|Year : 2002 | Volume
| Issue : 4 | Page : 171-173
Mycobacterial research in India: Successes and challenges
Central JALMA Institute for Leprosy (ICMR), PO Box 101, Taj Ganj, Agra - 282 001, India
Central JALMA Institute for Leprosy (ICMR), PO Box 101, Taj Ganj, Agra - 282 001, India
|How to cite this article:|
Katoch V M. Mycobacterial research in India: Successes and challenges. Indian J Med Microbiol 2002;20:171-3
Tuberculosis and leprosy, which are major mycobacterial diseases, have always been important public health problems in India. The exact status of infections due to non-tuberculous mycobacteria (NTM) and human infections due to Mycobacterium bovis is not known with certainty. While there has been major decline in the prevalence of leprosy recently, the problem remains significant in several parts of India, indicating possible continued transmission. The scenario in case of tuberculosis is quite alarming and has been further complicated by the spread of HIV as well as increasing drug resistance in improperly treated cases. It is well known that Indian workers have contributed significantly in unraveling the biology of mycobacteria and development of several effective drug regimens for treatment of these infections. It would be interesting to look at the current scene from different angles to know our strengths and limitations.
Major effort has been made in understanding the genetic structure and function of M.tuberculosis, M.leprae and other important mycobacteria. While the complete genome sequence of M.tuberculosis and Mycobacterium leprae has already been published, Indian laboratories have contributed in identifying some unique gene sequences of practical importance such as LSR protein gene of M.leprae and repeat sequences in M.tuberculosis. Studies on the transcriptional apparatus of mycobacteria have provided significant information about the promoter elements which are being exploited for developing improved vehicles for recombinant vaccines. In Indian laboratories, studies have been progressing to identify possible virulence genes. Differentially expressed genes in virulent versus avirulent strains such as devR and devS have been identified which can be considered as important leads to investigate these aspects further.
Slow growth of M.tuberculosis and inability of M.leprae to grow in any acceptable in-vitro system, have been the major handicaps in investigating and treating these diseases. During the last two decades many alternate methods for early detection of growth of M.tuberculosis and other non-tuberculous mycobacteria (NTM) have been described from the western laboratories. These include commercially available techniques such as BACTEC, Septi-Chek, MGIT tubes, E-test, phage reporter assays etc. There are reports of development of simpler procedures for direct sensitivity testing for M.tuberculosis. Overall in India, the emphasis is more on studies pertaining to application of already developed methods for determination of viability and drug screening. The number of well established clinical mycobacteriology laboratories using conventional and newer techniques has significantly gone up. A national Mycobacterial Repository for Indian strains has been established.
There has been significant progress with regard to the development of methods for faster identification of mycobacterial isolates. By incorporation of NAP (nitro alpha acetylamino beta hydroxy propiophenone) in the BACTEC, Septichek etc, quick classification into M.tuberculosis and NTM can be made. Further, the identity of isolates can be rapidly confirmed by analysis of lipid profiles by TLC, HP-TLC and HPLC. These lipid based techniques are being used for identification of mycobacteria. In India, methods and schemes based on isoenzyme and protein electrophoregrams and immunological relatedness of enzymes such as superoxide dismutases have been developed. Besides the phenotypic methods, gene probes for identification of M.tuberculosis and other NTM have been developed and are also available from commercial companies. For M.leprae, probes based on rRNA gene sequences have been developed in India and have been found to be useful for identification of M.leprae in the lesions. These probes have been further developed into semi-quantitative hybridization microdensitometric scanning technique for assessment of load of viable organisms in the lesions. These assays can be useful for monitoring the progress of leprosy cases especially for the purpose of making decisions about the adequacy / need of re-treatment. These techniques need to be put into practice in India and depending upon the experience, Indian laboratories may have to modify and or develop newer probe based methods suitable for Indian strains.
Recent years have witnessed a rapid progress in development of gene amplification techniques for identification of mycobacterial specific (genus, species) gene sequences in the culture isolates as well as clinical specimens. These amplification assays include techniques based on polymerase chain reaction (PCR) as well as isothermal amplification strategies. A number of PCR assays for detection of M.tuberculosis have been described in India. Some of these techniques have been evaluated in multi-centric trials and found to be highly sensitive as well as specific. Gene amplification techniques for detection of M.leprae have also been developed in India by targeting LSR gene and rRNA. The usefulness of these methods will be known after sufficient experience on clinical specimens from different parts of the country.
Emergence of drug resistance in tuberculosis has become a serious problem in several parts of the world including India. Newer information about the target genes for many antituberculosis drugs have provided better tools to undertake surveillance studies on the incidence and prevalence of drug resistant strains. There has been a major global effort in understanding the molecular basis of susceptibility/ resistance in mycobacteria. Target gene(s) for several antimycobacterial drugs have been identified in M.tuberculosis and rapid methods for identification of these mutants have been developed. Several Indian laboratories are engaged in investigating the molecular mechanisms of drug resistance. In the multi-centric studies carried out in India the absence of known mutations in a proportion of MDR strains (to different drugs including rifampicin, isoniazid, quinolones) and presence of novel mutations associated with varying degrees of rifampicin resistance has been observed. These findings indicate that there is need to develop indigenous probes suitable for Indian isolates and first test the usefulness of new molecular methods in a large number of strains from different geographical areas. Such probes are in various stages of development and testing.
In the recent years, several genomic techniques for identification of strain types for tracing the transmission have been developed for mycobacteria. These are of particular importance for mycobacterial infections as other phenotypic methods have not been found to be much useful for mycobacteria. Most important among them are DNA fingerprinting / restriction fragment length polymorphism (RFLP) methods. RFLP techniques for characterization of strains of M.tuberculosis, M.leprae, M.avium, M.partatuberculosis have been developed by several investigators from different parts of the world. In India, main contribution has been in the area of RFLP techniques based on direct repeats, fluorescent amplified fragment length polymorphism (FAFLP) based whole genome scan methods for M.tuberculosis as well as ribosomal RNA gene for M.tuberculosis, M.leprae and other mycobacteria. Though at present there is insufficient data available, such clustering of drug resistant strains has not been observed in the initial Indian studies among isolates (from non-AIDS cases) collected from different parts of the country. While the exogenous sources were clearly identified in studies in USA, the reactivation of endogenous infection is believed to be more important for developing infections which harbor large human pool with latent infection. By using direct repeat probes for RFLP, endogenous reactivation was shown to be the main reason for relapses in south India. More studies to investigate the epidemiology of tuberculosis and leprosy (where the incidence continues to be high) using these molecular methods are needed at this point of time.
In India, special efforts to develop vaccines for leprosy and tuberculosis have been made. For leprosy, many candidate vaccines using cultivable mycobacteria (ICRC and Mw) have undergone clinical trials and have shown promise. These have already shown clear immunotherapeutic potential. For tuberculosis, search has been continuing to identify antigens with potential role as immunomodulators. Though the information about relevant antigens is quite sketchy, some potentially useful antigens have been identified. Recombinant vaccines are in different stages of development and testing in Indian laboratories.
While the efforts and progress in the area of mycobacterial research almost match the global trends, major gap appears to be in case of testing and wider application of modern molecular and immunological techniques to improve the diagnostic capabilities, understanding the dynamics of transmission and underlying mechanism(s) of increase in drug resistance. Practically very few attempts have been made to understand relevant antigens of M.tuberculosis and M.leprae etc. to which the Indian population respond and as a result our attempts at development of immunodiagnostics / vaccines are weak from the beginning. Undoubtedly, these studies should receive due focus. The application of micro-arrays is also expected to be useful for investigating all the above important areas.
It would be justifiable to conclude that Indian investigators continue to contribute significantly towards improving the understanding of basic biology of pathogenic mycobacteria as well as developing and applying new generation methods for diagnosis and management of these diseases at different levels. While the research on M.bovis is picking up, other mycobacterial organisms are yet to receive adequate attention. It would be important not to ignore other mycobacteria such as M.bovis, M.paratuberculosis and other NTM. In addition, it would also be important to strengthen the drive towards adequate evaluation and wider application of modern methods (developed in India or elsewhere) for the benefit of the patient and community.