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 Table of Contents  
Year : 2019  |  Volume : 3  |  Issue : 2  |  Page : 87-91

Second-line drug susceptibilities of multidrug- and rifampicin-resistant Mycobacterium tuberculosis isolates in Delhi

1 School of Life Sciences, Jaipur National University, Jaipur, Rajasthan; New Delhi Tuberculosis Centre, New Delhi, India
2 New Delhi Tuberculosis Centre, New Delhi, India
3 Office of Delhi State Health Mission, Delhi State Revised National Tuberculosis Control Programme, New Delhi, India
4 School of Life Sciences, Jaipur National University, Jaipur, Rajasthan, India

Date of Submission23-Mar-2019
Date of Decision27-Apr-2019
Date of Acceptance28-Apr-2019
Date of Web Publication17-Jun-2019

Correspondence Address:
Dr. M Hanif
New Delhi Tuberculosis Centre, JLN Marg, Delhi Gate, New Delhi - 110 002
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/bbrj.bbrj_53_19

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Background: Multidrug-resistant tuberculosis (MDR-TB) is a threat to the accomplishments of the World Health Organization's (WHO) End TB Strategy. The treatment of MDR-TB is less effective and more toxic; additional resistance to second-line drugs makes the treatment more difficult. India accounts for one-fourth of the global TB burden. In 2015 alone, 2.8 million cases of TB were diagnosed of which 79,000 cases had MDR/rifampicin-resistant TB (RR-TB). This study was conducted to analyze the baseline susceptibility pattern of MDR/RR-TB isolates against second-line drugs. Methods: A total of 374 culture-positive MDR and rifampicin-resistant M. tuberculosis isolates were tested for susceptibility against capreomycin (CAP), kanamycin, levofloxacin, moxifloxacin, clofazimine, and linezolid. Results: Of the total 374 isolates, 236 (63.10%) strains were susceptible to all drugs, whereas 138 (36.89%) were resistant at least to one of the second-line drugs. One hundred and sixteen (31.01%) strains were identified as preextensively drug-resistant (XDR) (MDR isolates with additional resistance to either fluoroquinolones or second-line injectables) and 22 (5.88%) of the isolates tested were identified as XDR. Conclusion: India is a signatory to the WHO's “The End TB Strategy” with the aim of ending the global TB epidemic; intensified efforts for early detection and treatment of drug-resistant cases from both public and private sectors are required to accelerate the rate at which TB incidence falls and accomplish the desired results.

Keywords: Drug-susceptibility testing, extensively drug resistant, multidrug resistant, Mycobacterium tuberculosis, rifampicin-resistant tuberculosis

How to cite this article:
Sidiq Z, Hanif M, Chopra K K, Khanna A, Jadhav I, Dwivedi KK. Second-line drug susceptibilities of multidrug- and rifampicin-resistant Mycobacterium tuberculosis isolates in Delhi. Biomed Biotechnol Res J 2019;3:87-91

How to cite this URL:
Sidiq Z, Hanif M, Chopra K K, Khanna A, Jadhav I, Dwivedi KK. Second-line drug susceptibilities of multidrug- and rifampicin-resistant Mycobacterium tuberculosis isolates in Delhi. Biomed Biotechnol Res J [serial online] 2019 [cited 2022 Jan 29];3:87-91. Available from: https://www.bmbtrj.org/text.asp?2019/3/2/87/260485

  Introduction Top

The emergence of antimicrobial resistance has become one of the dominating, and most pressing, global concerns in public health. Yet, the epidemic of tuberculosis (TB), the world's number one infectious killer disease, often goes largely unnoticed and neglected. Drug-resistant TB (DR-TB) has jeopardized global TB control achievements and threatened the accomplishments of the World Health Organization's (WHO) End TB Strategy. Multidrug-resistant TB (MDR-TB, defined as resistance to, at least, rifampicin and isoniazid) and rifampicin-resistant TB (RR-TB) are especially devastating. Patients with MDR-TB and RR-TB (MDR/RR-TB) require radical changes in treatment compared to those with drug-susceptible TB. Moreover, once fluoroquinolones and injectable agents – leading components in second-line treatment regimens – are compromised by additional drug resistance (extensively DR TB (XDR-TB), defined as MDR-TB plus additional resistance to at least the two most important groups of second-line medicines: the fluoroquinolones and the injectable agents (kanamycin, amikacin, and capreomycin), treatment becomes extremely difficult.

According to the latest WHO's Global TB Report, there were an estimated 600,000 incident cases of MDR/RR-TB in 2016, with cases of MDR-TB which accounted for 82% (490,000) of the total.[1] Drug resistance surveillance data show that globally 4.1% of the new TB cases and 19% of the previously treated cases had MDR/RR-TB.[1] By the end of 2016, XDR-TB has been reported by 123 WHO Member States. Combining their data, the average proportion of MDR-TB cases with XDR-TB was 6.2%.[1]

India accounts for one-fourth of the global TB burden. About 2.4% of all the new TB patients and 11.6% of previously treated TB cases in India have MDR-TB.[2] At the end of 2015, India had 79,000 cases of MDR/RR-TB[3] which is 11% >2014. Moreover, no data from patients treated in the public sector are available. It is estimated that 2.2 million cases of TB were treated in public sector in 2016 which could be anything between 1.9 million and 5.24 million by 2024. These projections were made based on the data from the sale of drugs containing rifampicin, the main anti-TB drug.[4]

Untreated or inadequately treated patients of MDR/RR-TB are at increased risk of spreading their disease in the community, which could lead to outbreaks in vulnerable populations. Accurate and reliable laboratory drug-susceptibility testing (DST) data to second-line antituberculosis drugs (SLDs) are the basic requirement of the Directly Observed Treatment Short course-plus strategy, as they will support clinical decision-making and help to prevent the emergence of further drug resistance in patients with MDR-TB.[5] The aim of this study was to analyze the resistance to second-line anti-TB drugs of MDR and RR strains of M. tuberculosis isolated from presumptive DR-TB patients attending various chest clinics in Delhi.

  Methods Top

The study was conducted at New Delhi Tuberculosis Centre (NDTC), Jawaharlal Nehru Marg, New Delhi. Started as a model clinic in 1940, NDTC is functioning as State Tuberculosis Training and Demonstration Center as well as Intermediate Reference Laboratory providing diagnostic and treatment follow-up services to 17 of the 25 Revised National Tuberculosis Control Program-designated TB districts (also called as chest clinics) in Delhi. The center is also responsible for the overall supervision of the external quality assurance activities of the entire state and is going through regular rounds of proficiency testing for smear microscopy as well as culture and DST by the National Reference Laboratory, National Institute of Tuberculosis and Respiratory Diseases, New Delhi, India.

A total of 372 culture-positive MDR and RR M. tuberculosis isolates were included in this study. All of them had been isolated from individual presumptive DR-TB patients (failures of new TB cases, contacts of known MDR-TB case, smear-positive previously treated pulmonary TB cases at diagnosis, and any follow-up smear positive in new or previously treated cases) attending various chest clinics in Delhi between January 1, 2017, and August 31, 2017. The identification of MDR/RR-TB was done by screening the sputum samples/culture isolates of these patients for the presence of most common mutations conferring resistance to isoniazid and rifampicin using the Genotype MTBDRplus assay version 2.0 (Hain Life Sciences, Nehran, Germany). All clinical strains had been identified initially as M. tuberculosis using the immunochromatographic Capilia test.

The drugs used for susceptibility testing of these MDR and RR MTB isolates were capreomycin (CAP), kanamycin (KAM), levofloxacin (LVX), moxifloxacin (MOX), clofazimine (CFZ), and linezolid (LZD). All test drugs were obtained in a chemically pure form M/s Sigma Aldrich. Except for CFZ and LZD, which are considered self-sterilizing, all stock solutions were filtered in a sterile manner with a 0.22 μm pore size polycarbonate filter. All stock solutions were stored at −80°C in small aliquots. Frozen drug solutions were thawed once before use and the leftover was then discarded.

The DST of MDR/RR MTB isolates was performed by 1% proportionate method using BACTEC Mycobacteria Growth Indicator Tube (MGIT) 960 system (Becton Dickinson Diagnostic Instrument Systems, Sparks, MD, USA) following the standard protocol.[6] Briefly, culture suspensions for inoculation were first well dispersed with no large visible clumps. After thorough mixing and homogenization of the culture suspensions, the tubes were allowed to rest for at least 15 min, and the supernatant was used to inoculate the drug-containing MGIT with OADC supplement. The tubes were then placed in the DST set carrier and entered into the MGIT 960 instrument as “unknown drugs” using the DST entry feature.[6] The final drug concentrations used were as follows: 2.5 μg/ml for CAP, 2.5 μg/ml for KAN, 1.5 μg/ml for LVX, 0.5 μg/ml and 2.0 μg/ml for MOX, 1.0 μg/ml for LZD, and 0.5 μg/ml for CFZ.

  Results Top

A total of 3000 GenoType MTBDRplus tests were conducted. Of these, 2898 were done directly on sputum samples and 102 indirectly using culture isolates. Of the 3000 tests conducted, 287 (9.5%) were found to have mutations conferring resistant to RIF and INH (MDR), whereas 95 (3.16%) had mutations conferring resistance to RIF only (RR). The detailed results are provided in [Table 1]. Among the 382 MDR and RR samples identified, SLDs were performed in 372 (97.3%) samples. The remaining 10 (2.7%) samples were either culture negative or contaminated. These 374 MDR/RR MTB isolates were tested for susceptibility against KAN, CAP, LVX, MOX (0.5 μg/ml), MOX (2.0 μg/ml), CFZ, and LZD. Susceptibility patterns identified are presented in [Table 2]. Two hundred and thirty-six (63.10%) strains were susceptible to all drugs, whereas 138 (36.89%) were resistant at least to one of the second-line drugs. Results showed that 18 (4.8%) of the 374 isolates tested were resistant to KAN, 24 (6.4%) to CAP, 127 (33.9%) to LVX, 4 (1.0%) to LZD, 86 (22.91%) to MOX (0.5 μg/ml), and 10 (2.6%) to MOX (2.0 μg/ml). Monoresistance to LVX, CAP, KAN, and MOX (0.5 μg/ml) was detected in 36 (9.6%), 3 (0.8%), 1 (0.2%), and 1 (0.2%) strains, respectively. Monoresistance to MOX (2.0 μg/ml), CFZ, and LZD was not detected. Resistance to two drugs in different combinations was found in 69 (18.5%) strains, resistance to three drugs was found in 25 (6.7%) strains, resistance to four drugs was found in 6 (1.6%) strains, and resistance to five drugs was found in only 1 (0.2%) strains. One hundred and sixteen (31.01%) strains were identified as, pre-XDR, most of the strains (108 [93.1%]) had additional fluoroquinolone resistance. Twenty-two (5.88%) of the isolates tested were identified as XDR.
Table 1: Resistance profile of study population for isoniazid and rifampicin

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Table 2: Susceptibility patterns of multidrug-resistant/rifampicin-resistant tuberculosis isolates against second-line antituberculosis drugs

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  Discussion Top

The present study attempted to systematically investigate the prevalence of second-line drug resistance among the pulmonary MDR and RR M. tuberculosis strains from presumptive DR-TB patients in Delhi who had not been previously exposed to second-line drugs in the form of antituberculous therapy. The increased knowledge of second-line drug resistance could be valuable for selection of an effective combination of second-line drugs for the treatment of MDR-TB. Resistance to KAN, CAP, LVX, MOX, CFZ, and LZD was analyzed in this study.

Among all the SLDs tested, at 33.9%, LVX resistance was highest, followed by 22.9% in MOX (0.5). Previous studies from various parts of India have reported high levels (3% to 35%) of fluoroquinolone resistance among MDR-TB patients.[7],[8],[9],[10] Eight-five (66.9%) of LVX-resistant strains were also resistant to MOX (0.5). This result is not surprising since cross-resistance between LVX and MOX has been reported in various studies.[11],[12] Two strains resistant to LVX were also resistant to CAP and one strain was resistant to LZD. According to the WHO's Global TB Report, among the 40 countries with a high TB or MDR-TB burden, the proportion of MDR-TB/RR-TB cases with resistance to any fluoroquinolone for which testing was done was 20%.[1]

Thus, this study concurred with higher level of FQ resistance reported in the city and is slightly higher than the international data. This can be explained by the fact that fluoroquinolones are also the most widely prescribed class of antibiotics in the world today. Their broad spectrum of antibacterial activities and convenient dosing make them frequent choices for treating a variety of infections caused by susceptible organisms and for empiric therapy of common infections, such as pneumonia or sinusitis, where a causative organism has not been identified. A recent meta-analysis concluded that there is a threefold greater risk of FQ resistance among TB patients who are exposed to FQ before diagnosis of TB.[13]

Resistance to KAN in this study was found to be 4.8%. CAP, the basic peptide antimicrobial agent with a mechanism of action similar to that of the aminoglycosides, is used in combination therapy for the treatment of MDR-TB.[14],[15] In the present study, the proportion of strains resistant to CAP (6.4%) was slightly higher than resistance to the KAN.

In most of the previous studies, across the world, a high rate of susceptibility to aminoglycosides among MDR-TB cases has been reported.[16],[17] However, some studies from Russia, Argentina, and India have reported higher rates of resistance to KAN, which might be partially explained by the administration of these drugs or analogs to previously treated patients as monotherapy.[18] The low levels of KAN resistance in the present study may be due to its limited use in the private sector perhaps because of its availability in injectable form.

CFZ and LZD are now recommended as core SLDs in the MDR-TB regimen according to recent classification.[19] Limited or no data from India are available on resistance of these two drugs. In the present study, CFZ was active against all (100%) the MDR and RR strains of M. tuberculosis, whereas 4 (1.0%) strains were resistant to LZD. Studies across the world support the results of this study.[20] Three of the LZD-resistant strains also had LVX resistance, whereas one had KAN and CAP resistance.

The proportion of XDR-TB isolates in this study was found to be 5.8% (22/372). The prevalence of XDR-TB among MDR-TB in India in various studies has been reported ranging from 0.89% to 33%.[4],[9],[10]

  Conclusion Top

According to the present study, KAN was active against 95.2% of the strains, whereas CAP was active against 93.6% strains. The fluoroquinolones (LVX and MOX), on the other hand, were active against 66.1% and 74.3% strains, respectively. Alternative drugs are necessary for treating patients harboring resistance to LVX and MOX. The use of high-dose MOX for treating MDR/RR-TB patients is a suitable alternative as 97.4% strains were susceptible when tested at higher concentration (2.0 μg/ml) of MOX. The decision of adding CFZ and LZD to the core SLD group is supported by the results of this study as these two drugs were active against 100% and 99.0% of the strains, respectively. In addition, XDR-TB detected at the rate of 5.88% in the study despite being less the previous reports is still a matter of concern. India is a signatory to the WHO's “The End TB Strategy” with the aim to end the global TB epidemic and leave no-one behind; all efforts should be made to accelerate the rate at which TB incidence falls, together with an effective crisis response to contain the DR-TB epidemic, while it is still possible. This calls for early detection of such cases both from the public and private sector so that effective treatment can be started at the earliest and prevents the amplification and transmission of resistance in the community.

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Conflicts of interest

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  References Top

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  [Table 1], [Table 2]


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