| Abstract|| |
Detection of drug resistance in Mycobacterium tuberculosis by conventional phenotypic drug susceptibility testing methods requires several weeks. Therefore, molecular diagnostic tests for rapid detection of multidrug resistance tuberculosis (MDR-TB) are urgently needed. Early diagnosis helps in initiating optimal treatment which would not only enable cure of an individual patient but also will curb the transmission of drug resistance in the community. Line probe assay (LPA) has shown great promises in the diagnosis of MDR-TB. All MDR suspect patients from ten-linked districts were asked to deposit sputum samples at peripheral designated microscopy centers. The district TB officers facilitated the transport of samples collected during February 2014–December 2014 to our laboratory. The detection of rpoB gene mutations for rifampicin (RIF) and katG and inhA genes for isoniazid (INH), respectively, was performed on 663 samples by LPA. A total of 663 sputum samples from MDR suspects were received of which 321 (50.8%) were found to be MDR. Missing of WT8 along with mutation in codon S531 L was the most common pattern for RIF-resistant isolates (80.8%) and missing WT along with mutation in codon S315T1 of k atG gene was the most common pattern for INH-resistant isolates (91.3%).The MDR-TB in Eastern Uttar Pradesh, India, was found to be 50.8%. The common mutations obtained for RIF and INH in the region was mostly similar to those reported earlier.
Keywords: Line probe assay, multidrug resistance tuberculosis, mutation detection
|How to cite this article:|
Tripathi R, Anupurba S. Multidrug-resistant tuberculosis detection and characterization of mutations in mycobacterium tuberculosis by genotype MTBDRplus. Indian J Pathol Microbiol 2017;60:239-42
|How to cite this URL:|
Tripathi R, Anupurba S. Multidrug-resistant tuberculosis detection and characterization of mutations in mycobacterium tuberculosis by genotype MTBDRplus. Indian J Pathol Microbiol [serial online] 2017 [cited 2019 Jun 20];60:239-42. Available from: http://www.ijpmonline.org/text.asp?2017/60/2/239/208397
| Introduction|| |
India is the highest tuberculosis (TB) burden country in the world. There is a rise in incidence of multidrug-resistant TB (MDR-TB; resistance to at least isoniazid [INH] and rifampicin [RIF]), and emergence of extensively drug resistance TB (XDR-TB) which is resistance to first-line drugs as well as fluoroquinolones and one of the injectable second-line drugs (kanamycin, capreomycin, and amikacin). The global incidence of MDR-TB is 630,000 cases with India constituting one-tenth of the global burden with 64,000 cases, presently. Although the conventional drug susceptibility testing (DST) is considered the “Gold standard” for the detection of MDR and XDR-TB, however, it is time consuming about 6–8 weeks. Hence, there is a need for the introduction of rapid diagnostic tools to detect MDR-TB. Molecular assays to detect gene mutations that signal drug resistance such as the line probe assays (LPAs) are being widely recognized nowadays as best suited for rapid diagnosis for the detection of both INH and RIF drug resistance. The commercially available GenoType MTBDRplus assay version 2.0 (Hain Lifescience, Nehren, Germany) has been used in our study. This assay detects both the presence of MTB complex as well as the presence of INH and RIF resistance and can be done directly on clinical samples (smear-positive sputum). In GenoType MTBDRplus assay, a multiplex PCR is followed by a reverse hybridization of the obtained DNA amplicons to membrane-bound probes. RIF resistance is detected by the presence of mutations in the 81-bp hotspot region of the rpoB gene while INH resistance is detected by the presence of mutations in 315 codon region (high level) and mutations in the inhA promoter region (low level).,,,,, The aim of this study was to report the prevalence of MDR-TB and mutations in rpoB gene, katG gene, and inhA gene in Eastern Uttar Pradesh and confirmation of Mycobacterium other than tuberculosis (MOTTs) detected by MTBDRplus by culture.
| Materials and Methods|| |
Sputum samples were received from MDR-TB suspects from ten-linked districts. All the sputum samples that were smear positive for acid-fast bacilli (AFB) were tested by LPA. This is a retrospective analysis of the data generated from the results of our mandated diagnostic workflow. The study was approved by the institutional ethical committee. A total of 1027 diagnostic sputum samples from MDR suspect patients were received during February 2014–December 2014. The selection criteria for this report were failures of antituberculosis treatment (ATT), previously ATT-treated smear-positive cases or known contacts of MDR cases. Any new cases of TB were excluded from the study. A total of 663 AFB-positive samples were processed by NALC-NaOH decontamination method. DNA was extracted from the decontaminated samples using Genolyse ® kit (Hain Lifescience GmbH, Nehren, Germany) as per manufacturer's instructions. The extracted DNA was processed by the LPA using GenoType ® MTBDRplus (Hain Lifescience GmbH, Nehren, Germany) for detection of MTB complex and RIF and/or INH resistance according to the manufacturer's instructions.
The run was considered valid if the negative controls showed the presence of conjugate control and amplification control bands only. A positive Mycobacterium tuberculosis control (TUB) band indicated the presence of members of the M. tuberculosis complex in the sputum sample. Processed specimens of TUB negative cases (n = 31) were inoculated on plain Lowenstein–Jensen (LJ) and p-nitro benzoic acid (PNB) containing LJ to rule out the presence of MOTT. The rpoB, katG, and inhA gene loci each have a control band the presence of which is mandatory for interpretation of results. The presence of rpoB gene locus predicts RIF resistance while katG predicts high level and inhA low-level INH resistance. Absence of wild type and/or presence of mutant band signify resistance to a particular drug. The product insert was further referred for interpretation of banding patterns and troubleshooting.
| Results|| |
Obtained banding pattern of LPA is depicted in [Figure 1]. Of the total 663 smear-positive patients, 429 were males and 234 were females with male to female ratio of 1.8:1. Most patients (514 of 663, 77.5%) belonged to the age group of 15–45 years which included 352 (53.0%) in the age group of 15–30 years and 162 (24.0%) in age group of 31–45 years; 117 patients were more than 45 years, whereas 32 were <15 years. A total of 632 (413 males and 219 females) were found positive for M. tuberculosis complex [Figure 1]. However, 31/663 (4.6%) were found negative for TUB which was confirmed by culture growth on PNB-LJ. A total of 321/632 (50.8%) patients were diagnosed as MDR, 34/632 (5.4%) were diagnosed as Mono RIF resistant, 40/632 (6.3%) were found Mono INH resistant, and 226/632 (35.8%) were found as sensitive for both the drugs [Figure 1] and [Table 1]. A total 11/632 (1.7%) were found indeterminate for RIF. The indeterminate cases were referred for phenotypic DST to certified culture and DST laboratory as per the program recommendations. A total of five MDRs and three Mono RIF-resistant cases had shown heteroresistance for RIF. As RIF resistance is the surrogate marker for MDR, so we have done further Chi-square analysis to compare RIF resistant (MDR + Mono RIF-resistant cases) and RIF sensitive (sensitive by both drugs + Mono INH-resistant cases) cases in female versus male group and found an insignificant difference [Table 2]. Similarly, we found an insignificant difference for the occurrence of RIF resistance with the age group of <40 years and >40 years in two sexes (P = 0.24, 95% confidence interval 0.85–1.81).
|Figure 1: Banding pattern of GenoType MTBDRplus. Strip 1: Mono Rifampicin resistant with unknown mutations in rpoB gene (absence of WT7 but no corresponding mutation in rpoB gene), no mutation in katG and inhA gene. Strip 2 and 3: Multidrug resistance strain (absence of WT8 and presence of MUT3 in rpoB gene, absence of KatG WT, and presence of KatG MUT1 band). Strip 4: Sensitive strain for both the drugs (presence of all WT in all three genes). Strip 6: Master Mix negative control. Strip 7: DNA extraction negative control. Strip 8: Positive control (H37Rv)|
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We have categorized resistance due to known and unknown mutations of rpoB gene, katG gene, and inhA genes. The details of mutations are depicted in [Table 3]. We found 44/355 (12.4%) cases were RIF resistant due to unknown mutations. However, in common mutations, S531 L contributes maximum (80.8%). D516V, H526Y, and H526D mutations were found in 3.4% (12/355) cases, 5/355 (1.4%), and 6/355 (2.0%) cases, respectively.
|Table 3: Pattern of gene mutations detected by GenoType MTBDRplus assay in drug resistant Mycobacterium tuberculosis|
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Similarly, INH resistant due to known and unknown mutation was calculated by dividing number by MDR plus Mono INHr cases. The INH resistance due to S315T1 mutation in katG gene was found in 330/361 (91.3%) cases, whereas unknown mutations were found in 6/361 (1.7%) cases [Figure 1]. Two patients showed mutations due to a complete katG gene deletion. Furthermore, 27/361 (1.2%) INH-resistant cases were harboring mutation in both genes.
| Discussion|| |
In the recent years, a significant progress has been made in our understanding for the molecular basis of M. tuberculosis drug resistance. The World Health Organization endorsed the use of molecular LPAs for the screening of MDR-TB in June 2008 following which the GenoType MTBDRplus assay is being used routinely in different countries.,,
Data show 50.8% of MDR; this is higher than national observation of 12%–17% MDR. The reason could be selection bias of the patients as nine-linked districts were in criteria A and one district in criteria B for the MDR testing. The result could be nearer to national observation when we test in criteria C for all the linked districts. Similar findings were also reported by other authors. Singhal et al. have found 55% MDR (181 out of 328 valid LPA results) and Raizada et al. have also found 51% RIF resistant (127 out of 248 valid LPA results). However, Kumar et al. reported 25.8% of MDR.
Among all the gene mutations conferring RIF resistance, codon S531 L was found to be the most frequently encountered mutation (80.8%) which is comparable to the Indian study by Raveendran et al. in 2012 (84.6%), Mohan et al. in 2014 (81.8%) but higher than that reported in various other studies across the world (South Africa, Vietnam, and Mongolia)., The presence of mutations in codon 526 was 3.0% which is lower than the study conducted in South Africa (8.6%), Europe (15%), India (19%), Pakistan (22.5%), and Iran (45.6%),,,, but higher than the study conducted in Vietnam (1.8%). Mutations in the rpoB gene at the D516V codon were 3.4% in our study which is very low compared to the studies from South Africa (9.6%) and Europe (44%)., We also saw a 12.4% of unknown mutations in our study which could be attributed to mutations in other regions of the rpoB gene locus or mutations other than those on the strip [Figure 1] - strip 1].
The resistance of INH due to mutation in katG gene was found maximum in the S315T1 codon region amounting to 91.3%. This is in accordance with Huyen et al.'s study from Vietnam and Van Rie et al.'s study from South Africa., A high prevalence of katG mutations has been reported in high TB-prevalent countries to attribute for INH resistance and much lower prevalence in low TB-prevalent countries. This could be attributed to the on-going transmission of the strains in the high-burden settings. Two patients showed mutations due to complete katG gene deletion which is a quite possible and our results are as per the assumptions. We observed that the distribution of RIF-resistant cases into male and female groups and results suggest that male and female are equally contributed in precipitation of RIF resistance. Our results suggest that INH drug resistance is higher level due to mutation in katG gene in 94% cases. Furthermore, cases representing heteroresistant for RIF and INH due to the presence of all WT and at least one mutant band is possibly due to having mixed population of bacilli (including high lever and low level of INH resistance) in the same patient. The specimens which did not give TUB band and were able to grow on PNB-LJ medium indicate that negative TUB band could give an indication of the isolate being MOTT. This shows that smear-based diagnosis of T B could be erroneous as patients infected with NTM may wrongly be advised to take ATT. In conclusion, the heteroresistance should be reported as resistant and rapid diagnosis of TB and identification of MTB are advisable before initiation of ATT.
We acknowledge the support of PATH for infrastructure, FIND India for providing the laboratory consumables, and RNTCP for implementing the program.
Financial support and sponsorship
This study was financially supported by the Revised National Tuberculosis Control Programme.
Conflicts of interest
There are no conflicts of interest.
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Department of Microbiology, Institute of Medical Sciences, Banaras Hindu University, Varanasi - 221 005, Uttar Pradesh
Source of Support: None, Conflict of Interest: None
[Table 1], [Table 2], [Table 3]