The prevalence of folP1 mutations associated with clinical resistance to dapsone, in Mycobacterium leprae isolates from South Korea

Since 1953 the synthetic sulphone dapsone (4,4-diaminodiphenylsulphone) has been used successfully for the treatment of leprosy in South Korea (Chae, 2000). In the 1980s, however, the increasing number of dapsoneresistant strains of Mycobacterium leprae recorded in many other areas of the world Qi, 1985) prompted the World Health Organization (WHO) to recommend multidrug therapy (MDT), using dapsone, rifampicin, and clofazimine, for the treatment of leprosy worldwide (WHO, 1982). MDT has therefore been used for leprosy control in South Korea since 1982 (Chae, 2000), although dapsone remains the first-line drug for the treatment of leprosy as well as a key component in the chemotherapeutic regimens of MDT. The results of recent studies on the molecular mechanism of dapsone resistance in M. leprae demonstrated that the target of dapsone is the enzyme dihydropteroate synthase (DHPS). Dapsone resistance appears to be the result of mutations in the JolP I gene encoding for DHPS in M. leprae (Kai et al., 1999; Williams et al., 2000). Although many dapsone-resistant cases have been detected in South Korea (Kim, 1983), no study has examined the molecular characteristics of the dapsone-resistant strains or the status of dapsone resistance in the country. The aim of the present study, based on PCR, single strand conformation polymorphisms (SSCP) and a direct DNA sequencing assay, was to determine the prevalence of folP I mutations in M. leprae isolates from clinical cases of dapsoneresistant leprosy from South Korea. The isolates were collected from 50 leprosy patients, attending the Institute for Leprosy Research of the Korean Leprosy Control Association (in Seoul) or The Leprosy Mission Jesus Hospital (in Daegu), who had each shown a persistent or increased bacterial index (BI) in skin smears produced during or after routine treatment with dapsone or MDT. A dapsone-susceptible strain of M. leprae, Thai-53 (a kind gift from K. Kohsaka of the Leprosy Research Center, National Institute of Infectious Disease, Higashimurayama, Tokyo, Japan) was used as a control. The M. leprae DNA was isolated (Woods and Cole, 1989) and then examined in a PCR-SSCP assay, using two primers, FP-F (5'-TACTTACTGTAATCCCCTGTGCTG-3') and FP-R (5'TTGATCCTGACGATGGCTGTC-3'), that amplified a folP I fragment containing the sulphone-resistance-determining region (SRDR). These primers were designed using version 5.0 of the OLIGO software programme (National Bioscience Inc., Plymouth, MN) and the genomic sequence of the fol PI gene from the Thai-53 strain (GenBank accession number = AB028656). PCR was performed in 30 cycles of denaturation at 94°C for 50 s, annealing at 57°C for 90 s, and extension at 72°C for 90 s. The amplified DNA was then denatured (at 95°C for 5 min) and loaded onto a SSCP gel (FMC Mutation Detection Enhancement System; lntermount Scientific, Kaysville, UT). Electrophoresis was carried out for 12-14 h at 800 V and 10 mA. After electrophoresis, the gels were transferred to 3-MM paper (Whatman, Maidstone, U.K.) and autoradiographed using X-OMAT film (Eastman Kodak, Rochester, NY). In order to sequence the DNA fragments of cases with altered SSCP profiles, DNA bands showing mobility shifts were cut out of the dried gels and amplified using the same primer set. Sequencing of these PCR products was performed using a commercial sequencing kit (ABI Prism® dRhodamine Dye Terminator Cycle Sequencing Kit; Applied Biosystems, Foster City, CA) and an automated sequenc-