Tuberculosis is an infectious disease that kills lakhs of Indians every year. Early detection of the disease is key to administering treatment; however, this has been hampered by the fact that current diagnostic techniques are often costly and time-consuming. Now, researchers from the Indian Institute of Science, Bengaluru, have come up with an inexpensive paper-based diagnostic device for tuberculosis detection.
Tuberculosis (TB), caused by the bacteria Mycobacterium tuberculosis, kills more people every year than any other infectious disease. India has the highest TB burden in the world with about 2.2 lakh reported deaths each year. Despite being curable, detecting TB cases has been difficult as current diagnostic tests are expensive and time-consuming and difficult to access. There are only about 3000 GeneXpert machines across India, which makes it difficult for a population as large as India’s to access the test.
“The WHO-endorsed rapid molecular diagnostic test called GeneXpert is prohibitively expensive for many and there are too few of them available in the country, given the massive disease load,” Toley says. Using his past experience in developing technologies that enable sophisticated chemical reactions to be carried out on paper strips (paper-based microfluidics), Toley decided to address the TB diagnosis problem.
The paper-and-plastic device developed by the team enables rapid detection of tuberculosis at the point-of-care. It selectively amplifies (increases the number of copies of) the DNA of Mycobacterium tuberculosis (Mtb) from the DNA isolated from patient sputum. The presence of this DNA indicates TB.
The test consists of a few simple steps. First, one must add the DNA of the patient into a spot on the device (test-zone) and seal the device with an adhesive strip that comes with the kit. This set up must be kept at a temperature of 63°C for an hour. After this, one must add a fluorescent dye that binds to MtbDNA, flash ultraviolet light with a UV torch, and take a picture using a cell phone. Green fluorescence in the test zone indicates the presence of Mtb DNA, hence a positive diagnosis for TB. The technology is called Fluorescent Isothermal Paper-and-Plastic Nucleic Acid Amplification Test (FLIPP-NAAT).
“With FLIPP-NAAT, we have tried to address the challenges of affordability, accessibility and user-friendliness,” says Navjot Kaur, first author of the paper. While the GeneXpert test costs about Rs. 1800, a FLIPP-NAAT test has the potential to bring down the cost substantially – the team is aiming at bringing the cost down to less than Rs. 300 per test. “It is quite an important study,” says Debjani Paul, Associate Professor, Indian Institute of Technology, Bombay (IIT‑B), who was not involved in the study, “The World Health Organisation (WHO) has been pushing for a molecular test for TB that is suitable for primary healthcare centres. This certainly takes one step towards that goal.”
Coming this far was not easy. Speaking of the challenges she faced in the process, Kaur says, “The DNA amplification technique that we use in FLIPP-NAAT is a difficult technique to handle and standardize.” If the protocols were not followed religiously and the reagents not handled with utmost care, it would cost the researchers a lot of time and resources to figure out what was going wrong and to fix them. It took them almost a year to learn how to handle the technique and to understand the factors that affected the results. But once they had understood the factors that controlled the test performance, the technique was robust and reproducible.
Although this is a significant step forward in the rapid diagnosis of TB, some challenges remain – overcoming false positives and developing a rapid DNA extraction technique. Of the 30 patient samples that were tested using FLIP-NAAT and compared with the GeneXpert test, there were five false positives. But the group is working on these aspects. “These are very initial results and there is a long way to go for it to be comparable with the existing molecular tests,” Debjani Paul says. The device also lacks an integrated DNA extraction step, which is a crucial step for this test for TB.
“The main focus of future work would be to reduce the false positives generated by the test. We currently have multiple approaches in mind to tackle this issue. Our lab is also working on methods to make TB DNA extraction portable and low-cost,” Toley says.
Once these hurdles are crossed, FLIPP-NAAT could be a game-changer in the rapid diagnosis of TB in India. Besides its application for the diagnosis of TB in this device, it could be modified to detect other diseases as well.
Toley is careful in his claims, however. “One must be cautious about making these predictions too soon,” he says, “because technology is just one link in the chain of delivering quality healthcare. For this technology to reach the masses, many other pieces need to fall in place, e.g. a commercial partner to manufacture the prototype and market it, large-scale clinical trials, and inclusion of the test in the diagnostic algorithm of the national tuberculosis control program. We are trying to take one step at a time towards this ultimate goal.”
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