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ORIGINAL ARTICLE
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Assessment of resistance to second line anti-tubercular drugs by line probe assay at a tertiary care hospital


1 Department of Microbiology, Armed Forces Medical College, Lavale, India
2 Department of Microbiology, AIIMS, Jodhpur, Rajasthan, India
3 Department of Microbiology, Symbiosis Medical College for Women, Lavale, India
4 Department of Pulmonary Medicine, Army Institute of Cardio-thoracis Sciences, Pune, India
5 Department of Microbiology, Army Institute of Cardio-thoracis Sciences, Pune, India

Date of Submission28-May-2021
Date of Decision28-Jul-2021
Date of Acceptance07-Aug-2021

Correspondence Address:
Santosh Karade,
Department of Microbiology, Armed Forces Medical College, Pune - 411 040, Maharashtra
India
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/mjdrdypu.mjdrdypu_392_21

  Abstract 


Context: Emergence and spread of extensively drug resistance (XDR) tuberculosis (TB) is a challenge in resource-limited settings. Although India accounts for one-fourth burden of multi-drug resistant cases of TB, the prevalence of XDR TB varies widely. Timely detection of resistance to anti-tubercular drugs is important for optimal case management. Aims: Characterization of resistance mutations to second-line anti-tubercular therapy (ATT) using line probe assay (LPA). Materials and Methods: In this cross-sectional, conducted at a Tertiary Care Teaching Hospital in Western Maharashtra, we assessed resistance to second-line antitubercular drugs in 100 consecutive sputum specimens from patients failing first-line ATT as per national guidelines by LPA. Results: Of the 100 specimens studied, a total of 94 assay results were valid. Of these, no resistance was seen in 52 specimens. Resistance to fluoroquinolones (FLQ) was detected in 30% of specimen with a predominance of gyrA gene mutations. A total of 12% isolates were resistant to aminoglycosides with mutations observed in rrs gene. Conclusions: The study demonstrates the utility of LPA as a tool to support the diagnosis of drug-resistant TB in clinical setting.

Keywords: Fluoroquinolones, line probe assay, multidrug-resistant tuberculosis, mutations



How to cite this URL:
Thosani P, Agarwal A, Karade S, Sen S, S. Katoch C D, Jindamwar P, S. Shergill S P. Assessment of resistance to second line anti-tubercular drugs by line probe assay at a tertiary care hospital. Med J DY Patil Vidyapeeth [Epub ahead of print] [cited 2022 Dec 7]. Available from: https://www.mjdrdypv.org/preprintarticle.asp?id=336821




  Introduction Top


Tuberculosis (TB) is one of the top ten causes of death and the leading cause from a single infectious agent.[1] In 2018, an estimated 10 million people developed TB and another 1.2 million deaths were reported.[1] This public health problem is further compounded by emergence and spread of resistance to commonly available anti-tubercular drugs.

Drug resistance in Mycobacterium TB (MTB) occurs due to chromosomal mutations in selected genes, which emerge owing to lack of adequate treatment, inappropriate regimens, or poor patient compliance.[2] Multidrug-resistant TB (MDR TB), defined as resistance to isoniazid and rifampicin, the two most efficacious first-line TB drugs, is estimated to be 3.4% among new cases and 18% in re-treatment cases.[3] Extensively drug-resistant TB (XDR TB) is defined as MDR cases, which in addition are resistant to one of the fluoroquinolones (FLQ) and one of the second-line injectable agents (amikacin, kanamycin, and capreomycin). Prior study indicated 6.2% of MDR cases to be XDR TB.[4] In addition, Pre XDR cases are also increasing, which are MDR cases with resistance to at least one of the second-line agents, either a FLQ or an injectable aminoglycoside.

FLQ and aminoglycosides (AG) are important therapeutic options in second-line anti-tubercular therapy (ATT), are the principle second-line agents for TB. Resistance to FLQ arises due to point gyrA and gyrB genes, whereas resistance to AG occurs due to mutation in 16S rRNA coding rrs gene. There is a need to assess burden of resistance to these second-line drugs against MTB.

The management of XDR TB is a major challenge as it involves prolonged treatment, expensive drugs, adverse effects, and poor compliance. Phenotypic methods for the diagnosis of drug resistance are culture-based and take several weeks.

Line probe assay (LPA) is one molecular-based rapid diagnostic method which can detect resistance mutations and heteroresistance. Heteroresistance, defined as a phenotype of an isolate that has a mixed population of both sensitive and resistant organisms. It is recommended by the WHO in 2016 for the diagnosis of resistance to second-line drugs.[9] As LPA is recently introduced in the diagnostic algorithm of MDR-TB, there are few LPA-based studies from India that have analyzed mutations conferring resistance to second-line anti-tubercular drugs. Thus, the aim of this study was to characterize resistance mutations against second-line antitubercular drugs using LPA.


  Materials and Methods Top


This cross-sectional study was carried out at a tertiary care teaching hospital of Western Maharashtra involved in the care of TB patients. Considering the prevalence of MDR-TB of 17% in reinfection cases, a confidence level of 95% and acceptable margin of error of 8%, a minimum sample size of 85 first-line failure cases was estimated. A total of 100 sputum specimens from patients failing first-line ATT were collected between October 2016 and December 2019. Growth was obtained on liquid culture and then samples were stored at −70°C for further studies. In the present study, stored MTB liquid culture specimens were brought to room temperature and directly subjected to LPA as per national guidelines, for the identification of resistance mutations to second-line anti-tubercular drugs. Those isolates with any resistance detected to first-line anti-tubercular agents were included in the study. Atypical mycobacterial isolates and contaminated specimens were excluded from the study.

Mycobacterial DNA extraction was carried by manual method with GenoLyse® Kit (Hains Life Science, Germany) as per the manufacturer's instructions.[10] All procedures were performed as per standard microbiological protocols in a biosafety cabinet Type II. Polymerase chain reaction was performed as per the instructional manual of Genotype MTBDRsl version 2.0 LPA kit.

Amplified products were subjected to reverse hybridization using kit in a Hains TwinCubator. MTBDRsl detects mutations in gyrA, gyrB gene for FLQ resistance, rrs gene mutation for amikacin, capreomycin, and kanamycin resistance, and eis gene mutations for low-level kanamycin resistance.[3]

MTB strain H37Rv was tested with each batch as quality control. The membrane strip of the LPA also has internal controls in form of conjugate control and amplification control band. Besides the test strips has control bands specific for MTB complex and respective gene locus of gyrA, gyrB, rrs, and eis genes. Results were interpreted as shown in [Figure 1] by comparing mutation band (MUT) and wild-type (WT) band pattern with reference bands provided with the kit.
Figure 1: (a) LPA strip depicting zones of gyrA, gyrB,rrs and eis genes. This image shows absence of gyrA wild type WT2 band and presence of gyrA MUT2 band indicative of S91P mutation. (b). Inferred resistance is indicated when WT2 failed to amplify but there is no mutant band seen. (c). LPA strip showing hetero-resistance: Wild type band of gyrA as well as mutated gyrA MUT1 band is seen.

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Heteroresistance is indicated by the presence of MUT band as well as WT. Inferred resistance is indicated by the absence of one of the WT bands without any presence of MUT band.

Ethical consideration

Ethical approval from the Institutional Ethics Committee for the use of stored sample for resistance testing was obtained as a part of Armed Forces Medical Research Project No: 5075/2018.


  Results Top


Of the 100 stored liquid culture specimens subjected to LPA, 94 results were valid. Resistance to one or more second-line drugs was detected in 42 specimens. Of these 42, 30 specimens were resistant to FLQ and 12 specimens to AG. None of the isolates had detectable resistance to both the drugs. Fifty-two specimens showed no mutation to any of the second-line drugs. Six specimens showed invalid results.

Resistance to fluoroquinolones

GyrA gene mutations responsible for FLQ resistance were observed in 30 specimens (71.4%). No mutation was detected in gyrB. Mutation pattern and frequency observed in various isolates are shown in [Table 1]. Heteroresistance and inferred resistance to FLQ was observed in 6 (20%) and 2 (6.67%) specimens, respectively.
Table 1: Frequency and pattern of mutations observed in gyrA gene

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Resistance to aminoglycosides

In this study, resistant to AG in all 12 specimens (28.6%) was observed due to mutation A1401G in rrs gene. The details are shown in [Table 2]. No mutation was observed in eis gene.
Table 2: Pattern and frequency of mutation observed in rrs gene

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  Discussion Top


TB continues to be a major health concern in resource-limited settings. Inappropriate treatment and poor compliance are major reasons for emergence of MDR and XDR cases.[11] Exposure to antitubercular drugs in suboptimal dosage leads to chromosomal mutations in MTB. These mutations alter the structure of drug binding target site, leading to resistance. LPA is a user-friendly method for genotypic detection of these common resistance-causing mutations. Conventional culture-based methods take around 6–8 weeks for the detection of resistance to first and second-line anti-tubercular drugs. This delay leads to continuation of empirical or ineffective regimen leading to increased morbidity and mortality. This inevitable delay is one of the major factors for rapid transmission of the infection to community. LPA, a molecular method for rapid detection of susceptibility to various anti-tubercular drugs, is a simple technique that can be easily adopted in laboratories.

As there are scarce data on the mutations responsible for resistance to second-line agents in India, in this study, we identified resistance patterns to second-line agents by LPA on 100 clinical samples which were known to be resistance to first-line agents. Among second-line agents, FLQ found to be more resistant than AG. This may be due to high prevalence of latent TB in the community and the use of FLQ antibiotic for respiratory infections.

A90V mutation, of gyr A gene, conferring resistance to FLQ was the most frequent mutation found in 7 specimens (23.3%). S91P mutation is an uncommon mutation in India was observed in this study in 4 specimens (13.3%). Codon 94 is the most affected with most mutations, D94G is the most common mutation in Indian setting, was observed in 4 specimens (13.3%), whereas D94A was observed in 3 specimens (10%). D94N/Y and D94H observed in 2 specimens (6.7%). Mutations A90V, S91P, D94A confer low-level resistance to moxifloxacin and in higher concentrations, it helps to overcome the resistance, Whereas D94G, D94N/Y, and D94H implies complete resistance to FLQs. In case of AG, A1401G was observed 12 specimens (100%) which causes complete resistance. Comparison of mutation profile with prior studies is shown in [Table 3].[12],[13],[14],[15],[16]
Table 3: Review of recent studies on resistance to second-line antitubercular drugs

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Early detection of resistant mutation by LPA helps clinicians to omit the resistant drug and initiate target treatment leading cure and prevention of transmission.[9],[17] Not all mutations confer complete resistance. Gene mutations may be responsible for resistance to a drug but not to others within the same group. As an example, AG are known to have variable cross-resistance between the drugs and the resistance is mutation specific.[17] For instance, Low-level Kanamycin resistance is observed because of eis promoter mutations, but these same mutations do not confer resistance to amikacin. Thus, complete cross-resistance is observed only with some specific mutations and so the importance of identifying the mutations is essential for specific treatment.

Employing commercial LPAs (INNO-LiPA and Hain) for the rapid detection of MDR TB has been recommended by the WHO.[9] Our national program also recommends it in diagnostic algorithm. Data suggest that commercial LPAs are economically viable in some high-burden settings.

However, LPAs come with its own limitations as this technique can identify only the known common mutations. Although rare, any mutations occurring outside the region amplified by LPA would be missed by this assay. With continuous evolution of MTB under selective drug pressure, the number of mutations will increase. Thus, this assay should also upgrade with time. Furthermore, for inferred resistance, gene sequencing is required to identify specific mutations. Nevertheless, prior studies indicated good sensitivity of LPA on sputum samples.


  Conclusions Top


LPA is user-friendly assay for rapid identification of DR TB cases that allows early initiation of appropriate therapy, infection control and prevents further spread of drug resistance. Widespread implementation of the LPA by the national TB elimination program would help in containment of MTB drug resistance.

Financial support and sponsorship

AFMRC Project 5075/2018.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

1.
WHO. Global Tuberculosis Report 2018. Geneva: World Health Organization; 2018. 2018. p. 174. Available from: http://apps.who.int/iris.  Back to cited text no. 1
    
2.
Hameed HM, Islam MM, Chhotaray C, Wang C, Liu Y, Tan Y, et al. Molecular targets related drug resistance mechanisms in MDR-, XDR-, and TDR-Mycobacterium tuberculosis strains. Front Cell Infect Microbiol 2018;8:114.  Back to cited text no. 2
    
3.
Hain-Lifescience. GenoType MTBDRsl VER 2.0-Instructions for Use. Vol. 2, Document IFU-317A-01; 2015. p. 8-14. Available from:http://www.hain-lifescience.de/en/instructions-for-use.html. [Last accessed on 2021 May 20].  Back to cited text no. 3
    
4.
World Health Organization, Geneva. MDR TB/RR TB Factsheet TB 2017. World Heal Organ; 2017. p. 1-2. Available from: https://www.who.int/tb/challenges/mdr/MDR-RR_TB_factsheet_2017.pdf. [Last accessed on 2021 Mar 20].  Back to cited text no. 4
    
5.
World Health Organization, Geneva. (↱2016)↱. The use of molecular line probe assays for the detection of resistance to second-line anti-tuberculosis drugs: policy guidance. World Health Organization. Available at https://apps.who.int/iris/handle/10665/246131. [Last accessed on 2021 Mar 20].  Back to cited text no. 5
    
6.
Hain Lifescience GmbH, Geno Lyse- Kit for isolation of genomic bacterial DNA from patient specimens. Nehren, Germany. Available from https://www.hain-lifescience.de/en/products/dna-isolation/genolyse.html. [Last accessed on 2021 Mar 20].  Back to cited text no. 6
    
7.
Centers for Disease Control and Prevention (CDC). Emergence of Mycobacterium tuberculosis with extensive resistance to second-line drugs--worldwide, 2000-2004. MMWR Morb Mortal Wkly Rep 2006;55:301-5.  Back to cited text no. 7
    
8.
Singhal R, Reynolds PR, Marola JL, Epperson LE, Arora J, Sarin R, et al. Sequence analysis of fluoroquinolone resistance-associated genes gyrA and gyrB in clinical Mycobacterium tuberculosis isolates from patients suspected of having multidrug-resistant tuberculosis in New Delhi, India. J Clin Microbiol 2016;1:2298-305.  Back to cited text no. 8
    
9.
Gardee Y, Dreyer AW, Koornhof HJ, Omar SV, da Silva P, Bhyat Z, et al. Evaluation of the GenoType MTBDRsl version 2.0 assay for second-line drug resistance detection of Mycobacterium tuberculosis isolates in south Africa. J Clin Microbiol 2017;55:791-800.  Back to cited text no. 9
    
10.
Li Q, Gao H, Zhang Z, Tian Y, Liu T, Wang Y, et al. Mutation and transmission profiles of second-line drug resistance in clinical isolates of drug-resistant Mycobacterium tuberculosis from Hebei province, China. Front Microbiol 2019;10:1838.  Back to cited text no. 10
    
11.
Kateete, D.P., Kamulegeya, R., Kigozi, E. et al. Frequency and patterns of second-line resistance conferring mutations among MDR-TB isolates resistant to a second-line drug from eSwatini, Somalia and Uganda (2014–2016). BMC Pulm Med 19, 124 (2019).  Back to cited text no. 11
    
12.
Siddiqi N, Shamim M, Hussain S, Choudhary RK, Ahmed N, Prachee, et al. Molecular characterization of multidrug-resistant isolates of Mycobacterium tuberculosis from patients in North India. Antimicrob Agents Chemother. 2002;46:443-50.   Back to cited text no. 12
    
13.
Kiet VS, Lan NT, An DD, Dung NH, Hoa DV, van Vinh Chau N, et al. Evaluation of the MTBDRsl test for detection of second-line-drug resistance in Mycobacterium tuberculosis. J Clin Microbiol 2010;48:2934-9.  Back to cited text no. 13
    
14.
Zaunbrecher MA, Sikes RD Jr., Metchock B, Shinnick TM, JE. Overexpression of the chromosomally encoded aminoglycoside acetyltransferase EIS confers kanamycin resistance in Mycobacterium tuberculosis. Proc Natl Acad Sci USA 2009;106:20004-9  Back to cited text no. 14
    
15.
World health organization (WHO), Geneva. Molecular Line Probe Assays for Rapid Screening of Patients at Risk of MDR-TB. Policy statement, 27 Jun 2008:1-9. Available from: https://www.who.int/tb/features_archive/policy_statement.pdf [Last accessed 25 Mar 2021].  Back to cited text no. 15
    
16.
Central TB Division, Ministry of health and family Welfare, New Delhi. Indian TB Report-2020; March 2020. Available from: http://www.tbcindia.gov.in [Last accessed 25 Apr 2021].  Back to cited text no. 16
    
17.
Hillemann D, Rüsch-Gerdes S, Richter E. Feasibility of the GenoType MTBDRsl assay for fluoroquinolone, amikacin-capreomycin, and ethambutol resistance testing of Mycobacterium tuberculosis strains and clinical specimens. J Clin Microbiol 2009;47:1767-72.  Back to cited text no. 17
    


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