Analysis of gene probes and gene amplification techniques for diagnosis and monitoring of treatment in childhood leprosy.

Nucleic acid sequences of Mycobacterium leprae were detected using gene probes hybridizing with targeting ribosomal RNA (16S rRNA), ribosomal DNA (16S rDNA) and gene amplification techniques (PCR) in skin lesion of paediatric leprosy patients and the effect of treatment on the by these methods. Eighty paediatric leprosy patients were included in the study. Most cases (79%) were between 9 and 16 years of age. Cases were divided into three groups according to treatment status, viz. untreated (30), undergoing treatment (30), and at the end of treatment (20). Clinical and slit smear examination for acid fast bacilli (AFB) was performed and nucleic acids were extracted and fractionated from skin biopsies. M. leprae specific 16S rRNA and 16S rDNA was detected by hybridization with gene probes whereas the 36 kDa gene sequence was detected by a gene amplification assay (PCR). The cases were classified as paucibacillary (PB) and multibacillary (MB) by the standard criteria of WHO (1988). Positivity of 16S rRNA in PB cases decreased from 60% in untreated to 10.5% after 4-8 months of treatment whereas for 16S r DNA, it decreased from 50% to 21%, for PCR from 70% to 36.8% for the same specimen, and all became negative at 1 year. Similar trends were seen in MB cases where positivity in smear positive untreated cases decreased from 100% to 56.2% with 16S rRNA and 42.8% with 16S rDNA and PCR, respectively, after 9-12 months of treatment and all became negative at 2 years, except one case which remained positive with PCR. Similar results were observed in smear negative MB cases, 100% positivity detected by 16S r RNA and PCR, 75% detected by 16S rDNA decreased to zero after 9-12 months of therapy. This study suggests the potential usefulness of gene probes targeting 16S rRNA and 16S rDNA and PCR as supportive molecular tools for diagnosis of smear negative evolving MB disease and also monitoring the response to treatment, these observations however, needs to be validated in prospective follow up studies.