52 MDR isolates had no mutations at katG315 region [Table 2] and [Table 3]. No mutations were found in the studied region of the katG gene in the 4 INHs isolates. Further, 1 MDR-TB isolate showed no mutation in the studied segment of rpoB and katG genes.
Sensitivity and specificity
Of the 52 culture-proven MDR-TB isolates, 25 and 33 isolates were identified as MDR by PCR-SSCP and DNA sequencing, respectively. Of 33 DNA sequencing confirmed MDR-TB isolates, only 12 were detected as MDR by PCR-SSCP. All of the 49 non-MDR isolates defined by culture were classified as non-MDR isolates by PCR-SSCP [Table 2] and [Table 3]. The sensitivity of the PCR-SSCP method for MDR detection as compared to PM and DNA sequencing was found to be 48% and 52%, respectively, while specificity was 100%.
The sensitivities of the PCR-SSCP in comparison to conventional DST for the detection of RIF and INH resistance was found to be 71% and 75%, respectively. Specificities were observed to be 100% for both the drugs. Further, the sensitivities of the PCR-SSCP in comparison to DNA sequencing for the detection of rpoB and katG mutations were found to be 64% and 69% respectively and specificities were observed to be 100% for both the drugs.
Association of MDR-TB isolates with clinical and epidemiological characteristics of TB patients
We compared the patient characteristics associated with MDR, such as age, sex, treatment history, HIV status and bacteriological and radiological findings [Table 4]. Multidrug-resistant-TB isolates were significantly higher in HIV sero-negative patients than HIV sero-positive patients (94.23% Vs 79.59%; OR, 0.239; P = 0.028; 95% CI, 0.048-1.037), as well as in the previously treated patients in comparison to newly diagnosed patients (67.31% vs. 32.69%; OR, 5.147; P = 0.000; 95%CI, 2.036-13.236). Further, significant differences (P = 0.003) were observed between different age groups (Mean ± SD, 28.6 ± 11.77) of the patients and MDR-TB isolates. Positive smear remained associated with MDR-TB, but the association was not statistically significant (OR 0.750; CI 95% 0.255-2.182). However, there were no significant association found between MDR-TB isolates, gender and radiological findings of the patients [Table 4].
|Table 4: Association of MDR - TB isolates with clinical and epidemiological characteristics of TB patients |
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| ~ Discussion|| |
Sensitivity and specificity
Our data showed that PCR-SSCP was highly specific (100%) with acceptable sensitivity for detecting mutations in the rpoB and katG gene in RIF and INH-resistant isolates of M. tuberculosis i.e., 71 and 75%, respectively, in comparison to conventional DST and 64% and 69%, respectively, in comparison to DNA sequencing. In addition, sensitivity of the PCR-SSCP method for MDR detection as compared to PM and DNA sequencing was found to be 48% and 52%, respectively, with 100% specificity. In comparison to our findings the study of Tavakoli et al., demonstrated high specificity (93.8%) and sensitivity (95.2%) of PCR-SSCP method for the detection of RIF-resistant mutations. Lee et al.,  found 75% sensitivity similar to our study. Cheng et al., developed a multi-PCR-SSCP method and found sensitivities and specificities of 80%, 81.8% and 100%, 92% for INH and RIF, respectively. Contrary to these studies Bobadilla-del-Valle et al., showed very low sensitivity of the assay (31.4%); however, the specificity was 100%.
In our study, results of DNA sequencing combined with MAS-PCR assay for 52 MDR-TB isolates showed the presence of mutations at five different codons of rpoB gene with the frequencies of 35 (67.31%), 8 (15.38), 2 (3.85%), 1 (1.92%) and 2 (3.85%) at 531, 526, 516, 521 and 511 codons, respectively, while no mutations were detected in RRDR region of 4 RIF-resistant isolates. Most frequent mutation was observed at codon 531 (35/48). In a study from USA,  13 different types of missense mutations were identified in 32 RIF-resistant strains at codons 509, 511, 516, 522, 526, 531, 533, 550 and 572 and the most frequent mutation was observed at codon 531 (Ser to Leu, 42.1%). Further, Veronika Slizen  from Belarus found 20 types of mutations in her study. Mutations were detected in codons 507, 508, 510, 512, 516, 520, 521, 523, 525, 526 and 531. Most of the mutations were located in 510, 526, 523, 531 codons in 47.7, 45.5, 38.6, 29.5% of M. tuberculosis isolates correspondingly. Interestingly in a study from Belarus by Bostanabad et al., 60 mutations and 13 microdeletions were identified in 29 RIF-resistant M. tuberculosis (85%). Among 60 mutations, six silent (codon 507) and 54 missense were identified. Missense mutations produced 23 types of amino acid substitutions. Most frequent mutations were detected in codons 523 and 526.
In our study, partial katG gene sequencing revealed mutations at codon 315 in 48/52 (92.31%) MDR-TB isolates while four of 52 MDR isolates had no mutations at katG315 region. Similar results were reported in Latvia and Iran. , However, the frequency of katGS315T mutation may vary according to geographical area, even within a single country. A study from India has reported 64% of 70 isolates with the same mutation.  Similarly, 62% of 79 isolates from Spain and 67% of 37 isolates from the North of Mexico have been reported. ,
Association of MDR-TB isolates with clinical and epidemiological characteristics of TB patients
In the present study, MDR TB was significantly associated with previously treated patients with TB and among those without HIV infection than with HIV infection. In contrary, a study from Netherlands found MDR-TB to be significantly more frequent among previously untreated patients with TB and HIV infection than among those without HIV infection. Murase et al.  found that the patients with MDR-TB were significantly more likely to be previously treated. In a study by Quy et al.,  the cure rate was 87% among new patients compared to only 73% among previously treated patients. Failure was associated with multidrug resistance but not with HIV infection. Mortality was significantly associated with HIV infection, multidrug resistance and other resistance to two or more drugs.
In our study, significant differences (P = 0.003) were observed between different age groups (Mean ± SD, 28.6 ± 11.77), with high proportion in the age group between 0 and 20 years, of the patients and MDR-TB isolates. Positive smear remained associated with MDR-TB, but the association was not statistically significant (OR 0.750; CI 95% 0.255-2.182). A study from Tokyo, Japan found that the patients with MDR TB were significantly more likely to be younger, for age range of 21-40 years. All MDR-TB patients had pulmonary disease, and these patients were significantly more likely to have cavitary lesions and to have positive sputum smear test results.  Similar to our finding Flament-Saillour et al.,  also observed the association of positive smear with MDR-TB, but the association was not statistically significant.
It has been reported that the Beijing family is frequently associated with drug resistance.  In one of our previous studies (Unpublished data) we found that 21.15% (11/52) of the MDR isolates were Beijing genotype of M. tuberculosis. However, Murase et al.  found a higher proportion of Beijing family genotype (62%, n = 34) in MDR-TB cases.
| ~ Conclusions|| |
Routine surveillance of resistance to anti-TB drugs will improve timely recognition of MDR TB cases and help prevent further transmission in our region. The information obtained will provide a baseline data that can be used to design intervention programmes for prevention of MDR-TB not only in India but also in other resource-limited countries with high burden of TB.
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[Table 1], [Table 2], [Table 3], [Table 4]