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| Year : 2013 | Volume
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| Issue : 2 | Page : 139-143 |
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| Evaluation of GenoType® MTBDRplus assay for rapid detection of drug susceptibility testing of multi-drug resistance tuberculosis in Northern India |
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Anand Kumar Maurya1, Jyoti Umrao2, Amresh Kumar Singh2, Surya Kant3, Ram Awadh Singh Kushwaha3, Tapan N Dhole2
1 Department of Microbiology, Sanjay Gandhi Postgraduate Institute of Medical Sciences; Department of Pulmonary Medicine, King George Medical University, Lucknow, Uttar Pradesh, India
2 Department of Microbiology, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow, Uttar Pradesh, India
3 Department of Pulmonary Medicine, King George Medical University, Lucknow, Uttar Pradesh, India
Click here for correspondence address and email
| Date of Web Publication | 23-Sep-2013 |
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 Abstract | | |
Background: The problem of multi-drug resistance tuberculosis (MDR-TB) is growing in several hotspots throughout the world. Rapid and accurate diagnosis of MDR-TB is crucial to facilitate early treatment and to reduce its spread in the community. The aim of the present study was to evaluate the new, novel GenoType® MTBDRplus assay for rapid detection of drug susceptibility testing (DST) of MDR-TB cases in Northern India. Materials and Methods: A total of 550 specimens were collected from highly suspected drug resistant from pulmonary and extra-pulmonary TB cases. All the specimens were processed by Ziehl- Neelsen staining, culture, differentiation by the GenoType® CM assay, first line DST using BacT/ALERT 3D system and GenoType® MTBDRplus assay. The concordance of the GenoType® MTBDRplus assay was calculated in comparison with conventional DST results. Results: Overall the sensitivity for detection of rifampicin, isoniazid and MDR-TB resistance by GenoType® MTBDRplus assay was 98.0%, 98.4% and 98.2% respectively. Out of 55 MDR-TB strains, 45 (81.8%), 52 (94.5%) and 17 (30.9%) strains showed mutation in rpoB, katG and inhA genes respectively (P < 0.05). The most prominent mutations in rpoB, katG and inhA genes were; 37 (67.3%) in S531L, 52 (94.5%) in S315T1 and 11 (20%) in C15T regions respectively (P < 0.05). Conclusions: Our study demonstrated a high concordance between the GenoType® MTBDRplus assay resistance patterns and those were observed by conventional DST with good sensitivity, specificity with short turnaround times and to control new cases of MDR-TB in countries with a high prevalence of MDR-TB. Keywords: GenoType® MTBDRplus assay, multi-drug resistance tuberculosis, tuberculosis
How to cite this article:
Maurya AK, Umrao J, Singh AK, Kant S, Kushwaha RA, Dhole TN. Evaluation of GenoType® MTBDRplus assay for rapid detection of drug susceptibility testing of multi-drug resistance tuberculosis in Northern India. Indian J Pathol Microbiol 2013;56:139-43 |
How to cite this URL:
Maurya AK, Umrao J, Singh AK, Kant S, Kushwaha RA, Dhole TN. Evaluation of GenoType® MTBDRplus assay for rapid detection of drug susceptibility testing of multi-drug resistance tuberculosis in Northern India. Indian J Pathol Microbiol [serial online] 2013 [cited 2023 Dec 1];56:139-43. Available from: https://www.ijpmonline.org/text.asp?2013/56/2/139/118681 |
| Introduction | |  |
Tuberculosis (TB) is one of the major global health problems and the prevalence of TB is high among the developing world. A major concern is the presence of multi-drug resistant TB (MDR-TB), which is defined as resistant to both isoniazid (INH) and rifampcin (RIF). [1] World Health Organization (WHO) estimates current MDR-TB rates in new and previously treated cases globally at 2.9% and 15.3% respectively, with 57% of MDR-TB cases from three high burden countries (China, India and Russia). [2] The diagnosis of MDR-TB is based on the mycobacterial culture and drug susceptibility testing (DST) on liquid or solid media and the results are available in 4-8 weeks. [3] DST is only advised after 2-3 months of the first line anti-tubercular therapy and this may be the reason for a high burden in new cases with primary drug resistance TB. Automated liquid culture (BACTEC 460 TB, Becton Dickinson and Company, Sparks, MD, USA and BacT/ALRERT 3D system, bioMerieux, Durham, USA) have significantly shortened turnaround times compared with solid media, but still require isolation of mycobacterial cultures prior to DST. [3] Rapid detection of DST permits the establishment of an effective treatment regimen, reduces the risk of further resistance development and limits the spread of MDR-TB. [4] In June 2008, WHO recommended the use of molecular line probe assay for the diagnosis of MDR-TB. [5] The GenoType® MTBDRplus assay (Hain Lifescience, Nehren, Germany) is a commercially available assay that combines detection of Mycobacterium tuberculosis complex (MTBC) with prediction of resistance to RIF and INH. In this assay, a multiplex polymerase chain reaction (PCR) is followed by hybridization of the obtained deoxyribonucleic acid (DNA) amplicons to membrane-bound probes. The assay combines detection of MTBC with detection of mutation(s) in the 81-bp hotspot region of rpoB, at codon 315 of the katG gene and in the inhA promoter region. [6],[7],[8] The aim of this study was to evaluate the novel GenoType® MTBDRplus assay for rapid detection of DST of MDR-TB cases in Northern India.
| Materials and Methods | | |
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. An informed consent was taken from all the subjects enrolled in this study. All patients who were attending the out-patient department and indoor patients department of various wards/units of two tertiary care centers and referred cases from other peripheral health-care centers in Northern India were screened for MDR-TB from January 2011 to September 2012 as we mainly recruits treatment failures and complicated TB cases.
Criteria for inclusion
Patients included in the study were both new or previously treated suspected DR-TB cases from all age groups, in whom TB was confirmed by culture and in whom DST against MTBC strains had been performed. Those infected with mycobacteria other than TB (MOTT) were not included in this study.
Microbiological methods
All the clinical specimens were received and subjected to direct smear microscopy by Ziehl-Neelsen (ZN) staining method. [9] Specimens which contain commensal bacterial flora, were decontaminated by the standard N-acetyl-L-cysteine-NaOH method. [10] Specimens from sterile sites were centrifuged and the sediment was inoculated into the BacT/ALERT mycobacteria process (MP) vials of the BacT/ALERT 3D system (bioMerieux, Durham, USA) containing modified Middlebrook 7H9 with an antibiotic supplement (amphotericin B [0.018%, wt/vol], azlocillin [0.0034%, wt/vol], nalidixic acid [0.04%, wt/vol], trimethoprim [0.00105%, wt/vol], polymyxin B [10,000 U] and vancomycin [0.0005%, wt/vol]). The BacT/ALERT MP vials were monitored continuously by the BacT/ALERT 3D system. [11] All the positive vials were subjected to smear microscopy for the presence of acid fast bacilli (AFB). No growth after 6 weeks of incubation was considered as negative for mycobacteria. Positive cultures for mycobacterium were typed by niacin production, catalase activity at 68°C and pH 7 and susceptibility to p-nitrobenzoic acid. [12]
Identification of MTBC strains
The identification and differentiation of MTBC from MOTT were performed by the GenoType® CM assay as per the manufacturer's instructions (Hain Lifescience GmbH, Nehren, Germany). [13] The standard strain of MTBC, H37 Rv ATCC™ No. 27294 was used as a positive control.
BacT/ALERT DST by standard 1% proportion method
The BacT/ALERT MB susceptibility reagents and the BacT/ALERT MP bottles were procured from bioMerieux, Durham, USA. DST was performed for the first line anti-tubercular drugs as per the manufacturer's protocol. [11] Briefly, 0.5 ml of the lyophilized antibiotic solutions and 0.5 ml restoring fluid were added to the BacT/ALERT MP test bottles and the undiluted direct control bottle, respectively. The final drug concentrations in the test bottles were 0.9 mg/l for RIF, 0.4 mg/l for INH. Seeded inoculum 0.5ml was added to all BacT/ALERT MP test bottles. All the bottles were loaded into the BacT/ALERT 3D system simultaneously and the maximum test time was automatically limited to 15 days. The same standard strain of MTBC, H37 Rv ATCC™ No. 27294 was used as a positive control.
GenoType® MTBDRplus assays
All culture positive MTBC clinical isolates were subjected to the first line anti-tubercular resistance, concordance and mutational analysis by the GenoType® MTBDRplus assay as per manufacturer's instructions (Hain Lifescience GmbH, Nehren, Germany). 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 polymerase (QIAGEN, Hilden, Germany) and 5 μl of a preparation of chromosomal DNA in a final volume of 50 μl were used. The amplification protocol consisted of 15 min of denaturation at 95°C, followed by 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. Reverse hybridization and detection was performed in an automated washing and shaking device (Profiblot; Tekan, Maennedorf, Switzerland). The hybridization procedure was performed at 45°C for 0.5 h, followed by washing steps and the colorimetric detection of the hybridized amplicons. After a final wash, the strips were air-dried and fixed on paper provided with the kit.
The GenoType® MTBDRplus strip contains 17 probes, including amplification and hybridization controls to verify the test procedures. For the detection of rifampicin resistance, eight rpoB wild-type probes (probes WT1 to WT8) encompass the region of the rpoB gene encoding amino acids 509-533. Four probes (probes rpoB MUT D516V, rpoB MUT H526Y, rpoB MUT H526D and rpoB MUT S531L) specifically target the most common mutations conferring resistance to RIF. For the detection of INH resistance, one probe cover the wild-type S315 region of katG, while two others (probes katG MUTT1 and MUTT2) are designed to assess the AGC-to-ACC (S315T) and the AGC-to-ACA (S315T) mutations. Furthermore, the promoter region of the inhA gene is included on the new strip and encompasses the regions from positions -15 to -16 for the inhA WT1 probe and positions -8 for the inhA WT2 probe. Four mutations (-15C/T, -16A/G, -8T/C and -8T/A) can be targeted with the inhA MUT1, MUT2, MUT3A and MUT3B probes. Again, either the absence of one or more wild-type probe(s) or the presence/staining of mutant probes were indicative of the resistant strain [Figure 1]. | Figure 1: Representative deoxyribonucleic acid strip patterns obtained with the GenoType® MTBDRplus assay. The positions of the oligonucleotide probes are given on the right. Lane 1: Mycobacterium tuberculosis complex H37 Rv control strain (rpoB, katG, inhA WT). Lane 2: Absence of TUB (excluded from the study analysis). Lane 3: Fully susceptible strain (rpoB, katG, inhA WT). Lane 4: Multi-drug resistant tuberculosis (absence of WT) [3,4] and presence of MUT1 D516V in rpoB, katG presence of MUT1 S315T1 and absence of WT 1
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Data and statistical analysis
The sensitivity, specificity, positive predictive value and negative predictive value of GenoType® MTBDRplus assay results were compared with the conventional BacT/ALERT 1% proportion DST results for RIF, INH and MDR-TB. Data were analyzed using the SPSS 15.0 (Statistical Package for the Social Sciences, Chicago, IL, USA) for Windows. For comparison of data, P < 0.05 was considered to be statistically significant.
| Results | | |
Smear microscopy, culture and species identification
Among a total of 550 specimens collected from 423 (76.9%) pulmonary TB (PTB) and 127 (23.1%) extra-pulmonary TB (EPTB) patients of highly suspected cases of treatment defaulters, re-treatment and relapse cases, only 103 (18.7%) were AFB positive in ZN microscopy and 257 (46.7%) were positive for mycobacteria by BacT/ALERT 3D system. After using a panel of different biochemical and GenoType® mycobacterium CM assay; 209 (81.3%) strains confirmed as MTBC and 48 (18.7%) were confirmed as one of the species of MOTT.
Phenotypic DSTs by 1% proportional method
DST by 1% proportion method was performed on 209 MTBC strains, among which 55 (26.4%) cases were new and 154 (73.6%) were previously diagnosed and treated TB cases (P < 0.05). A total of 123 (58.5%) strains were identified as resistant to one or more than one anti-tubercular drug and 86 (41.5%) strains were pan-susceptible (SS). Out of 123 resistance cases, 55 (44.7%) strains were identified as MDR-TB and 68 (55.3%) strains were monoresistance to either INH or RIF.
Genotypic DSTs by GenoType® MTBDRplus assay
Among a total of 209 MTBC culture isolates, 206 (98.5%) MTBC isolates showed readable results by using GenoType® MTBDRplus assay and remaining 3 (1.5%) isolates were excluded from the study due to unreadable or having either no bands at all or very light/weak/unreadable bands in rpoB, katG and/or inhA sections. Among a total of 55 MDR-TB strains, 45 (81.8%), 52 (94.5%) and 17 (30.9%) strains harbored known mutation in rpoB, katG and inhA genes respectively. The most prominent mutations in rpoB, katG and inhA genes were 37 in S531L (67.3%), 52 in S315T1 (94.5%) and 11 in C15T (20%) region respectively (P < 0.05).
Concordance between conventional DST and GenoType® MTBDRplus assay
The concordance of the GenoType® MTBDRplus assay was calculated in comparison with conventional DST results in a total of 206 MTBC isolates tested. Considering the phenotypic proportion DST method is the gold standard, the sensitivity and specificity of the GenoType® MTBDRplus assay for RIF, INH and MDR-TB strains were 98.0% (95% confidence interval [CI]: 92.88-99.70%); 98.8% (95% CI: 93.59-99.80%), 98.4% (95% CI: 94.24-99.76%); 98.8% (95% CI: 93.67-99.81%) and 98.2% (95% CI: 90.24-99.70%); 100.0% (95% CI: 97.56-100.00%) [Table 1]. | Table 1: Concordance of GenoType® MTBDRplus assay for detecti on of RIF, INH and MDR-TB
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Analysis of disagreements between phenotypic DST and GenoType® MTBDRplus assay
Comparison of the results revealed some disagreement between two sets of data (molecular DST and phenotypic DST). In a group of strains phenotypically sensitive to RIF from culture isolates identified as resistant by using the GenoType® MTBDRplus assay (n = 2), no mutation in rpoB gene were found (ΔWT). In a group of strain phenotypically resistant to INH from culture isolates identified as sensitive by using GenoType® MTBDRplus assay. In third discrepant strain, RIF was resistant in phenotypic DST, but it was sensitive by the GenoType® MTBDRplus assay [Table 2]. | Table 2: Three discrepant RIF and INH resistance results using the GenoType® MTBDRplus assay and phenotypic DST
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| Discussion | | |
In the present study, we studied the concordance of the phenotypic DST and GenoType® MTBDRplus assay for the rapid detection of MDR-TB resistance among culture isolates from PTB and EPTB patients in the Northern India. The performance of the GenoType® MTBDRplus assay was correlated very accurately with DST by 1% proportion method using BacT/ALERT 3D system. Overall the sensitivity of the GenoType® MTBDRplus assay the detection of RIF, INH and MDR-TB was high at 97.98%, 98.37% and 98.2% respectively, which was similar to previously reported studies from different countries. [4],[6],[8],[14],[15],[16],[17],[18] Different studies have already demonstrated the feasibility of MTBDRplus assay as an effective tool in early detection of MDRTB and have good concordance with phenotypic DST. [8],[15],[19] In our study, the GenoType® MTBDRplus assay showed high sensitivity and specificity for the detection of RIF (97.98-98.82%) as compared with conventional DST. However, for detection of INH resistance, in comparison with the conventional DST, the sensitivity and specificity of MTBDRplus assay was 98.37-98.84%. Our result was with concordance with other different studies, in which they showed good concordance between conventional DST and the GenoType® MTBDRplus assay. [8],[15],[19] We found 3 (1.5%) unreadable results or having either no band(s) at all or very light/weak/unreadable bands in rpoB, katG and/or inhA sections by the GenoType® MTBDRplus assay. The reason may be explained by the fact that inhibitors in the specimen can lead to inhibition of amplification of MTBC genome in the specimen without any effect on the amplification control in the primer-nucleotide mix and probably due to the non-homogeneous distribution of bacilli in the specimen and absence of bacilli in the PCR mixture. [20] We found, rpoB gene detected very frequently 48 (81.8%) among RIF resistant strains, which was similar to finding from a recent South African study. [18] For INH resistance, katG gene was the most common mutational gene seen in 52 (94.5%) MTBC strains in our study, which was not in accordance with different previous studies in that it was less frequent (37.6-72%, P = 0.01). [18],[21],[22] We found mutation(s) in INH resistant strains, which carried a mutation in the inhA promoter region was 30.9% and was considerably lower than 40% in a different study (P = 0.007). [18],[23] Among three RIF and INH discrepant resistance strains, the reasons may be explained by the fact that; firstly by the presence of "heteroresistance" i.e., simultaneous presence of both drug resistant and sensitive MTBC bacilli in culture and secondly by the fact that all the responsible probe(s) were not included in the GenoType® MTBDRplus assay strips conferring resistance in MDR-TB strains. [4],[24] However, the molecular basis of falsely RIF and INH resistant isolates (n = 3) warrants further investigation by molecular sequencing; although, the number of discrepant strains (03) were small in this study.
The GenoType® MTBDRplus assay is a rapid DST method and turnaround times is one working day and it is easy to perform, less expensive and superior from phenotypic DST because it requires culture, which may require 4-6 weeks or a longer time. Phenotypic DST of the first line anti-tubercular drugs by conventional methods is difficult due to various technical reasons and the results are not always accurate. [24] However, automated liquid culture systems have significant shortened turnaround times as compared with conventional solid media. These systems may not be feasible in laboratories in low to middle income countries with a high burden of TB and MDR-TB due to lack of proper infrastructure and resources. The GenoType® MTBDRplus assay has been used as an effective tool for MDR-TB screening and has short turnaround times in a high-volume facility to control and prevent new cases of MDR-TB in developing countries.
To conclude, the GenoType® MTBDRplus assay is a rapid screening test with high sensitivity and specificity for simultaneous detection of RIF and INH resistance along with diagnosis of MDR-TB. It has a potential to substantially reduce the turnaround times over conventional DST methods. Our study demonstrated a high concordance between this assay resistance patterns and those observed by conventional DST method. This assay could be used for the accurate diagnosis and therapeutic management of TB patients in the community with a high burden of MDR-TB. This test may be a useful tool for WHO Global Task Force and Revised National Tuberculosis Control Program to control and prevent new cases of MDR-TB in the community.
| Acknowledgments | | |
This work was supported by a grant from Indian Council of Medical Research, New Delhi (Extramural ICMR Project Sanction No. 5/8/5/4/2007-ECD-I). The 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.
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Correspondence Address:
Tapan N Dhole
Department of Microbiology, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow-226 014, Uttar Pradesh
India
 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  | Check |
DOI: 10.4103/0377-4929.118681
[Figure 1]
[Table 1], [Table 2] |
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