AIIMS-led team de­vel­ops sen­si­tive tests for pul­monary, pleu­ral TB

Sen­si­tiv­ity for pul­monary TB and pleu­ral TB were 94% and 93%, re­spec­tively.

The Hindu - - SCIENCE & TECHNOLOGY - R. Prasad

highly sen­si­tive di­ag­nos­tic test for pul­monary TB and pleu­ral TB has been de­vel­oped by a multi-in­sti­tu­tional team led by Jaya Si­vaswami Tyagi from the Depart­ment of Biotech­nol­ogy at AIIMS. The di­ag­nos­tic test makes use of a DNA ap­tamer (a small sin­gle-stranded DNA mol­e­cule that binds to a spe­cific tar­get mol­e­cule) that shows high bind­ing affin­ity to a TB anti­gen. Spu­tum sam­ples were used for di­ag­nos­ing pul­monary TB while pleu­ral fluid was used for di­ag­nos­ing pleu­ral TB.

The sen­si­tiv­ity of the di­ag­nos­tic test for pul­monary TB and pleu­ral TB was 94% and 93%, re­spec­tively. Speci­ficity was 100% for pul­monary TB and 98% for pleu­ral TB. The results were pub­lished in the jour­nals and

ACS In­fec­tious Dis­eases An­a­lyt­i­cal Bio­chem­istry.

The lab­o­ra­tory-based ap­tamer test for di­ag­nos­ing pul­monary TB takes about five hours to turn in the results while the portable, rapid test takes just 30 min­utes. “The lab-based test has sev­eral steps and so takes time while the rapid test is an electrochemical sen­sor-based de­tec­tion which is a direct de­tec­tion and so takes less time,” says Surabhi La­va­nia from AIIMS and first au­thor of the pul­monary TB pa­per and coau­thor of the other pa­per. At 92.3% and 91.2%, the sen­si­tiv­ity and speci­ficity of the portable test is a bit in­fe­rior com­pared with the lab­based test.

In the case of pleu­ral TB, the ap­tamer-based lab di­ag­no­sis was far su­pe­rior to even Xpert. While the sen­si­tiv­ity was about 22% with Xpert, it was about 93% with ap­tamer. “Xpert re­lies on the pres­ence of bac­te­ria, which is nearly ab­sent in pleu­ral fluid, while the ap­tamer­based test­ing re­lies on bac­te­rial anti­gen and so has high sen­si­tiv­ity,” says Pooja Ku­mari from AIIMS and first au­thor of the pa­per on pleu­ral TB test.

The sen­si­tiv­ity of the lab­based and portable ap­tamer­based test for pul­monary TB is far su­pe­rior com­pared with smear mi­croscopy, X-ray and even ELISA.

Cost-ef­fec­tive

“The portable test is cheaper and can be used for screen­ing pul­monary TB and ac­tive case find­ing in high-risk groups,” says Prof. Tyagi.

The HspX anti­gen present in spu­tum sam­ples of peo­ple with pul­monary TB is made use of for di­ag­no­sis. The team had used the same anti­gen present in the cere­brospinal fluid sam­ples for di­ag­nos­ing TB menin­gi­tis (pub­lished in Septem­ber 2018 in The reA

Tu­ber­cu­lo­sis).

searchers made use of a par­tic­u­lar ap­tamer (H63SL2-M6) that de­tects the anti­gen in spu­tum sam­ples for pul­monary TB di­ag­no­sis.

“Spu­tum is a dif­fi­cult sam­ple to han­dle due to the pres­ence of mu­cus. The spu­tum should first be made into a uni­form sus­pen­sion be­fore ap­ply­ing the test. So [we] must pre­pare the sam­ple and this takes time,” says Prof. Tyagi.

In the case of the lab-based di­ag­no­sis, the spu­tum sam­ple is im­mo­bilised on a plate and the ap­tamer is added. If the sam­ple is pos­i­tive for pul­monary TB then it will con­tain the HspX anti­gen and the ap­tamer binds to it. An en­zyme (horse­rad­ish per­ox­i­dise) that binds to the ap­tamer (through bi­otin-strep­ta­vidin affin­ity) is then added fol­lowed by a sub­strate. A change in sub­strate colour is seen in­di­cat­ing that the sam­ple is pos­i­tive for pul­monary TB. If the sam­ple is neg­a­tive for pul­monary TB, then even the first step of the ap­tamer bind­ing to the sam­ple does not take place and the colour change does not oc­cur.

Re­ly­ing on HspX anti­gen to di­ag­nose pleu­ral TB in a lab set­ting was found be ef­fec­tive and highly sen­si­tive (93%).

Rapid di­ag­no­sis

To make the pul­monary TB di­ag­no­sis portable, the re­searchers bound the ap­tamer to an elec­trode coated with gold nanopar­ti­cles. “When the sam­ple con­tain­ing the HspX anti­gen is added, the ap­tamer binds to the anti­gen and un­der­goes a struc­tural change, which is read out in the elec­tri­cal sig­nal,” says Dr. Tarun Ku­mar Sharma from Trans­la­tional Health Sci­ence and Tech­nol­ogy In­sti­tute (THSTI), Faridabad, and the other cor­re­spond­ing au­thor of the pa­pers. “There is a drop in elec­tri­cal sig­nal if the sam­ple is pos­i­tive and no change in sig­nal when the sam­ple is neg­a­tive.”

The elec­trode is al­ready im­mo­bilised with the ap­tamer, so only the liq­ue­fied spu­tum has to be added and the re­ac­tion is in­stan­ta­neous. “Most time is taken to liq­uefy the spu­tum sam­ple than the test­ing per se,” says Prof. Tyagi. No sam­ple pro­cess­ing is needed in the case of pleu­ral TB sam­ples.

“We are also try­ing to adapt this ap­tamer on an elec­trode for rapid di­ag­no­sis of pleu­ral TB,” says Dr. Sharma.

“We have ap­plied for a patent for the ap­tamer reagent and have li­censed it to Ap­taBharat In­no­va­tion Pvt. Ltd, a start-up at THSTI. We soon plan to val­i­date the tests on well char­ac­terised panel of spec­i­mens and field eval­u­a­tion,” says Prof. Tyagi.

NISSAR AHMAD ■

Care­ful test: The spu­tum should first be made into a uni­form sus­pen­sion be­fore ap­ply­ing the test. (Photo used for il­lus­tra­tive pur­poses only.)

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