Comparison of two different PCR amplification products (the 18-kDa protein gene vs. RLEP repetitive sequence) in the diagnosis of Mycobacterium leprae.

To determine the best molecular method for diagnosing leprosy, two sets of Mycobacterium leprae-specific primers were compared. Fresh biopsies and slit skin smear samples were obtained from 67 leprosy patients and examined by touchdown (TD) PCR using primers amplifying either a 129-bp fragment of the RLEP repetitive sequence or a 360-bp fragment of the 18-kDa protein gene of M. leprae. Seventeen of 30 (56.7%) biopsy specimens and four of 37 (10.8%) slit skin smear specimens were positive using the primer for the 18-kDa protein gene, whereas 24 of 30 (80%) biopsy and 27 of 37 (73%) slit skin smear samples showed detectable PCR products in the RLEP repetitive sequence. Twenty-one of 31 cases (67.7%) with a bacterial index of zero were PCR positive for the primer RLEP repetitive sequence. These results demonstrate that detection of M. leprae using PCR with primers to a RLEP sequence is more sensitive and specific than PCR with the 18-kDa protein gene primers and also slit smears with acid fast staining. PCR of RLEP repetitive sequences is therefore a useful means of detecting M. leprae DNA even when it is present at very low levels.

Comparison of two different PCR amplification products (the 18-kDa protein gene vs. RLEP repetitive sequence) in the diagnosis of Mycobacterium leprae

To determine the best molecular method for diagnosing leprosy, two sets of Mycobacterium leprae-specific primers were compared. Fresh biopsies and slit skin smear samples were obtained from 67 leprosy patients and examined by touchdown (TD) PCR using primers amplifying either a 129-bp fragment of the RLEP repetitive sequence or a 360-bp fragment of the 18-kDa protein gene of M. leprae. Seventeen of 30 (56.7 per cent) biopsy specimens and four of 37 (10.8 per cent) slit skin smear specimens were positive using the primer for the 18-kDa protein gene, whereas 24 of 30 (80 per cent) biopsy and 27 of 37 (73 per cent) slit skin smear samples showed detectable PCR products in the RLEP repetitive sequence. Twenty-one of 31 cases (67.7 per cent) with a bacterial index of zero were PCR positive for the primer RLEP repetitive sequence. These results demonstrate that detection of M. leprae using PCR with primers to a RLEP sequence is more sensitive and specific than PCR with the 18-kDa protein gene primers and also slit smears with acid fast staining. PCR of RLEP repetitive sequences is therefore a useful means of detecting M. leprae DNA even when it is present at very low levels.

Typing of clinical isolates of Mycobacterium leprae and their distribution in Korea.

Although there is no genetic diversity in isolates of Mycobacterium leprae, the variance of tandem repeats in the rpoT gene was recently demonstrated. We have typed clinical isolates of M. leprae in Korea using difference of the tandem repeats in the rpoT gene. Among 69 patients, 65 Korean isolates (94.2%) demonstrated four copies of the 6 bp tandem repeat (GACATC) in the rpoT gene, and incidences of three copies were found in only two Koreans and two foreigners (2.9%, respectively).

Detection of Toll-like receptor 2 (TLR2) mutation in the lepromatous leprosy patients.

Toll-like receptor 2 (TLR2) is critical in the immune response to mycobacterial infections and the mutations in the TLR2 have been shown to confer the susceptibility to severe infection with mycobacteria. To define this, we screened the intracellular domain of TLR2 in 131 subjects. Groups of 45 lepromatous and 41 tuberculoid leprosy (TT) patients and 45 controls were investigated. Ten subjects among the lepromatous leprosy (LL) patients had a band variant detected by single-stranded conformational polymorphism. DNA sequencing detected a C to T substitution at nucleotide 2029 from the start codon of the TLR2. The mutation would substitute Arg to Trp at amino acid residue 677, one of the conserved regions of TLR2. In our results, the mutation was involved in only LL, not TT and control. Thus, we suggest that the mutation in the intracellular domain of TLR2 has a role in susceptibility to LL.

Mycobacterium leprae typing by genomic diversity and global distribution of genotypes.

The genetic diversity and related global distribution of 51 Mycobacterium leprae isolates were studied. Isolates were obtained from leprosy patients from 12 geographically distinct regions of the world and two were obtained from nonhuman sources. Polymerase chain reaction (PCR) followed by DNA sequencing was performed targeting the rpoT gene of M. leprae. Isolates were classified into two groups based on the number of tandem repeats composed of 6 base pairs in the rpoT gene. Isolates from Japan (except Okinawa) and Korea belonged to one group, while those from Southeast Asian countries, Brazil, Haiti and Okinawa in Japan belonged to a second genotype. M. leprae obtained from two nonhuman sources (an armadillo and a mangabey monkey) revealed the latter genotype. These results demonstrate the genetic diversity of M. leprae and the related genotype-specific distribution in the world.

Gamma interferon in experimental leprosy.

We have shown that preactivated MACs are able to cope with newly acquired leprosy bacilli, and a high intracellular burden of live M. leprae induces a refractory response of the host MAC to activation by IFN-gamma. Our studies underscore the fact that MAC function in LL is dependent on localized conditions, influenced by the high intracellular burden of leprosy bacilli and, in part, involving the production of prostanoids. These findings preclude inferences about the function of granuloma MAC s in leprosy based on the responses of MACs from other easily accessible anatomical compartments such as the peritoneal cavity or peripheral blood. We feel that the clearance of bacilli from the LL lesion as a consequence of local immunotherapeutic measures or chemotherapy likely depends on the influx of new competent MAC rather than the activation of resident lepromatous MACs.