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ORIGINAL ARTICLE
Year : 2020  |  Volume : 151  |  Issue : 4  |  Page : 361-370

Genotype analysis of ofloxacin-resistant multidrug-resistant Mycobacterium tuberculosis isolates in a multicentered study from India


1 Division of Molecular Biology, ICMR-National AIDS Research Institute, Pune, Maharashtra, India
2 Department of Microbiology, Institute of Medical Sciences, Banaras Hindu University, Varanasi, Uttar Pradesh, India
3 Department of Bacteriology, ICMR-National Institute for Research in Tuberculosis, Chennai, Tamil Nadu, India
4 Department of Immunology, ICMR-National Institute for Research in Tuberculosis, Chennai, Tamil Nadu, India

Date of Submission09-Mar-2018
Date of Web Publication12-May-2020

Correspondence Address:
Dr Vijay Nema
Division of Molecular Biology, ICMR-National AIDS Research Institute, 73 G MIDC, Bhosari, Pune 411 026, Maharashtra
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijmr.IJMR_493_18

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   Abstract 

Background & objectives: Drug resistance surveillance offers useful information on trends of drug resistance and the efficacy of control measures. Studies and reports of drug-resistant mutations and phenotypic assays thus become important. This study was conducted to investigate the molecular characteristics of ofloxacin (OFX)-resistant, multidrug-resistant tuberculosis (MDR-TB) isolates from different geographical regions of India and their association with strains of different genotypes. Further, the nitrate reductase assay (NRA) was tested against Mycobacteria Growth Indicator Tube
(MGIT) for the determination of OFX resistance as an alternative and cost-effective method.
Methods: A total of 116 Mycobacterium tuberculosis isolates were used to assess the mutations in the gyrA, gyrB genes and resistance levels to OFX. Mutational analysis in gyrA and gyrB genes and genotype analysis of M. tuberculosis isolates was done by gene-specific polymerase chain reaction (PCR) followed by DNA sequencing and spoligotyping, respectively.
Results: Three (6.25%), 12 (44.44%) and 12 (29.27%) MDR-TB isolates from western, northern and southern India, respectively, were found to be OFX-resistant MDR-TB isolates. OFX resistance was observed to be significantly higher in MDR-TB cases for all study regions. Beijing genotypes from northern India were observed to be associated with OFX-resistant MDR-TB cases (P <0.05). Among 35 (30.15%) phenotypically OFX-resistant isolates, 22 (62.86%) had mutations in the gyrA gene and two (5.71%) isolates had mutations in the gyrB gene.
Interpretation & conclusions: These results caution against the PCR-based prediction of OFX resistance patterns and highlight the need for searching other genetic loci for the detection of mutations conferring resistance to OFX in M. tuberculosis. Our study also showed the usefulness of NRA as an alternative method to detect OFX resistance.

Keywords: Drug resistance - gyrase - multidrug-resistant tuberculosis - Mycobacterium tuberculosis - nitrate reductase assay - ofloxacin resistance - spoligotyping


How to cite this article:
Gupta A, Sinha P, Rathod S, Shanmugam SK, Uma Devi K R, Anupurba S, Nema V. Genotype analysis of ofloxacin-resistant multidrug-resistant Mycobacterium tuberculosis isolates in a multicentered study from India. Indian J Med Res 2020;151:361-70

How to cite this URL:
Gupta A, Sinha P, Rathod S, Shanmugam SK, Uma Devi K R, Anupurba S, Nema V. Genotype analysis of ofloxacin-resistant multidrug-resistant Mycobacterium tuberculosis isolates in a multicentered study from India. Indian J Med Res [serial online] 2020 [cited 2020 May 27];151:361-70. Available from: http://www.ijmr.org.in/text.asp?2020/151/4/361/284218

Fluoroquinolones (FQs) such as ofloxacin (OFX), levofloxacin or moxifloxacin are important drugs for the management of multidrug-resistant tuberculosis (MDR-TB), and their resistance is associated with the condition of extensively drug-resistant (XDR)-TB. XDR-TB is defined as MDR-TB with additional resistance to any FQs and a second-line injectable drug such as kanamycin, amikacin, or capreomycin[1]. Dispersed but scanty reports are available from India about XDR-TB[2],[3],[4],[5], but are not enough to estimate the extent and magnitude of XDR-TB. Reasons primarily include the lack of facilities for second-line drug susceptibility determination, availability of standardized protocols with desired facilities and our focus on primary care of TB patients. It is important to monitor resistance to these drugs, especially FQs like OFX. OFX remains the backbone of second-line treatment along with amikacin, important for the detection of XDR cases and crucial for overall management and control of TB[2]. Inaccurate testing of TB also facilitates the emergence of drug resistance (DR). Therefore, the accurate and rapid detection of TB and DR-TB is imperative for timely and adequate treatment to interrupt the transmission of DR/MDR- and XDR-TB[6]. Several molecular and commercial tools are available for susceptibility testing of Mycobacterium tuberculosis[7]. It is, however, difficult for a low resource setting to have all these sophisticated techniques and infrastructure. Earlier, the nitrate reductase assay (NRA) for the detection of first-line anti-TB drug resistance was evaluated[8],[9]. NRA is a rapid, inexpensive, phenotypic method that can easily be incorporated into existing facilities of a low resource setting and is based on the metabolic ability of M. tuberculosis to reduce nitrate to nitrite[10]; it has been endorsed by the WHO for the rapid detection of MDR-TB[11].

This study was undertaken to do genotypic analysis of OFX-resistant and MDR-TB isolates from different regions of India to determine molecular mutations conferring drug resistance. We further evaluated NRA to detect OFX-resistant MDR-TB isolates using MGIT960 as the gold standard.


   Material & Methods Top


Mycobacterium tuberculosis isolates from the ICMR-National AIDS Research Institute (ICMR-NARI), Pune; ICMR-National Institute for Research in Tuberculosis (ICMR-NIRT), Chennai; State TB Testing and Demonstration Centre (STDC), Pune, and Institute of Medical Sciences, Banaras Hindu University (BHU), Varanasi, were included. Information on demographic characteristics of patients and sputum mycobacteriology studies were collected from the medical files and compliance charts of the centres involved except for the southern part of India. The study was approved by the Ethics Committee of ICMR-NARI, Pune.

A total of 116 clinical isolates of M. tuberculosis collected from three different geographical regions of India namely Pune (western India), Varanasi (northern India) and Chennai (southern India) during 2013-2014 were studied. All isolates were freshly sub-cultured on LJ (Löwenstein-Jensen) medium (HiMedia, Mumbai) before use.

OFX was purchased from Sigma-Aldrich, USA. The stock solution was prepared at a concentration of 2 μg/ml in 0.1 N NaOH, filter sterilized and preserved at −20°C for not more than one month. To prepare Griess reagent 50 per cent (vol/vol) concentrated hydrochloric acid (HCl), 0.2 per cent (wt/vol) sulphanilamide, and 0.1 per cent (wt/vol) n-1-naphthylethylenediamine dihydrochloride were prepared in small volumes and mixed shortly before use in the ratio1:2:2, respectively.

Drug susceptibility testing (DST) against first-line drugs: BACTEC™ MGIT™ 960 Mycobacterial Detection System (Becton, Dickinson and Company, USA) using SIRE kit was used as per manufacturer's instructions for the susceptibility testing against streptomycin (STR), isoniazid (INH), rifampin (RIF) and ethambutol (EMB). M. tuberculosis H37 Rv (ATCC 27294) was used as the susceptible control.

MGIT960 for ofloxacin-drug susceptibility testing (OFX-DST): For susceptibility testing against OFX, blank tubes of medium were used wherein the final concentration of OFX was 2 μg/ml as per the Clinical and Laboratory Standards Institute (CLSI) standards[12]. The inoculum was prepared from a fresh LJ medium by following the methods used by Devasia et al[13]. A growth control containing no antibiotic and a sterile control without inoculation were used with every set of experiments.

Nitrate reductase assay for OFX-DST: The assay was performed as described earlier[14]. Briefly, the 7H11 medium was prepared by adding OFX at the concentration of 2 μg/ml and 1 mg/ml of sodium nitrate (NaNO3). The inoculum turbidity was adjusted to a 1 McFarland tube, and the inoculum was diluted 1:10 vol/vol in phosphate-buffered saline. For each isolate, 200 μl of the undiluted suspension was inoculated into the OFX containing tube, and 200 μl of the 1:10 dilution was inoculated into the drug-free tube. The tubes were incubated at 37°C. After seven days, 500 μl of the Griess reagent mixture was added to one drug-free tube. If any colour appeared, the reagent mixture was added in all the tubes; otherwise, the tubes were re-incubated and the procedure was repeated at days 9 and 14. An isolate was considered resistant if there was a colour change in the antibiotic tube greater than that in the 1:10-diluted growth control.

PCR amplification/DNA sequencing for detection of ofloxacin resistance determinants: Genomic DNA was extracted from M. tuberculosis isolates using the CTAB (cetyltrimethylammonium bromide)-NaCl method[15] and PCR assay was performed to detect mutations in gyrA and gyrB genes. Primers for gyrA and gyrB were designed by taking NC_000962 as a template which also covers the quinolone resistance determining region (QRDR) of the open reading frame (ORF). The gyrA gene was amplified with the forward primer (5-CCCTGCGTTCGATTGCAAA-3) and reverse primer (5-CTTCGGTGTACCTCATCGCC-3). The gyrB gene was amplified using the forward primer (5-ACGCGAAAGTCGTTGTGAAC-3) and reverse primer (5-CGCTGCCACTTGAGTTTGTAC-3). The amplification reaction volume of 25 μl contained 1x HiFi Buffer (Kapa Biosystems Inc., USA), 2 mM MgCl2, 200 μM each of dNTP (Fermentas, USA), 10 pmol each of forward and reverse primers, 1 U of KAPA HiFi HotStart DNA Polymerase (Kapa Biosystems Inc., USA), 10 ng of DNA template and water to make the final volume. Amplification conditions were used as suggested by the manufacturer of KAPA HiFi HotStart DNA Polymerase with 66°C as annealing temperature for gyrA and 62°C as annealing temperature for gyrB. PCR products of gyrA and gyrB were 423 and 520 base pairs (bp) respectively in size. Automated sequencing was performed using the BigDye Terminator kit v3.1 (Applied Biosystems, USA) as per manufacturer's protocol. DNA sequence analysis and comparisons of gyrA (n=116) and gyrB (n=116) were carried out with SeqScape® v2.5 software from Applied BioSystems, USA.

Spoligotyping and data analysis: Spoligotyping was performed using the standard method[16] at ICMR-NIRT, Chennai. Results were matched with the SITVIT web database to get spoligotypes matched with available profiles (http://www.pasteur-guadeloupe.fr:8081/SITVIT_ONLINE/), and major lineages were determined.

Statistical analysis: MedCalc-free statistical calculators (https://www.medcalc.org/calc/) were used to calculate the statistical measures of sensitivity, specificity, accuracy, positive predictive value (PPV) and negative predictive value (NPV) of the NRA using the MGIT 960 system and DNA sequencing as the reference tests. Fisher's exact test was used to calculate exact probability.


   Results Top


In the present study, a total of 116 M. tuberculosis isolates, obtained from 116 adult patients with pulmonary TB were used. Amongst 116 isolates, 45 (38.79%) and 27 (23.28%) were identified as MDR and OFX-resistant MDR-TB, respectively. There were 48 isolates from western India; 27 from northern India and 41 from southern India. The clinical characteristics of study population are given in [Table 1].
Table 1: Clinical characteristics of the study population among phenotypically ofloxacin (OFX)-resistant and -susceptible Mycobacterium tuberculosis isolates from different parts of India

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Performance of NRA for OFX-DST: An excellent agreement was obtained between NRA and the reference method MGIT 960 system with 100 per cent sensitivity, specificity, NPV and PPV. When the results of NRA were compared with DNA sequencing for OFX resistance detection, 90 per cent sensitivity and 97.67 per cent specificity were found with excellent agreement between the two methods.

Frequency and association of ofloxacin resistance with clinical characteristics of TB patients: OFX resistance was observed to be significantly higher in MDR-TB cases in all study regions [Table 2]. There were no significant differences found between OFX resistance and age, sex, HIV status and treatment history of patients based on the available data. However, a focused study to specifically assess these characteristics and their association with the development of OFX resistance may be taken up with a larger sample size. The frequency of OFX resistance in the studied isolates from the north and south India was higher compared to western India (Table II). The frequency of OFX-resistant MDR-TB cases was three (6.25%), 12 (44.44%) and 12 (29.27%) in western, northern and southern regions of India, respectively.
Table 2: Calculation of significance for the observation of ofloxacin (OFX) resistance in multidrug-resistant (MDR) and non-MDR cases (using MedCalc online calculator software)

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Frequency of gyrA and gyrB gene mutations in OFX-resistant and sensitive clinical isolates: Our results revealed that among 35 (30.15%) phenotypically OFX-resistant isolates, 22 (62.86%) had mutations in the gyrA gene and two (5.71%) isolates had mutations in the gyrB gene. The single-nucleotide mutation sites were in codons GAC500GCC and GAC472GTC. The changes at this position are reported earlier (GAC-GCC), but the one in the present study was different GAC (codon 472 and nucleotide position 1415) to GTC [Table 3]. Both the isolates were from southern India. No gyrB mutations were observed in northern and western Indian isolates. Six of the 48 (12.5%), 16 of the 27 (59.26%) and 13 of the 41 (31.7%) isolates, from western, northern and southern India, respectively, were resistant to OFX. The most predominant mutation occurred at codon D94G with nucleotide substitution from GAC-GGC in 2/6 (33.33%), 10/16 (62.5%) and 2/13 (15.38%) OFX-resistant isolates from western, northern and southern India, respectively. Further, two and one among OFX-resistant isolates from northern and southern India were found to have mutations at codons A90V (GCG-GTG) and D94N (GAC-AAC), respectively. One isolate from southern India had double mutations at codons A90V and D94G (GCG-GTG, GAC-GGC) [Table 3]. Four (1 each from western, southern and 2 from northern India) OFX-susceptible isolates were found to be false positive and had a mutation at codon A90V (GCG-GTG). Most of the clinical isolates (105/116, 90.52%) had an AGC-ACC and GAG-CAG polymorphisms at codons 95 and 21, respectively, but were not related to OFX resistance.
Table 3: Phenotypic quinolone resistance and polymorphisms in gyrA and gyrB of Mycobacterium tuberculosis

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Prevalent genotypes and drug resistance: Central Asian strain (n=18, 37.5%), Beijing (n=9, 33.33%) and East African Indian (n=11, 26.83%) genotypes were predominant in western, northern and southern India, respectively [Table 4] and [Table 5]. The distribution of DR according to M. tuberculosis genotype and geographical regions has been summarized in Table V. Only Beijing genotypes from northern India were observed to be associated with OFX-resistant MDR-TB cases (7/12, 58.33%, P=0.021).
Table 4: Distribution of different family strains in northern, western and southern regions of India

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Table 5: Frequency of genotypes among drug-resistant tuberculosis patients in different geographical regions of India

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   Discussion Top


The present study was conducted with two major objectives. One being the evaluation of the performance of NRA for OFX-DST, and the other one was to find the level of concordance between genotypic assays and phenotypic assays for drug resistance. The NRA has earlier been evaluated in some settings for the detection of OFX resistance[13],[17],[18],[19] including one study from India[20]. We obtained 100 per cent concordance of NRA with MGIT960-based assay, thus, NRA could be proposed as a promising tool to detect MDR-/XDR-TB isolates in low-resource settings. NRA is accurate and easy to implement in clinical diagnostic laboratories, making it a good option for rapid screening for MDR- and XDR-TB.

Earlier studies reported that the majority (50-90%) of FQ-resistant M. tuberculosis isolates carry mutations in the QRDR of the gyrA and a small number (3-7%) have mutations in the gyrB gene[21],[22]. The analysis of gyrA and gyrB gene mutations in OFX-resistant and sensitive clinical isolates of M. tuberculosis revealed various point mutations at single or more than one position. Some mutations were found at earlier reported positions in the sequenced genes but had a different nucleotide. This showed a different amino acid on translation. The change in amino acid may have nominal or significant effect on protein structure and functions. In some cases, the observed mutations could not be correlated with phenotypic results or the phenotypes did not match the genotypes. The reason for this could be the presence of a compensating mechanism working elsewhere in the genome. In the present study, the association between gyrA/B mutation and OFX resistance was significant (odds ratio (OR)=32.58; P=0.001; 95% confidence interval (CI)=8.639-135.132). Our data suggested that though gyrA/B was a sensitive marker for OFX resistance study as suggested earlier[20],[21], it was not the only causative agent of OFX resistance. This was because in our study, only 68.57 per cent (24/35) of OFX-resistant isolates had mutations in gyrA/B gene. This situation has been reported previously also with FQs resistance[23] and can be explained by a mutation outside of the sequenced regions or another molecular alteration decreasing the accumulation of the drug inside the cell, such as an efflux mechanism[23],[24]. In addition to other substitutions, we got a new substitution at gyrA codon 15 and 25 (nucleotide position 43 and 73, respectively) in two and three isolates, respectively, along with a large number of isolates harbouring the polymorphisms at codons 95 and 21. These substitutions were not found to be related to OFX resistance and might have evolutionary significance.

Overall, the OFX resistance was observed to be significantly higher in MDR-TB cases in all study regions and could be a surrogate marker for the emergence of OFX-resistant MDR-/XDR-TB cases. Therefore, OFX resistance may also be included along with other four first-line drugs for the initial screening of DR-TB. The frequency of OFX resistance in the studied isolates was observed to be higher in isolates from northern and southern India compared to western India. The reason for this was not clear from the data we had and a study with a significantly higher number of isolates would be able to explain the causality of this. In all, OFX resistance was higher in all regions studied, which was in line with previous studies[5],[25],[26].

Our data imply that molecular techniques targeting only QRDR region to detect FQ resistance may be less specific in areas with a high prevalence of FQ-resistant MDR-TB. Larger patches of sequences covering the whole gene and regulatory regions along with other molecular alterations decreasing the accumulation of the drug inside the cell, such as an efflux mechanism should be considered. Available information on genotypes of OFX-resistant MDR-TB isolates from different geographic regions of India is scanty. Our study showed a predominance of Beijing/CAS and EAI genotypes and an association between MDR and OFX resistance-conferring mutations and the Beijing genotype. A study from north India also observed Beijing as dominant genotype along with CAS_Delhi among OFX-resistant MDR-TB isolates[5]. While correlating the association of gyrA mutations in various lineages another study from north India found that the association of Beijing isolates was significantly high (P=0.0006) with gyrA gene mutations[27]. A study conducted in Vietnam observed a significant association of Beijing genotype of M. tuberculosis with high-level quinolone resistance[28] while in China, Beijing genotype showed no association with FQ resistance[29],[30]. These findings may be useful for the establishment of rapid molecular diagnostic methods to be implemented in India and countries with high OFX resistance. Studies with a higher number of patients over long periods are required to see the pattern of OFX resistance and to understand the epidemiology of OFX-resistant MDR- and XDR-TB in India.

One of the limitation of our study was that spoligotyping was used instead of 24 loci MIRU for genotyping of M. tuberculosis but the information obtained using spoligotyping could be useful as it is a first-line discriminatory test and the gold standard for the identification of Beijing strains of M. tuberculosis. Further, OFX-resistant MDR-TB could actually be the XDR-TB in many cases which not studied but data of DST against the injectables i.e., kanamycin, capreomycin and amikacin were not available. The DST only to OFX and not against aminoglycosides may not be enough to estimate the situation of XDR-TB in India. Further efforts are required to take care of these limitations with a larger number of sample.

Acknowledgment: Authors thank Shrimati Shilpa Balgam and other officers of State TB Testing and Demonstration Centre (STDC), Pune, Shri Mycal Periera, ICMR-National AIDS Research Institute (ICMR-NARI), Pune and Dr Gomathi Sekar from ICMR-National Institute for Research in Tuberculosis (ICMR-NIRT), Chennai, for providing isolates used in this study. Authors also thank Late Ms Glory Francis, Ms Namrata Yadav, Shrimati Divya Pandey and Shrimati Najneen Akbar, ICMR-NARI, for their support in handling isolates.

Financial support & sponsorship: This work was supported by the Indian Council of Medical Research, New Delhi (extramural grant no. AMR/45/2011-ECD-I).

Conflicts of Interest: None.



 
   References Top

1.
World Health Organization. Definitions and Reporting Framework for Tuberculosis - 2013 Revision. Geneva: WHO; 2013.  Back to cited text no. 1
    
2.
Mondal R, Jain A. Extensively drug-resistant Mycobacterium tuberculosis, India. Emerg Infect Dis 2007; 13 : 1429-31.  Back to cited text no. 2
    
3.
Porwal C, Kaushik A, Makkar N, Banavaliker JN, Hanif M, Singla R, et al. Incidence and risk factors for extensively drug-resistant tuberculosis in Delhi region. PLoS One 2013; 8 : E55299.  Back to cited text no. 3
    
4.
Michael JS, John TJ. Extensively drug-resistant tuberculosis in India: A review. Indian J Med Res 2012; 136 : 599-604.  Back to cited text no. 4
    
5.
Singhal P, Dixit P, Singh P, Jaiswal I, Singh M, Jain A. A study on pre-XDR& XDR tuberculosis their prevalent genotypes in clinical isolates of Mycobacterium tuberculosis in north India. Indian J Med Res 2016; 143 : 341-7.  Back to cited text no. 5
    
6.
Kaswa MK, Aloni M, Nkuku L, Bakoko B, Lebeke R, Nzita A, et al. Pseudo-outbreak of pre-extensively drug-resistant (Pre-XDR) tuberculosis in Kinshasa: Collateral damage caused by false detection of fluoroquinolone resistance by GenoType MTBDRsl. J Clin Microbiol 2014; 52 : 2876-80.  Back to cited text no. 6
    
7.
Balasingham SV, Davidsen T, Szpinda I, Frye SA, Tønjum T. Molecular diagnostics in tuberculosis: Basis and implications for therapy. Mol Diagn Ther 2009; 13 : 137-51.  Back to cited text no. 7
    
8.
Gupta A, Anupurba S. Direct drug susceptibility testing of Mycobacterium tuberculosis against primary anti-TB drugs in northern India. J Infect Dev Ctries 2010; 4 : 695-703.  Back to cited text no. 8
    
9.
Gupta A, Sen MR, Mohapatra TM, Anupurba S. Evaluation of the performance of nitrate reductase assay for rapid drug-susceptibility testing of Mycobacterium tuberculosis in North India. J Health Popul Nutr 2011; 29 : 20-5.  Back to cited text no. 9
    
10.
Angeby KA, Klintz L, Hoffner SE. Rapid and inexpensive drug susceptibility testing of Mycobacterium tuberculosis with a nitrate reductase assay. J Clin Microbiol 2002; 40 : 553-5.  Back to cited text no. 10
    
11.
World Health Organization. Strategic and Technical Advisory Group for Tuberculosis (STAG-TB): Report of the Ninth Meeting. Geneva: WHO; 2009.  Back to cited text no. 11
    
12.
Woods GL, Brown-Elliott BA, Conville PS, Desmond EP, Hall GS, Lin G, et al. Susceptibility testing of Mycobacteria, Nocardiae, and other aerobic Actinomycetes. 2nd ed. Wayne (PA): Clinical and Laboratory Standards Institute; 2011.  Back to cited text no. 12
    
13.
Devasia RA, Blackman A, May C, Eden S, Smith T, Hooper N, et al. Fluoroquinolone resistance in Mycobacterium tuberculosis: An assessment of MGIT 960, MODS and nitrate reductase assay and fluoroquinolone cross-resistance.J Antimicrob Chemother 2009; 63 : 1173-8.  Back to cited text no. 13
    
14.
Martin A, Imperiale B, Ravolonandriana P, Coban AY, Akgunes A, Ikram A, et al. Prospective multicentre evaluation of the direct nitrate reductase assay for the rapid detection of extensively drug-resistant tuberculosis. J Antimicrob Chemother 2014; 69 : 441-4.  Back to cited text no. 14
    
15.
van Embden JD, Cave MD, Crawford JT, Dale JW, Eisenach KD, Gicquel B, et al. Strain identification of Mycobacterium tuberculosis by DNA fingerprinting: Recommendations for a standardized methodology. J Clin Microbiol 1993; 31 : 406-9.  Back to cited text no. 15
    
16.
Kremer K, van Soolingen D, Frothingham R, Haas WH, Hermans PW, Martín C, et al. Comparison of methods based on different molecular epidemiological markers for typing of Mycobacterium tuberculosis complex strains: Interlaboratory study of discriminatory power and reproducibility. J Clin Microbiol 1999; 37 : 2607-18.  Back to cited text no. 16
    
17.
Imperiale BR, Morcillo NS, Palomino JC, Vandamme P, Martin A. Predictive value of direct nitrate reductase assay and its clinical performance in the detection of multi- and extensively drug-resistant tuberculosis. J Med Microbiol 2014; 63 : 522-7.  Back to cited text no. 17
    
18.
Abilleira F, Brum C, von Groll A, da Silva PE. Evaluation of direct microplate nitrate reductase assay as a rapid method for the detection of multiple and extensively tuberculosis drug resistance. Biomedica 2015; 35 : 285-91.  Back to cited text no. 18
    
19.
Shrivastava N, Set R, Bankar S, Shastri J. Detection of ofloxacin resistance by nitrate reductase assay Mycobacterium tuberculosis isolates from extrapulmonary tuberculosis. Indian J Med Microbiol 2017; 35 : 69-73.  Back to cited text no. 19
    
20.
Visalakshi P, Arora J, Behera D. Evaluation of nitrate reductase assay for rapid detection of resistance to second-line drugs in Mycobacterium tuberculosis in a tertiary care hospital. Diagn Microbiol Infect Dis 2010; 68 : 236-40.  Back to cited text no. 20
    
21.
Maruri F, Sterling TR, Kaiga AW, Blackman A, van der Heijden YF, Mayer C, et al. A systematic review of gyrase mutations associated with fluoroquinolone-resistant Mycobacterium tuberculosis and a proposed gyrase numbering system. J Antimicrob Chemother 2012; 67 : 819-31.  Back to cited text no. 21
    
22.
Singh P, Jain A, Dixit P, Prakash S, Jaiswal I, Venkatesh V, et al. Prevalence of gyrA and B gene mutations in fluoroquinolone-resistant and -sensitive clinical isolates of Mycobacterium tuberculosis and their relationship with MIC of ofloxacin. J Antibiot (Tokyo) 2015; 68 : 63-6.  Back to cited text no. 22
    
23.
Von Groll A, Martin A, Jureen P, Hoffner S, Vandamme P, Portaels F, et al. Fluoroquinolone resistance in Mycobacterium tuberculosis and mutations in gyrA and gyrB. Antimicrob Agents Chemother 2009; 53 : 4498-500.  Back to cited text no. 23
    
24.
Pasca MR, Guglierame P, Arcesi F, Bellinzoni M, De Rossi E, Riccardi G. Rv2686c-Rv2687c-Rv2688c, an ABC fluoroquinolone efflux pump in Mycobacterium tuberculosis. Antimicrob Agents Chemother 2004; 48 : 3175-8.  Back to cited text no. 24
    
25.
Pradhan N, Desai S, Kagal A, Dharmashale S, Bharadwaj R, Ghorpade S, et al. Patterns of TB drug-resistance in a tertiary care facility in Pune, India. Clin Microbial 2013; 2 : 123.  Back to cited text no. 25
    
26.
Selvakumar N, Kumar V, Balaji S, Prabuseenivasan S, Radhakrishnan R, Sekar G, et al. High rates of ofloxacin resistance in Mycobacterium tuberculosis among both new and previously treated patients in Tamil Nadu, South India. PLoS One 2015; 10 : e0117421.  Back to cited text no. 26
    
27.
Rufai SB, Singh J, Kumar P, Mathur P, Singh S. Association of gyrA and rrs gene mutations detected by MTBDRsl V1 on Mycobacterium tuberculosis strains of diverse genetic background from India. Sci Rep 2018; 8 : 9295.  Back to cited text no. 27
    
28.
Duong DA, Nguyen TH, Nguyen TN, Dai VH, Dang TM, Vo SK, et al. Beijing genotype of Mycobacterium tuberculosis is significantly associated with high-level fluoroquinolone resistance in Vietnam. Antimicrob Agents Chemother 2009; 53 : 4835-9.  Back to cited text no. 28
    
29.
Zhao LL, Chen Y, Liu HC, Xia Q, Wu XC, Sun Q, et al. Molecular characterization of multidrug-resistant Mycobacterium tuberculosis isolates from China. Antimicrob Agents Chemother 2014; 58 : 1997-2005.  Back to cited text no. 29
    
30.
Zhang Z, Lu J, Wang Y, Pang Y, Zhao Y. Prevalence and molecular characterization of fluoroquinolone-resistant Mycobacterium tuberculosis isolates in China. Antimicrob Agents Chemother 2014; 58 : 364-9.  Back to cited text no. 30
    



 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5]



 

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