Identification of novel hsp65 RFLPs for Mycobacterium leprae.

Leprosy or Hansen's disease is a chronic infectious disease caused by an acid-fast bacillus, Mycobacterium leprae (M. leprae). The bacilli proliferate in macrophages infiltrating the skin and gain entry to the dermal nerves via the laminar surface of Schwann cells where they replicate. After entry, the Schwann cells proliferate and then die. Conclusive identification of M. leprae DNA in a sample can be obtained by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) for the heat shock 65 gene (hsp65). Molecular epidemiology will make it possible to study the global distributions of M. leprae, explore the relationship between genotypes-incidence rates, mode of transmission, and the type of disease (tuberculoid vs. lepromatous). We amplified DNA using PCR for the hsp65 gene from 24 skin lesions from patients diagnosed with various types of leprosy. Fifteen out of 24 were positive for the hsp65 gene. Digestion with HaeIII-PAGE for the RFLP confirmation of the presence of M. leprae DNA showed the typical pattern in 5 out of 24 and 2 novel patterns in 10 out of 24 patients. We confirmed the presence of M. leprae DNA by sequencing the genes for gyraseA or B and folP, which contained only M. leprae specific single nucleotide polymorphisms (SNPs). Thus, we describe novel hsp65 RFLPs for M. leprae found in a high frequency making them ideal for future epidemiology and transmission studies.