A PROFILE OF THE ROUTINE AND RESEARCH ACTIVITIES OF THE TUBERCULOSIS LABORATORY
Ø Development of improved diagnostic tools for early diagnosis and monitoring of infection and disease, due to drug-sensitive and drug-resistant strains of M. tuberculosis.
Ø Research, including the development of new molecular tools, to assess the factors influencing, the occurrence, distribution, and transmission of TB and multidrug-resistant TB.
Ø Clinical testing of prophylactic and therapeutic anti-TB regimens and evaluate the effectiveness of treatment, especially against multi-drug resistant tuberculosis.
Tuberculosis is a cause for growing concern in India where out of 844 million people, we encounter 3 million sputum positive cases and 12 million radiologically active abacillary pulmonary tuberculosis cases. The prevalence of extrapulmonary tuberculosis in India is recognised though the exact figures have not been compiled. With a mortality rate of 50-80/10,000 population (300,000-500,000 annually) and an immense economic burden in terms of man-hours lost and cost incurred in treating patients, the Dept. of Microbiology pioneered the use of molecular tools and rapid techniques for improving detection of Mycobacterium tuberculosis from clinical samples.
The All India Institute of Medical Sciences is a tertiary care national hospital where drug resistant pulmonary tuberculosis patients and those suffering from extrapulmonary tuberculosis are referred. In 1989, we developed a polymerase chain reaction assay based on the immunogenic protein, MPB64 encoding gene (Shankar et al., 1990). After demonstrating the diagnostic ability of this assay for pulmonary and extrapulmonary samples (Shankar et al., 1991; Manjunath et al., 1991), we evaluated the efficacy of the system for diagnosis of TBM (Seth et al.,1996) including tuberculoma, pulmonary tuberculosis (Dar et al., 1998) and infertility arising due to tuberculosis. Since 1995, PCR is being offered as a routine diagnostic service. We report about 400 extrapulmonary samples every month.
ANTITUBERCULOUS DRUG SUSCEPTIBILITY TESTING
Besides the routine smear and culture done on samples, we perform sensitivity testing for first and second line antituberculosis drugs for all M.tuberculosis complex cultures isolated in the laboratory, according to the CDC guidelines using proportionate method. We are also determining susceptibility patterns of clinical isolates to the first and second-line antibacterial drugs on BACTEC 460 TB system.
Using BACTEC 460TB system, we report isolation of mycobacteria within 10-15 days; we differentiate M.tuberculosis complex and nontubercular mycobacteria using NAP differentiation kits, easing the decision of the physician regarding the treatment regimen.
There are few reports from India about the prevalence of NTM. In our laboratory, all cultures isolated are defined to the species level, using a battery of biochemical tests. We are also sequencing the 16S rRNA gene segment, besides PCR-REA of hsp65 gene segment and 16S rRNA gene segment.
Diagnostic PCR for tuberculosis is being maintained for the past 9 years. This proves to be of great utility especially in paucibacillary extrapulmonary samples.
Antitubercular Drug Susceptibility testing by culture based methods is available for routine use and is being carried out regularly on patient samples.
Rapid culture of Mycobacteria and Antitubercular Drug susceptibility for first lineas well as second line drugs for the treatment of Tuberculosis is being carried out on Bactec 460TB (Becton & Dickinson).
Molecular epidemiology can be helpful in recognising outbreaks, detecting transmissions and monitoring control programmes. In the Tuberculosis Laboratory, newer molecular technologies viz., spoligotyping, and double-repetitive element polymerase chain reaction (DRE-PCR)have been inducted in addition to the traditional IS6110-RFLP,for identifying unsuspected incidents of transmission.
We have generated a baseline data on the genotype of M.tuberculosis strains circulating in and around Delhi, using IS6110-RFLP and spoligotyping. We found only 1.3% strains lacking IS6110. There was also an absence of ongoing transmission due to one genotype, in the community. We are publishing the results of this study shortly. We also have a rapid, simple and low-cost option for typing strains of M.tuberculosis, the double-repetitive element (DRE)-PCR ready for use in outbreak situations.
Using spoligotyping, isolates of Mycobacterium tuberculosis grown from pulmonary tuberculosis patients were found to belong to a cluster. In an attempt to apply spoligotyping directly to multibacillary sputum specimens, cultures and respective samples were analysed. Mostly, the samples and their respective cultures yielded concordant patterns. An instance of suspected cross-contamination during sample processing was also investigated by spoligotyping on serial samples which had acid-fast bacilli. Varied patterns were obtained ruling out cross contamination and assuring the quality control.
Autorad showing the RFLP patterns of clinical M. tuberculosis isolates
Autorad showing stained spoligopatterns of clinical M.tuberculosis isolates. Beijing pattern is the most prevalent in countries.
Double-Repetitive Element Polymerase Chain Reaction (DRE-PCR) was evaluated in M.tuberculosis isolated characterized by IS610-RFLP and spoligotyping. It was found to be a discriminative genotyping tool and was useful for initial screening of isolates. This technique is being used for routine typing of strains as well as in samples when cross-contamination is suspected.
RAPID DETECTION OF DRUG RESISTANCE
A new molecular technique was incorporated based on the same principle as spoligotyping,(reverse hybridization), Resistance genotyping by line probe assay can detect drug resistance mutations rapidly to aid the clinicians in rapid recognition of multidrug resistant Mycobacterium tuberculosis strains.
In a community-based study, it was found that rpoB resistance genotyping was useful in determining phenotypic resistance.
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Research is ongoing to look for mutations in the rpoB gene by PCR and sequencing. Though these mutations have been very well defined for strains isolated in the West, little work has been done in the Indian isolates. Knowledge about novel mutations and their relevance to clinical resistance would enable us to modify the reverse-line blot genotyping to include all rpoB mutations attributing to rifampicin resistance in Indian isolates. Besides, we have collaborated in the development and validation of a resistance genotyping scheme based on reverse line blotting for rpoB, ahpC, inhA katG, rrn, rpsL and embB gene mutations. We are evaluating the utility of this system in the Revised National Tuberculosis Control Program (RNTCP).