Indigenous TB test can enhance accuracy, speed up testing

The rt-LAMP assay is a molecular test with high sensitivity and specificity
| Photo Credit: CDC/ Ray Butler, MS

Researchers at the Thiruvananthapuram-based Sree Chitra Tirunal Institute for Medical Sciences and Technology (SCTIMST) have developed and tested a novel, cost-effective, real-time LAMP (rt-LAMP) assay for early diagnosis of TB. Much like GeneXpert and Truenat, the rt-LAMP assay too is a molecular test with high sensitivity and specificity. The rt-LAMP assay was able to detect TB DNA even when only 10 copy numbers were present per microlitre in a sample. 

The main handicap that prevented the use of LAMP for TB diagnosis so far was the inability of using any dye, as dyes generally inhibit reaction leading to false negatives. Researchers at SCTIMST have overcome this challenge by turning to a fluorescent dye Syto 16, which is routinely used in labs for studying cells and other biological samples. And unlike RT-PCRs that require three different temperature settings to complete a test, the rt-LAMP test works at a single temperature. Since a fluorescent dye is used, the amplification can be measured not at the end of the run but every minute. 

“With six primers used for amplification compared with two in the case RT-PCRs, the rate of amplification is high, and hence results of positive samples can be obtained in 10-20 minutes,” says Dr. Anoopkumar Thekkuveettil from SCTIMST and the corresponding author of a paper published in Scientific Reports. “Since all six primers need to bind to the DNA for amplification to begin, the rt-LAMP assay has very high specificity.” Since no probe is used unlike in the case of RT-PCR tests, and as the dye and primers are inexpensive, the rt-LAMP test becomes highly affordable. 

The assay was tested on 350 presumptive pulmonary TB sputum samples. The rt-LAMP kit was evaluated against a microbiological reference standard (MRS), GeneXpert, and smear test. The study was conducted from October 2019 to March 2020 and from January 2023 to March 2024. The rt-LAMP assay demonstrated slightly higher sensitivity and specificity than the GeneXpert assay. Compared with MRS, rt-LAMP showed 89.36% sensitivity and 94.06% specificity. 

“The rt-LAMP assay kit has been developed as an open platform system which allows existing RT-PCR machines to be repurposed for TB diagnosis. This can be done by programming an existing RT-PCR machine to run at one temperature setting instead of three,” Dr. Anoopkumar says. “It is possible to process up to 96 or 384 tests in a single run. Thus the assay facilitates high throughput testing, allowing for a large number of patient samples to be processed in a single run.”

“The technology has been licensed to industry, has received approval from CDSCO, and is currently being validated by ICMR. The WHO Health Technology Access Pool program is currently evaluating the technology and is waiting for ICMR validation,” he says.

With nearly 79% of presumptive TB cases in India diagnosed using sputum smear microscopy and only 21% tested using molecular assay as recently as 2023, India continues to over-rely on smear microscopy for TB diagnosis. Despite an increase in molecular testing facilities from 5,090 in 2022 to 6,496 in 2023, it is nowhere close to meeting the National Strategic Plan 2017-2025 of reducing the reliance on smear microscopy.