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 ~  Abstract
 ~ Introduction
 ~  Materials and Me...
 ~ Results
 ~ Discussion
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BRIEF COMMUNICATION
Year : 2016  |  Volume : 34  |  Issue : 1  |  Page : 92-94
 

Detection of rifampicin resistance in tuberculosis by molecular methods: A report from Eastern Uttar Pradesh, India


Department of Microbiology, Institute of Medical Sciences, Banaras Hindu University, Varanasi, Uttar Pradesh, India

Date of Submission11-Feb-2015
Date of Acceptance20-Jul-2015
Date of Web Publication15-Jan-2016

Correspondence Address:
S Anupurba
Department of Microbiology, Institute of Medical Sciences, Banaras Hindu University, Varanasi, Uttar Pradesh
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0255-0857.174122

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 ~ Abstract 

Diagnosis of drug resistance tuberculosis (TB) by the gold standard method is labour intensive and time consuming. Hence, there is an urgent need for introduction of rapid diagnostic techniques. Line probe assay (LPA) and cartridge-based nucleic acid amplification test (CBNAAT) have been introduced in India under Revised National Tuberculosis Control Program. Spot and morning sputum samples of previously treated patients by anti-TB drugs were subjected to LPA or CBNAAT. Total 682/1253 (54.4%) were diagnosed as rifampicin-resistant. The patients could be diagnosed early by molecular methods and put on second line treatment.


Keywords: Cartridge-based nucleic acid amplification test, drug resistance tuberculosis, line probe assay


How to cite this article:
Tripathi R, Sinha P, Kumari R, Chaubey P, Pandey A, Anupurba S. Detection of rifampicin resistance in tuberculosis by molecular methods: A report from Eastern Uttar Pradesh, India. Indian J Med Microbiol 2016;34:92-4

How to cite this URL:
Tripathi R, Sinha P, Kumari R, Chaubey P, Pandey A, Anupurba S. Detection of rifampicin resistance in tuberculosis by molecular methods: A report from Eastern Uttar Pradesh, India. Indian J Med Microbiol [serial online] 2016 [cited 2019 Oct 17];34:92-4. Available from: http://www.ijmm.org/text.asp?2016/34/1/92/174122



 ~ Introduction Top


India is the highest tuberculosis (TB) burden country in the world. The global incidence of multidrug-resistant TB (MDR-TB) is 630,000 cases. India constitutes one tenth of the global burden with 64,000 cases, presently.[1] Though the conventional drug susceptibility testing (DST) is considered the “Gold standard” for the detection of drug-resistant TB, it is time-consuming taking about 6–8 weeks.[2]

Hence, there is need of introduction of rapid diagnostic tools to detect MDR-TB. Cartridge-based nucleic acid amplification test (CBNAAT) is based on real-time amplification and detection of Mycobacterium tuberculosis (MTB), as well as drug sensitivity to rifampicin in a clinical specimen. Line probe assay (LPA) is based on multiplex polymerase chain reaction that is used to detect MTB complex as well as drug sensitivity to rifampicin and isoniazid. In both the tools, rifampicin resistance is detected by the presence of mutations in the 81-bp hotspot region of the rpoB gene.[3] The aim of this study is to report the utility of rapid molecular tools in diagnosis of drug resistance TB.


 ~ Materials and Methods Top


TB culture and DST laboratory, IMS-BHU is LPA certified for TB in North India, with a mandate to diagnose drug-resistant TB. The specimens of MDR-TB suspects from 10 linked districts of eastern Uttar Pradesh were packed as per Programmatic Management of Drug Resistance Tuberculosis guidelines [4] and couriered to our laboratory. All the sputum samples since January 2013 to January 2014 were subjected to CBNAAT for the diagnosis of rifampicin resistance. After establishment and the certification of LPA Laboratory in February 2014, the specimens were subjected to smear preparation, Zeal-Neelsen staining and microscopy. Smear positive sputum samples for acid fast bacilli (AFB) were tested by LPA from February 2014 to August 2014. In this report, we have presented the CBNAAT data since January 2013 to August 2014 and LPA since February to August 2014. This is a retrospective analysis of the data generated from the results of our mandated diagnostic workflow. The ethical approval has been taken retrospectively.

A total of 1253 diagnostic sputum samples from MDR suspect patients were tested during January 2013 to August 2014 of which 893 (71.3%) samples were subjected to CBNAAT and 360 (28.3%) were processed for LPA. CBNAAT was done by supplies with Xpert ® MTB/RIF kit (Cepheid AB, Sweden) as per manufacturer's specification.[5] Automated results generated by CBNAAT and were communicated online.

A total of 360 AFB positive samples were processed by NALC-NaOH decontamination method. DNA was extracted from decontaminated samples using Genolyse ® kit (Hain Lifescience GmbH, Nehren, Germany) as per manufacturer's instructions.[6],[7]

The extracted DNA was processed by the LPA using GenoType ® MTBDRplus (Hain Lifescience GmbH, Nehren, Germany) for detection of MTB complex and rifampicin and isoniazid resistance. A positive MTB control (TUB) band indicated the presence of members of the MTB complex in the sputum sample.[8] Samples with no TUB band by LPA (n = 16) were cultured on p-nitro benzoic acid (PNB) containing media and plain Lowenstein-Jensen (LJ) media for further confirmation as per Revised National Tuberculosis Control Program (RNTCP) guidelines.[9] DNA was extracted from colonies of those samples that showed growth of AFB and LPA were carried out on the extracted DNA as above.


 ~ Results Top


A total of 682/1253 (54.4%) patients were resistant and 449/1253 (35.8%) were sensitive for rifampicin. In a total of 112/1253 (8.9%) cases MTB was not detected; two cases were diagnosed as error and eight showed indeterminate for rifampicin by CBNAAT and LPA [Table 1]. Specimens of 106 patients that were negative for MTB (by CBNAAT), showed error and indeterminate by CBNAAT and LPA were requested to send their samples to certified culture DST laboratory for phenotypic DST as per the program recommendation. Out of 682,208 cases were resistant by LPA whereas 474 cases were rifampicin-resistant by CBNAAT. Rifampicin resistance has seen to be (57.7%) in LPA and (53.0%) in CBNAAT. Of total 360 diagnosed cases by LPA 129 were sensitive and seven patients showed indeterminate for rifampicin. A total of 16/360 (4.4%) showed negative for MTB by LPA. Out of 16 cultures, thirteen cultures turned positive (8 cultures showed positive on plain and PNB, 5 were positive on plain only), two cultures were negative and one was contaminated. On further DNA extraction and LPA of culture positive samples, all showed negative for MTB.
Table 1: Status of drug resistance detected by CBNAAT and LPA

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 ~ Discussion Top


Recently, significant progress has been made in our understanding for the molecular basis of MTB drug resistance. World Health Organisation (WHO) endorsed the use of molecular-based CBNAAT and LPA for screening of MDR-TB,[5],[7] following which these two assays are being used routinely in different countries.[3],[5],[7]

We are providing a diagnosis of drug-resistant TB to MDR suspected patients under RNTCP. We found 54.4% of drug resistance to rifampicin which is considered as a surrogate marker for MDR in previously treated patients. This is higher than national observation of 12–17% in MDR suspects.[9] 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. The high level of rifampicin resistance could also be due to non-availability of rapid diagnostic techniques as a result of which the patients did not have access, and there was a large pool of undiagnosed cases. Our results are correlated with Singhal et al., who reported 55.2% of MDR [10] and Raizada et al.[11] have also found 51% rifampicin-resistant. However, Kumar et al.[12] and Sharma et al.[13] reported 25.8% and 22% of MDR, respectively. A total of 8.9% were negative for MTB detected by both the techniques. This may be due to: (1) Most of them may be having a bacillary load lower than the detection limit of CBNAAT. (2) Few of them could be truly negative for MTB and suspected of non-TB mycobacterium (NTM). Two smear positive turned out culture negative, possibly due to the presence of dead bacilli in sputum samples. Out of the 16 samples which did not give TUB band, 13 were able to grow on LJ medium. However, none of these turned out to be MTB, which indicates that negative TUB band could give an indication of the isolate being NTM.

Diagnosis of drug resistance in TB by established gold standard method (solid culture and DST), takes 8–16 weeks, whereas molecular methods could diagnose rifampicin resistance in 2–72 h. Consequently, early management of these patients has a potential of rendering them noninfectious and preventing transmission of MDR strains in the community. This data can also help us in monitoring the MDR level in this region of Uttar Pradesh.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

 
 ~ References Top

1.
WHO. Global Tuberculosis Report; 2012 Available from: http://www.apps.who.int/iris/bitstream/10665/75938/1/9789241564502_eng.pdf.  Back to cited text no. 1
    
2.
TB India 2012. RNTCP Annual Status Report, Central TB Division. Available from: http://www.tbcindia.nic.in/pdfs/TB%20India%202012-%20Annual%20Report.pdf.  Back to cited text no. 2
    
3.
Barnard M, Albert H, Coetzee G, O'Brien R, Bosman ME. Rapid molecular screening for multidrug-resistant tuberculosis in a high-volume public health laboratory in South Africa. Am J Respir Crit Care Med 2008;177:787-92.  Back to cited text no. 3
    
4.
Guidelines for Programmatic Management of Drug Tuberculosis (India). Available from: http://www.tbcindia.nic.in/docs/pdf.[Last updated on 2012 May].  Back to cited text no. 4
    
5.
Policy Statement. Automated Real Time Nucleic Acid Amplification Technology for Rapid and Simultaneous Detection of Tuberculosis and Rifampicin Resistance: Xpert MTB/RIF System. WHO/HTM/TB/2011.4.  Back to cited text no. 5
    
6.
GenoType MTBDR Plus, Ver. 2.0, Instructions for Use, IFU-304A-02, Hain Life Sciences; 2/2012. Available from: http://www.ipaqt.org/wp-content/uploads/2013/02/MTBDRplusV2_product-insert.pdf.[Last accessed on 2014 Mar 04].  Back to cited text no. 6
    
7.
Molecular Line Probe Assay for Rapid Screening of Patients at Risk of Multi Drug Resistant Tuberculosis (MDR-TB). Policy Statement. Geneva: World Health Organization; 2008. Available from: http://www.who.int/tb/features_archive/policy_statement.pdf.[Last accessed on 2014 Mar 04].  Back to cited text no. 7
    
8.
Training Manual for Mycobacterium tuberculosis Culture and Drug susceptibility testing 2009: Revised National TB Control Programme; Central TB Division, Directorate General of Health Services, Ministry of Health and Family Welfare, Nirman Bhawan, New Delhi. Available from: http://www.tbcindia.nic.in/pdfs/Training%20manual%20M%20tuberculosis%20C%20DST.pdf. [Last accessed on 2014 Mar 04].  Back to cited text no. 8
    
9.
Revised national tuberculosis Annual status report. TB India 2011. Available from: http://tbcindia.nic.in/../guidelines%20for%20.pdf.  Back to cited text no. 9
    
10.
Singhal R, Myneedu VP, Arora J, Singh N, Sah GC, Sarin R. Detection of multi-drug resistance and characterization of mutations in Mycobacterium tuberculosis isolates from North-Eastern States of India using GenoType MTBDRplus assay. Indian J Med Res 2014;140:501-6.  Back to cited text no. 10
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11.
Raizada N, Sachdeva KS, Chauhan DS, Malhotra B, Reddy K, Dave PV, et al. A multi-site validation in India of the line probe assay for the rapid diagnosis of multi-drug resistant tuberculosis directly from sputum specimens. PLoS One 2014;9:e88626.  Back to cited text no. 11
    
12.
Kumar P, Kumar P, Balooni V, Singh S. Genetic mutations associated with rifampicin and isoniazid resistance in MDR-TB patients in North-West India. Int J Tuberc Lung Dis 2015;19:434-9.  Back to cited text no. 12
    
13.
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.  Back to cited text no. 13
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