DETECTION OF MYCOBACTERIUM-LEPRAE BY GENE AMPLIFICATION - COMBINED ETHIDIUM-BROMIDE STAINING AND PROBE HYBRIDIZATION

Biopsy and skin-scraping specimens from 130 leprosy cases across the d isease spectrum (56 TT/BT/I, 73 BB/BL/LL, and I neuritic case) and 50 healthy contacts were studied to assess the application of gene amplif ication. The nucleic acids from these clinical specimens were extracte d by an integrated freeze-thawing-optimized lysozyme-/proteinase-k tre atment-purification and fractionation procedure. The nucleic acids fro m cultured organisms were isolated by the stepwise procedure earlier s tandardized at this laboratory, Gene amplification for a 360-bp fragme nt of the 18-kDa protein gene was carried out using primer and the pro cedure described by its developers, and a 360-bp fragment on Southern blot was taken as the yardstick of positivity. The polymerase chain re action product was analyzed by electrophoresis, ethidium-bromide (EB) staining, and blot (B) hybridization. Overall sensitivity ranged from 71% in specimens with undetectable acid-fast organisms to 100% in spec imens with demonstrable acid-fast bacilli. A positivity of 73% in TT/B T/I specimens and 93% in BB/BL/LL specimens was observed. Four combina tions were discerned: EB+, B+ (71%); EB-suspicious, B+ (14%); EB-, B(3%) and EB-, B- (12%). By combining the blot hybridization with EB st aining, the sensitivity could be significantly improved as compared to EB staining alone. The test was found to be absolutely specific by th e absence of any false positivity in control specimens as well as with purified DNAs from mycobacterial as well as non-mycobacterial organis ms grown from these specimens. It is recommended that for optimum sens itivity and specificity both EB staining and blot hybridization should be done.