| Abstract|| |
Aim: This study was performed for the rapid identification of Mycobacterium tuberculosis complex and its resistance to rifampicin and isoniazid, directly from the sputum samples of pulmonary tuberculosis patients. Materials and Methods: A commercially available genotype MTBDR plus assay was used for the identification and detection of mutations in Mycobacterial isolates. A total of 100 sputum samples of pulmonary tuberculosis patients were analyzed by using the genotype MTBDR plus assay. The MTBDR plus assay is designed to detect the mutations in the hotspot region of rpoB gene, katG and regulatory region of inhA gene. Results: The genotype MTBDR plus assay detected 22% multidrug resistant (MDR), 2% rifampicin (RMP) monoresistant and 1% isoniazid (INH) monoresistant isolates. In 22 MDR isolates, the codons most frequently involved in RMP-associated mutations were codon 531 (54.55%), 516 (31.82%) and 526 (13.63%), and 90.90% of MDR isolates showed KatG S315T mutations and 9.1% showed inhA C-15T mutations associated with INH resistance. Conclusion: The new genotype MTBDR plus assay represents a rapid, reliable tool for the detection of MDR-TB, wherein results are obtained in 5 h allowing early and appropriate treatment, which is essential to cut the transmission path and reduce the spread of MDR-TB. The genotype MTBDR plus assay can readily be included in a routine laboratory work for the early diagnosis and control of MDR-TB.
Keywords: MDR-TB, molecular diagnosis, genotype MTBDR plus assay
|How to cite this article:|
Sharma S, Madan M, Agrawal C, Asthana AK. Genotype MTBDR plus assay for molecular detection of rifampicin and isoniazid resistance in Mycobacterium tuberculosis. Indian J Pathol Microbiol 2014;57:423-6
|How to cite this URL:|
Sharma S, Madan M, Agrawal C, Asthana AK. Genotype MTBDR plus assay for molecular detection of rifampicin and isoniazid resistance in Mycobacterium tuberculosis. Indian J Pathol Microbiol [serial online] 2014 [cited 2019 Aug 17];57:423-6. Available from: http://www.ijpmonline.org/text.asp?2014/57/3/423/138738
| Introduction|| |
The emergence and spread of MDR-TB is an increasing public health problem in India, with an estimated number of 110,000 cases spread across the country (TB India, 2009).  Multidrug-resistant Mycobacterium tuberculosis (MDR-TB), defined as resistance to rifampicin and isoniazid, poses a serious threat to the treatment of tuberculosis (TB) worldwide.  For the treatment of MDR - TB isolates, more expensive and more toxic second-line drugs are required.  Rifampicin, first introduced in 1972 as an antitubercular drug, is extremely effective against MTB.  Rifampicin, along with INH, forms the backbone of short-course chemotherapy.  As rifampicin resistance is relatively rare, detection of rifampicin resistance serves as surrogate marker for detecting MDR-TB.  The mutations associated with rifampicin resistance are located in the rpoB gene; specifically, 95% of these mutations are found in the well-defined 81-bp core region, spanning codon 507-533, known as the rifampicin resistance-determining region (RRDR). , However, INH resistance in M. tuberculosis is more complex because it involves mutations in more than one gene or gene complex, , such as the katG, inhA, and kasA genes, and the intergenic region of the oxyR - ahpC complex.
The routine culture and drug susceptibility testing methods are time consuming, which would also delay the diagnosis of MDR-TB. A delay in the diagnosis of MDR-TB associated with standard drug susceptibility testing method is likely to contribute to the transmission of disease. Molecular genetic systems can be an alternative to reduce time to identification to 1 day and, additionally, DST for first-line and second-line drugs in case of acid-fast positive microscopy.
The genotype MTBDR plus assay enables a rapid result from the pulmonary patient specimen and from the culture material. The identification of rifampicin resistance is enabled by the detection of the most significant mutations of the rpoB gene (coding for the β-subunit of the RNA polymerase). For testing the high-level isoniazid resistance, the katG gene (coding for the catalase peroxidase) is examined and for testing the low-level isoniazid resistance, the promoter region of the inhA gene (coding for the NADH enoyl ACP reductase) is analyzed.
In the present study, we aimed to detect the rifampicin and isoniazid resistance in the clinical strains of M. tuberculosis directly from the smear-positive sputum samples of pulmonary TB patients.
| Materials and methods|| |
A total of 100 sputum samples were collected from the suspected pulmonary TB patients over a 1-year period from Dec 2010 to Dec2011 at the TB-Chest department of Subharti Medical College, Meerut, U.P., India.
Genotype MTBDR plus assay
In this study, we studied the sputum samples directly from the pulmonary TB patients. This assay was performed as per the standard protocol provided by the manufacturer (HainLifescience GmbH, Nehren, Germany). Briefly, it was performed in three steps.
- DNA extraction: A decontaminated smear-positive sputum sample was suspended in 500 μL of DNA-free water. Then, the mixture was centrifuged for 15 min at approx 10,000 x g to pellet the bacteria. The supernatant was discarded and the bacteria was resuspended in 100 μL of water by vortexing. This suspension was then heated at 95°C for 20 min and then incubated for 15 min in an ultrasonic bath. The content was spin down at 13,000 x g for 5 min and 5 μL of this supernatant was directly used for polymerase chain reaction.
- Amplification: Amplification mixture containing 35 μL of primer nucleotide mix, 5 μL of the 10x polymerase incubation buffer, 2 μL of 25 mM MgCl 2 , 0.2 μL of AmpliTaq, 3 μL of double-distilled water and 5 μL of DNA solution, for a final volume of 50 μL, was prepared. The reaction mixture were then subjected to initial denaturation at 95°C for 5 min and 2 min at 58°C, followed by 30 cycles comprising 25 s at 95°C, 40 s at 53°C and 40 s at 70°C, and then a final extension at 70°C for 8 min.
- Hybridization: Hybridization was performed as per the manufacturer's instructions (HainLifescience GmbH, Nehren, Germany). Twenty microliters of denaturation solution (DEN, blue) was dispensed in a corner of each of the wells used. After that, 20 μL of the amplified solution was added and pipetted up and down to mix well and then incubated at room temperature for 5 min. One milliliter of pre-warmed hybridization buffer (HYB, green) was carefully added and then gently shook the tray until the solution had a homogenous color. A membrane strip was placed in each well, which was completely covered by the solution. The tray was placed in a shaking waterbath/Twincubator and incubated for 30 min at 45°C. The hybridization buffer was completely aspirated out. One milliliter of stringent wash solution (STR, red) was added to each strip and incubated for 15 min at 45°C in a shaking water bath/twincubator. The following steps were performed at room temperature. Stringent wash solution was completely removed. Each strip was washed once with 1 mL of rinse solution (RIN) for 1 min on a shaking platform/twincubator and RIN was poured out after incubation. One milliliter of diluted conjugate was added to each strip and incubated for 30 min on a twincubator. The solution was removed and each strip was washed twice for 1 min with 1 mL of rinse solution and once for 1 min with 1 mL distilled water and solution was poured out each time. One milliliter of diluted substrate (1:100) dimethyl sulfoxide was added to each strip and incubated after properly protecting from light without shaking for approximately 10 min. The reaction was stopped by briefly rinsing twice with distilled water. Strips were removed by using tweezers from the tray and dried between two layers of absorbent paper.
| Results|| |
A total of 100 sputum samples were analyzed by using the genotype MTBDR plus assay. Of these samples, 22 strains were found to be MDR, two were RMP monoresistant and one was isoniazid monoresistant [Figure 1], [Figure 2], [Figure 3], [Figure 4]. In MDR isolates, 54.55% (12/22) strains showed an absence of rpoBWT8 (rpoB wild type probe) and presence of rpoBMUT1 (rpoB mutation probe) hybridyzation band on a membrane strip, which represents a mutation and amino acid change from serine to leucine associated with codon no.531 of the rpoB gene. Other mutations in the rpoB gene were associated with codon 516 was 31.82% (7/22) and codon 526 was 13.63% (3/22) [Table 1].
|Table 1: Mutations associated with RMP and INH resistance by the genotype MTBDR plus assay in MDR strains (n = 22)|
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|Figure 1: Mycobacterium tuberculosis, susceptible to rifampin (RIF) and isoniazid (INH)|
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|Figure 2: M. tuberculosis, INH monoresistant (katG S315T1 mutation and inhA C15T mutation)|
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|Figure 3: Multidrug-resistant tuberculosis (MDR-TB), rpoB S531L mutation and katG S315T1 mutation|
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|Figure 4: M. tuberculosis, RIF monoresistant (mutation in rpoB 530-533 region).|
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Mutation in katG codon 315 was detected in 20 MDR isolates, and 14 of these strains showed a katGMUT1 hybridyzation band and six showed a katGMUT2 hybridyzation band. Two of these MDR isolates showed a wild type hybridization pattern in the KatG 315 genetic code, which revealed a mutation pattern in the ribosome-binding site region of inhA.
| Discussion|| |
The resistance is attributed primarily to improper prescription or patient non-compliance. MDR-TB was associated with high death rates of 50-80%, spanning a relatively short time from diagnosis to death.  Drug resistance is a result of random genetic mutations in particular genes. Delay in the recognition of drug resistance results in a delay in initiation of treatment or effective chemotherapy, which is the major factor that contributes to MDR-TB outbreaks. Drug resistance now can be assessed rapidly within 1 day by a molecular assay. The genotype MTBDR plus assay is a rapid and reliable method for the specific detection of the most common and frequent mutation leading to RMP and INH resistance. Rifampicin monoresistance is relatively rare, and the detection of rifampicin resistance serves as a surrogate marker for detecting MDR-TB.  In our study, we found two rifampicin-monoresistant strains. RIF-resistance mechanism involves missense mutations in the RRDR region of the rpoB gene. Extensive studies showed that 95% of RIF-resistant strains harbor a mutation within the 81-bp region of the rpoB gene. , In our study, the S531L mutation was observed in 54.55% cases, followed by D561V in 31.82% cases and H526Y in 13.63% cases. A high frequency of the S531L mutation has also been reported in other studies performed in different countries. ,,,
Mutations in the katG and the inhA genes are associated with approximately 70% and 80% of INH-resistant MTB isolates.  Although isoniazid resistance in M. tuberculosis is more complex due to the implication of the number of genes, up to 95% of isoniazid resistance may be due to mutations in katG.  Twenty (90.90%) of the MDR isolates showed a mutation in the katG codon 315 and two (9.1%) of the MDR isolates showed a mutation in inhA gene. The prevalence of mutations in the katG and inhA genes seems to vary widely in different geographic locations. Ninety-seven percent of katG mutations and 24% of inhA mutations were found in the INH-resistant isolates from KwaZulu-Natal,  whereas Van Rie et al. reported 72% of katG mutations and 2% mutations in the inhA gene of INH-resistant isolates in the Western Cape province of South Africa.  Extensive studies from other countries have confirmed this variability in the contribution of different mutations to INH resistance. ,
The genotype MTBDR plus assay can detect the low level of isoniazid resistance associated with inhA gene, which was not possible with the previous version of the assay (genotype MTBDR assay), which could only detect the mutations in the katG gene.
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Department of Microbiology, Subharti Medical College, Swami Vivekanand Subharti University, Meerut - 250 005, Uttar Pradesh
Source of Support: None, Conflict of Interest: None
[Figure 1], [Figure 2], [Figure 3], [Figure 4]