As the conventional techniques are too lengthy, or lack sensitivity and specificity, it has been a long-standing need in India to develop a test which is rapid, sensitive and specific. A test based on polymerase chain reaction (PCR) fulfils all these criteria. Hence a PCR test, adapted to Indian conditions, was developed at BARC’s Radiation Medicine Centre (RMC) for the rapid diagnosis of tuberculosis. After successful validation of the test, both in PTB and EPTB, and development of an efficient DNA extraction method, a diagnostic kit was developed in collaboration with JONAKI, BRIT. The kit has been launched in the market, and will be of immense use in early diagnosis of TB.

Tuberculosis is a public-health problem of global importance, more so because TB has re-emerged as one of the leading causes of death, killing nearly 3 million people annually. The emergence of HIV infection and rising prevalence of multi-drug resistant tuberculosis have threatened the effectiveness of standard chemotherapy.

Prevailing conventional methods fall short of the requirement for rapid diagnosis that is both specific and sensitive. Though the conventional method, AFB smear microscopy, has high positive predictive value, it lacks the sensitivity. Culture technique is sensitive and is considered to be the Gold Standard, but is labour intensive and extremely slow in final diagnosis: it takes 3-6 weeks because of extremely slow growth rate due to long generation time of mycobacteria. Tuberculin test is another widely used supportive diagnostic test that lacks specificity; serological tests were found to be unsatisfactory both in sensitivity and specificity.

The break-through by RMC in rapid diagnosis with sensitivity and specificity has thus immense value in the battle against TB. Further on, this diagnostic break-through has been followed up by development of a TB-PCR kit in collaboration with JONAKI, BRIT, a unit of the Department of Atomic Energy (DAE). The kit has undergone trials and improvements and has been launched in the market directly, for widest usage. Its commercialisation could have restricted its availability.

It needs to be mentioned that the RMC diagnostic break-through was made possible by the invention of Polymerase Chain Reaction (PCR) in 1993, a Nobel Prize winning technology, by Kary Mulls. The invention of Polymerase Chain Reaction provided an exciting boost in the diagnosis of tuberculosis. As the PCR invention offered amplification of small amount of DNA, it was extensively evaluated for the detection of MTB from sputum samples for diagnosis of PTB.

However, it was not a straight jump from the Nobel Prize winning invention of Polymerase Chain Reaction to its application for developing a TB-PCR Kit for rapid TB diagnosis. A lot of research and technological development had to be done by RMC and BRIT in order to produce a kit that was fit for Indian conditions.

Avoiding medical jargon, let us say, the discovery of occasional MTB strains in India lacking the required sequence implied possibility of a few false negative results. To overcome these problems, a PCR test was developed keeping a specific gene of MTB involved.

These PCR conditions were optimized using standard MTB DNA. The analytical sensitivity and specificity of the test were established. Different DNA extraction protocols were standardized and compared for extraction efficiency. Standardized protocols were then used on clinical samples such as sputum from pulmonary TB cases and abdominal biopsies and Cerebro Special Fluids from patients with extra-pulmonary TB for validating the PCR test.

After satisfactory validation, it was decided to test the market, in a kit form. This was jointly undertaken by RMC and JONAKI, BRIT. The prototype kit produced jointly by RMC and JONAKI, BRIT when evaluated using clinical samples from TB patients and non-TB controls, showed sensitivity of 84% and specificity of 97%.

A small batch of kits was then given to different hospitals for evaluation. After satisfactory feed-back, a bulk production was carried out and a totally indigenous kit – which (to use medical jargon) contains both column based extraction as well as PCR reagents – was launched in the market a year ago.

In reflecting over a high value, great achievement: RMC, BARC and JONAKI, BRIT have successfully developed an in-house TB-PCR test for both pulmonary and extra-pulmonary tuberculosis, and have made it commercially available – after different levels of quality evaluation and validation. (IPA Service)