Managing drug-resistant tuberculosis: experiences from India

With an annual incidence of 1.9 million cases, India ranks 17th among 22 high-burden countries for TB [1]. After the directly observed treatment, short-course (DOTS) strategy was accepted globally, the government of India launched one of the world’s largest TB control strategies (the Revised National Tuberculosis Control Programme [RNTCP]) and the entire country has been covered under DOTS treatment [101]. As is well known, drug resistance has become a serious challenge for TB control programs all over the world [2,102]. India is also making significant efforts to tackle the problem of drug resistance [103]. Considering the magnitude and scale of TB, lessons learnt from India could be meaningful at a global level and, thus, may be a learning experience for many.

Magnitude of the problem

Emergence of multidrug resistance (MDR) and extensive drug resistance (XDR) has made the situation frightening both for the unfortunate victims and also the lay public. Different studies show that incorrect regimens, poor compliance and other related factors are contributing to the increase in resistance [2,102]. However, alarming reports do not necessarily reflect a true estimate of the magnitude of the problem. Most of these reports are based on drug sensitivity testing in patients attending the outdoor patient departments of major tertiary care hospitals. While the high prevalence of drug resistance in some of these reports deserves an adequate response, these hospital-based figures should not be extrapolated for epidemiological purposes. It has been estimated that India and China account for nearly 50% of the global burden of MDR cases, with India perhaps having the highest number [1]. According to this report, approximately 5% of all pulmonary TB cases in India may be MDR. At the microlevel, MDR rates are low in new, untreated cases, with figures ranging from 1 to 5% (mostly <3%) from different parts of India [3,4,103]. However, during the last decade, there has been an increase in reported incidences of drug resistance in Cat II cases, especially those treated with incorrect regimens and also in an irregular fashion. The figures for MDR rates in such cases vary from area to area and, overall, 12–17% of MDR rates have been estimated in well-conducted studies [103]. Several studies have been undertaken to determine the prevalence of drug resistance in Mycobacterium tuberculosis, but trends from the same population have been available in only a few settings and suggest that the situation has, by and large, remained stable over the last decade [3,4,103]. The government of India, through several national institutes such as the National Tuberculosis Institute (NTI; Bangalore) and Tuberculosis Research Centre (TRC; Chennai) have carried out drug susceptibility surveys in different parts of the country, especially in new, untreated cases to determine the primary drug resistance, which indirectly suggests the transmission of drug-resistant organisms. National JALMA Institute for Leprosy and Other Mycobacterial Diseases (NJILOMD, Agra) has carried out a study in three districts of Uttar Pradesh (UP), the most populated state of India [5]. The main purpose of the studies at NJILOMD is to determine the prevalence and drug-resistance profile in defined populations of several districts in UP. The second major objective of these studies is to carry out genotyping of isolates to trace the transmission chain in areas and populations where the impact of a control program can be closely monitored. Isolates of M. tuberculosis from pulmonary TB cases from these areas, covered by our Model Rural Health Research Unit (MRHRU) at Ghatampur, UP, and its satellite areas, have been included in these investigations. All the investigations on drug resistance by NJILOMD have been based on drug susceptibility testing (DST) using the proportional method. Other methods are used in selected cases for research/comparative purposes only. In the study carried out in the rural population of Kanpur Nagar district, the incidence of primary/initial MDR was found to be very low (0.5%, overall 3.3% for the entire district). Results of studies carried out in Agra and Banda districts of UP by NJILOMD show primary MDR rates to be in the range of 1.5–4%. MDR rates in previously treated cases (Cat II) were found to be higher with an average of approximately 15% in the Ghatampur area. It varied greatly in Agra and Banda as it was based on referred cases only. These studies have also identified other cases of polyresistance (not MDR), which also respond poorly to Cat II treatment. Another study in 35 districts of UP (Western UP and Bundelkhand regions; population 84.65 million in 2007) is ongoing, and is being jointly carried out by NJILOMD and the State TB Demonstration Centre, Agra. Various other studies carried out in other parts of India have also shown prevalence of MDR in the similar range [3,4,6].

Drug susceptibility testing

Determining the drug susceptibility profile of M. tuberculosis isolates accurately and rapidly is a challenging task. As currently recommended, the proportional method is a cumbersome, time-consuming and technically demanding approach; a variety of alterative techniques are being used for determination of DST [6]. The BACTEC method is perhaps the most common rapid technique being used worldwide but is gradually being phased out due to a radioactivity hazard. Several other methods based on nonradioactive detection strategy are being considered, including Mycobacterial Growth Indicator Tube, Septi-Check, PhaB Assay, Alamar Blue Assay, resazurin microtiter assay, Luciferase Reporter Phage Assay and E-test. Several other phenotype methods proposed for the rapid characterization of drug-resistant M. tuberculosis include ATP bioluminescence assay, flow cytometry and the colorimetric nitrate reductase-based antibiotic susceptibility test. Experience is growing about the usefulness and cost–effectiveness of these assays in the setting of India but no recommendations have yet been made.

Molecular techniques are also being extensively used for understanding the mechanisms of drug resistance in TB. Specificity and speed are major advantages of molecular assays. In case of TB, mutations in the target sites are considered the most important mechanisms of generating resistant isolates [7]. While DNA sequencing is the gold standard for the detection of mutations in the genes responsible for drug resistance, other simple approaches, such as line probe hybridization assay, PCR-single strand conformational polymorphism, PCR heteroduplex formation and PCR-restriction fragment length polymorphism, have been described for the detection of mutations [7]. Techniques such as line probe hybridization are simple to use and more suited for laboratories with a small infrastructure [6]. While a line probe assay (LPA) for rifampicin is commercially available (Inno-LIPA), improved versions have been developed in India and abroad (e.g., Haine’s test). A line probe-based strategy has been observed to be useful by several investigators for detection of mutations encoding for rifampicin and isoniazid hydrazide resistance. To take care of novel mutations in a section of Indian isolates, alternate molecular assay systems have been developed at several centers in India (e.g., All India Institute of Medical Sciences, New Delhi and National Hinduja Hospital, Mumbai) and are being evaluated. At present, after proper evaluation supported by the WHO and FIND, and supervised by the Central TB Division, government of India, LPA (Haine’s test) is being introduced across the country.

As the detection of mutations in the target sites is not proving successful for drugs other than rifampicin, there is an increasing focus on investigating other possible mechanisms such as permeability barriers, inactivation of drugs by enzymatic machinery of the bacteria and extrusion of drugs by efflux pumps. All of these mechanisms play varying roles in different isolates for various drugs and a comprehensive picture is yet to emerge. Unknown mechanisms can be better understood by microarray and also proteomic approaches. With microarray upregulation of several genes in isoniazid hydrazide resistance, involvement of new pumps in ofloxacin as well as MDR has been identified in M. tuberculosis[8]. These investigations have provided several leads that have potential applications in the development of new compounds and also new molecular methods for detection of drug resistance. However, this is all in the realm of the future and no clear recommendations can be made at the moment.

While the search for optimum techniques continues, India has embarked upon a major infrastructure development program of creating laboratories all over the country. Besides a supranational laboratory at TRC, the government of India has accredited four National References laboratories – NRLs at TRC, Chennai; NTI, Bangalore; NJILOMD, Agra; and Lala Ram Swaroop Institute of Tuberculosis and Respiratory Diseases, New Delhi. These NRLs are, in turn, helping in the development and supervision of 27 intermediate reference laboratories (IRLs) in the states, as well as laboratories in medical colleges and other institutions [103]. During the last 2–3 years, 12 IRLs have been accredited and five are expected to be accredited in the near future. The government of India has planned that by 2012 the country should have 43 laboratories with the capability for performing molecular methods (e.g., LPA) for detection of drug resistance, and two-thirds of IRLs should also be using liquid culture. Initial focus will be on Cat II cases and cases not responding to Cat I regimen, but by 2015, India plans to investigate all smear-positive cases for MDR diagnosis and management. The greatest difficulty in this development has been to obtain properly qualified and experienced manpower for mycobacteriology, which suggests the need to prepare long-term plans to cope with these public health challenges.

Treatment

There is a well-accepted strategy to manage TB at the global level. For the treatment of new, untreated cases of TB, DST is not recommended in India. This is based on the fact that the drug resistance (especially MDR) is low in new, untreated cases. Keeping in mind the available published literature and also unpublished data from India, DST appears to be quite important for previously treated cases, particularly in those showing poor response to Cat II DOTS regimen. India has already been listed as one of the countries from which XDR has been reported. However, the exact magnitude of XDR is not known in India. As part of the RNTCP of the government of India, different State Health Authorities have followed a well-planned strategy to face the challenge of drug resistance in TB. These measures are essentially preventive and include increasing coverage with DOTS, operational research to monitor the trends in drug resistance, research in methods for rapid detection of drug resistance and improving methods/regimens for prevention and management of drug-resistant cases.

India began its DOTS Plus program in 2005 and is being introduced in phases. While some empirical protocols based on current experience have been developed, it is accepted that the treatment will be based on DST. DOTS Plus has been initiated in nine states. It has been planned that by 2010 approximately 8000 MDR cases (5–10% of MDR estimated numbers) will be covered by DOTS Plus, which will be scaled-up to 30,000 or more from the year 2012 onwards. Thus, the challenge of providing diagnostic support and treatment to every MDR case is immense but the progress is continuous and sustained.

Besides effective surveillance programs and the use of proper DOTS, different methods have been considered to treat MDR and/or Cat II cases. In addition to the use of drugs based on DST (DOTS Plus), immunotherapy may be another approach that may be considered for the management of such cases. Promising reports concerning the use of Mycobacterium vaccae in the treatment of drug-resistant TB outside of India have been published [9]. One such immunomodulator Mycobacterium w (Mycobacterium indicus pranii), marketed by Cadilla Pharmaceuticals Ltd (Gujarat, India), is already undergoing clinical trials for Cat I and II cases in several institutions of the country and interim results are expected this year. These results will provide the basis for further action.

Tracing the transmission with molecular methods

In various Indian studies, genotyping of M. tuberculosis strains has been performed by IS 6110, spoligotyping and mycobacterial interspersed repetitive units–variable number of tandem repeats (MIRU-VNTR) methods. In some initial investigations, random amplified polymorphic DNA (NJILOMD) and amplified fragment length polymorphism (Centre for DNA Fingerprinting and Diagnostics) have also been used. Various DNA fingerprinting techniques have confirmed that the transmission of drug resistance is still low in India. Beijing strains have made a niche but, so far, do not yet appear to contribute significantly to transmission of disease and drug resistance. Molecular epidemiology studies carried out by us have shown that IS 6110 typing could be useful in 60–70% of Indian isolates [10]. Spoligotyping shows the predominance of CAS and EAI varieties, with the Beijing type being less than 10% (average ∼5%). Regional differences in strains have also been indicated in some of these studies. MIRU-VNTR typing has revealed very high diversity among North Indian strains of M. tuberculosis. Furthermore, the Beijing variety was not found to be linked with transmission of drug resistance in the North Indian population of Ghatampur [11]. These techniques can be used alone and/or as complementary approaches (preferable) to monitor the effect of DOTS and other interventions on transmission dynamics. In the present situation, these approaches will be used in model areas but their ultimate utility will become important when the problem is controlled to some extent and one will have to solve specific questions.

To conclude, India is following a multipronged strategy to tackle the problem of drug-resistant TB. Emphasis is on expanding the coverage of DOTS and DOTS Plus, by establishing a well-focused laboratory strengthening program, by developing and/or using rapid methods of detection of growth and DST for better patient care, and finally by encouraging researchers to develop better alternate therapeutic options.

Acknowledgements

Participation of several scientists, research scholars, laboratory and field staff of the Drug Resistance Study Group of the National JALMA Institute for Leprosy & Other Mycobacterial Diseases, Agra & MRHRU, Ghatampur, collaborators from AIIMS, CDFD as well as State Health Services of UP (Directors and staff of STBDC, Agra, State TB Officers, District TB Officers and Medical Officers of TB Centres) is gratefully acknowledged.

Financial & competing interests disclosure

Studies cited from NJILOMD, Agra have been financially supported by the Department of Biotechnology, Indian Council of Medical Research and Central TB Division, Govt. of India. The author has no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

No writing assistance was utilized in the production of this manuscript.