|Year : 2017 | Volume
| Issue : 2 | Page : 129-133
Genotypic characterization of rpoB, katG and inhA gene of multi drug tuberculosis isolates from extra pulmonary tuberculosis
Anand Kumar Maurya1, Vijaya Laskshmi Nag1, Surya Kant2, Ram Awadh Singh Kushwaha2, Tapan N Dhole3
1 Department of Microbiology, All Institute of Medical Sciences, Bhopal, Madhya Pradesh, India
2 Department of Pulmonary Medicine, King George's Medical University, Lucknow, Uttar Pradesh, India
3 Department of Microbiology, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow, Uttar Pradesh, India
|Date of Web Publication||23-Nov-2017|
Anand Kumar Maurya
Department of Microbiology, All India Institute of Medical Sciences, Bhopal - 462020, Madhya Pradesh
Source of Support: None, Conflict of Interest: None
Background: Multidrug-resistant tuberculosis (MDR-TB) has appeared public health concern worldwide. Circulating of drug resistance strains is rising problem in MDR-TB among extra pulmonary TB (EPTB) cases. The objective of this study was to the genotypic characterization of MDR-TB isolates from EPTB and correlate with a phenotypic MDR-TB pattern in this region. Methods: This was study conducted prospectively manner. One hundred and sixty-four M. tuberculosis complex isolates were processed for first-line phenotypic drug susceptibility testing to rifampicin, isoniazid (INH), streptomycin, and ethambutol. Phenotypic confirmed MDR-TB strains were further characterized by gene sequencing for genotypic analysis of rpoB, katG, and inhA. Results: Among 164 MTBC strains, 39.1% of strains were resistant to first-line antitubercular drugs, and 13.4% were MDR-TB along with EPTB cases. In one strain, katG and inhA gene were absent in sequencing analysis. S531 L (61.9%) and S315T (71.4%) mutations were the most predominant mutation in MDR-TB isolates among EPTB cases. Conclusion: Molecular drug resistance testing allows to improved diagnosis, reduces the risk of additional resistance and effective treatment of drug resistance TB. Molecular DST testing can help in the detection for MDR-TB which helps in the prompt initiation of effective antitubercular therapy.
Keywords: Extrapulmonary tuberculosis, multidrug resistance tuberculosis, tuberculosis
|How to cite this article:|
Maurya AK, Nag VL, Kant S, Singh Kushwaha RA, Dhole TN. Genotypic characterization of rpoB, katG and inhA gene of multi drug tuberculosis isolates from extra pulmonary tuberculosis. Biomed Biotechnol Res J 2017;1:129-33
|How to cite this URL:|
Maurya AK, Nag VL, Kant S, Singh Kushwaha RA, Dhole TN. Genotypic characterization of rpoB, katG and inhA gene of multi drug tuberculosis isolates from extra pulmonary tuberculosis. Biomed Biotechnol Res J [serial online] 2017 [cited 2019 May 19];1:129-33. Available from: http://www.bmbtrj.org/text.asp?2017/1/2/129/219105
| Introduction|| |
Tuberculosis (TB) is remaining serious public health problem worldwide. India is highest TB burden country probable numbers of TB cases which report for 80% of worldwide. multidrug-resistant (MDR)-TB is defined as resistance to two most important antimycobacterial drugs isoniazid (INH) and rifampicin (RIF), has also developed into a rising global public health crisis. The study reported that the prevalence of MDR-TB was 2.2% in new cases and 15% of previously treated patients in India., MDR-TB strain is creating a severe crisis in TB control program, and it needs to emphasize on the advance, speedy, consistent diagnostic techniques for drug susceptibility testing (DST) in clinical isolates., Conventional methods are available for DST by solid media and liquid medium, but they need more time to detection of MDR-TB and less sensitive if compare to molecular testing. The World Health Organization (WHO) approved that the applicability of molecular DST testing for the diagnosis of MDR-TB in 2008. Molecular DST testing continues transforming from conventional to molecular testing in the diagnosis of TB worldwide. Molecular DST testing is valid on the principle of nucleic acid amplification which allows a prompt and precise identification of the Mycobacterium species <24 h., Molecular DST testing should be detected of Mycobacterium tuberculosis complex among RIF and INH resistance. The mechanism of drug resistance in M. tuberculosis due to genomic mutations in the different codon of rpo B gene in RIF resistance and kat G gene for INH resistance., The aim of this study was to the genotypic characterization of MDR-TB isolates from extra pulmonary TB (EPTB) cases and correlate with a phenotypic MDR-TB pattern in this region.
| Methods|| |
Study design and setting
This is prospective study was undertaken over 3 years in the two tertiary care teaching and research institute in northern India. About 2–10 ml of specimens were collected from 756 specimens, nonrepeated specimens from suspected cases of EPTB. The specimens were included as lymph node aspirate and cold abscesses, pleural fluid, cerebrospinal fluid, synovial fluid, ascitic fluid, urine, gastric aspirate, pus, bone marrow aspirates, wound swabs, and biopsy materials. The study was approved by the Institutional Ethics Committee and written informed consent was obtained from the patients before enrolment into the study.
164 M. tuberculosis complex isolates were analysis for first line antitubercular DST by BACTEC method using 1% proportional methods for RIF, INH, streptomycin, and ethambutol. All steps were carried out in a Biosafety cabinet as per biosafety guidelines. The same standard strain of M. tuberculosis complex, H37 Rv ATCC™ No. 27294, was used as positive control.
Molecular characterization of multidrug-resistant isolates
Isolation of DNA from phenotypic MDR M. tuberculosis complex isolates by Quigen kits. The primers were designed from published genome sequence of M. tuberculosis which linked with drug resistance responsible for mutations in M. tuberculosis genes [Table 1]., For each multiplex polymerase chain reaction (PCR) reaction, a 20 μl reaction contained 4 μl 5X Phire Reaction Buffer (1.5 mM MgCl2) (Finnzymes, OY, Finland), 10Mm dNTPs of 0.4 μl, 0.4 μl Phire Hot Start II DNA Polymerase, 1 μl (25 pmole) of 6 primer (SBS Gentech Co. Ltd), 13.2 μl Water (Nuclease–free) and 1 μl of extracted DNA. Amplification was carried out in an XP Thermal Cycler (BIOER Technology Co. Ltd, TC-XP-D dual block Thermal Cycler, China). The thermo cycling setup as an initial denaturing at 98°C for 3 min, 35 cycles of 95°C for 5 s, primer annealing 50°C for 5 s, and 72°C for 10 s, and a final extension at 72°C for 1 min.
DNA sequencing of genotypically confirmed multidrug-resistant-tuberculosis strains
PCR Products of genotypically confirmed MDR strains were purified from gel using PCR purification Kit Genei PureTM Quick (Bangalore genei, India). The sequencing was done forward and reverse primer by Bigdye-Terminator kit and ABI PRISM® 3730 DNA Analyzers (Applied Biosystems, USA) by chromus Biotech, India and Omega Biotech, India. Sequence obtained was compared with give comparative rpo B, kat G and inh A sequences of H37Rv in the databases. Bio Edit Sequence Alignment Editor version 7.05.2 were used for alignment of nucleotide sequences. The amplified product was precipitated with 10% of 3M sodium acetate and 2.5 volumes of ethanol. The DNA was pelleted by centrifugation at room temperature at 14,000 rpm for 30 min, washed with 70% ethanol and air dried. DNA was resuspended in 18–20 μl of template suppressor reagent. The sample was denatured at 95°C for 5 min, and then, it was kept on ice for 5 min, and then transferred to 0.5 ml sequencing vials. The sequencing vials were placed in the sample tray and loaded into ABI PRISM® 3730 DNA Analyzers (Applied Biosystems, USA) for sequencing.
Study data were evaluated using SPSS version 20.0 (Statistical Package for the Social Sciences, Chicago, IL, USA). The difference was significant when P < 0.05.
| Results|| |
Out of 756 EPTB specimens, 71 (9.3%) were positive for acid-fast bacilli by Ziehl–Neelsen staining and 227 (30.1%) were positive for mycobacteria by culture. Out of 164 microbiologically proven M. tuberculosis complex from 227 mycobacterial culture positive, 97 (59.2%) cases were males, mean age of these patients was 39.6 ± 17.2 years and 122 (74.4%) were new and 42 (25.6%) were treated cases. Three (1.8%) cases were HIV positive, and all were on antiretroviral therapy. All the three cases also had a history of ATT in the past. We found the prevalence of MDR-TB 22 (13.4%) among EPTB among in this region. Out of 22 MDR-TB, 8 (19.1%) treated cases as compared to 14 (11.5%) new cases (P < 0.05). The pattern of MDR-TB was resistance to HR, HRS, HRE, and HRES were 7 (4.3%), 6 (3.6%), 3 (1.8%), and 6 (3.6%), respectively [Table 2].
|Table 2: Distribution of drug resistance pattern to first line antitubercular drugs (n=164)|
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MDR-TB was diagnosed in 22 (13.4%) of cases by a phenotypic method by which also confirmed by the genotypic method. 22 isolates were detected as MDR-TB by the phenotypic method, and 21MTBC strains were detected by the genotypic method (P < 0.05). In one case mutation in kat G and inh A gene was absent in sequencing analysis. A total of 21 strains where the sequencing was done partial kat G gene sequencing which appear mutations in 21 MDR-TB isolates whereas only 1 isolates had mutations in the inh A promoter region. Among 21 MDR-TB isolates among kat G gene mutation, 20 (95.2%) point mutations, and 1 (4.8%) deletion mutations. The sequencing revealed that mutation in partial kat G, 15 (71.4%) isolates had mutations in the kat G gene codon 315 (2922 G→C); 4 (19.1%) isolates had mutations in the kat G gene codon 275 (2801 A→C) and 1 (4.7%) were kat G deletion shown in [Table 3]. The result of partial inh A gene sequence for point mutations in the inh A gene codon 209 (647 A→G) were 2 (9.5%) [Table 3]. 304-bp central region of the rpo B associated point mutations in 21 MDR isolates. Prominent mutations were found on 531, 526, and 516 codons. Thirteen (61.9%) were detected in point mutations at codon 531 (2431C→T), 3 (14.3%) isolates were found at 526 codons (2416 A→T), 5 (23.8%) isolates were found at 526 codons (2415 C→T), 3 (14.3%) were at 516 codons (2385 G→T) and 2 (9.5%) isolates were found at 516 codons (2386 A→T) are shown in [Table 3].
|Table 3: Details of DNA sequencing of multidrug resistant tuberculosis strains (n=21)|
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| Discussion|| |
Phenotypic DST is very complex due to technical difficulties and consequences are not constantly precise., Phenotypic DST results may take up to 8 weeks, and hence the chance of transmission of TB strains is very high. Various molecular based methods contain have been assessed to get better the rapidity in the diagnosis of MDR-TB.,, Molecular DST is very helpful in detection of TB and especially MDR-TB as well as detects various mutations linked to exact drugs to defeat these problems. In the present study, molecular DST testing was used for rapid detection of MDR-TB among EPTB cases: We also focused most prominent mutations linked with resistance to INH and RIF. Molecular DST method performed was comparable to the phenotypic DST for INH and RIF by a conventional method in the study.
Molecular DST methods are appropriate for detection of drug resistance-TB in samples which gone capability and reducing the overall cost. These techniques could be sophisticated laboratories having specific instruments. However, these techniques are very useful to get directly detection from the sample where a low amount of bacterial load. This will also very useful for detection of MDR-TB patients along to control spread and decrease mortality.
In the present study, molecular DST method permits concurrent detection of M. tuberculosis resistance to antitubercular drugs for MDR-TB simultaneously. Multiplex PCR used for rapid detection of MDR-TB resistance linked different mutations.,,
In this study, 21 isolates were found different point mutation in 304-bp central region in rpoB gene by sequencing. The majority of mutations at 531 (61.9%) and 526 (14.7%), but less mutation were shown at codon 516 (9.5%). Twenty-one mutations had identified in this study which already reported by other authors.,,
In one of phenotypically diagnosed MDR strain, we could not find any mutation during genotyping. This finding was comparable to other published reports. A study done in Italy showed 6% of RIF-resistant isolates showing no mutation in the rpoB gene by genotyping; similar reports have been published in other studies.,, Various studies data already showed the frequent mutation in the hotspot position of rpoB gene.,,
The most prominent detected rpoB mutation pattern was Ser531 Leu (61.9%) in the present study, which was higher than Poland (37.5%), Vietnam (40.4%), Germany (41.7%), India (59%), and China (59.2%). The frequency of His526 Leu (23.8%) in the present study, but 22% in India and 23.5% in Shanghai., Various studies reported 50%–60% of INH resistance isolates contain missense mutations or deletions or insertions in katG gene and the most frequent katG 315 codon in INH-resistant strains., We found 21 MDR-TB isolates in the region of katG, 15 (71.4%) had mutations at codon 315 in katG; 15 (71.4%) had Ser315Thr substitutions, 4 (19.1%) had a Thr275Pro substitution, and 1 (4.7%) had complete katG mutations. The study results are strongly agreed with the study published. The study reported from Africa, 68% of INH resistance had Ser315Thr substitutions and another study also found that 96% INH resistance from Germany., The sequencing of inhA gene revealed point mutation in 2 (9.5%) at codon Arg209Gly. In comparison, 18% had an inhA mutation seen in China.
The WHO recommended that the applicability of TB liquid culture and DST still in resource limited laboratories to get better detection of TB. Nowadays, the manufacturers are introducing low price policy for automated liquid culture medium in lower income countries. This may be very appreciably reduced the time to detection and DST among MDR-TB cases. The clinician may also very helpful in timely modify therapeutic as per quick DST results.,
| Conclusion|| |
We found a high prevalence of MDR-TB among EPTB and the situation is alarming in this area. S531 L and S315T mutations were the predominant among MDR-TB cases in this region. The present study showed that use of molecular DST testing to improved detection and management of TB. In this means, the buildup of the result on circulating MDR-TB strains gives an essential clue about the reappearance of TB.
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.
Financial support and sponsorship
This study was funded by Indian Council of Medical Research, New Delhi.
Conflicts of interest
There are no conflicts of interest.
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[Table 1], [Table 2], [Table 3]