Quantitative real-time PCR analysis of Mycobacterium leprae DNA and mRNA in human biopsy material from leprosy and reactional cases.
Mycobacterium leprae, the causative agent of leprosy, is uncultivable in defined media. Development of new diagnostic tools which do not depend on growth of bacteria is needed for the early detection of M. leprae and for monitoring the effectiveness of chemotherapy. We used a real-time PCR-based assay to quantify the copy number of bacterial DNA and hsp18 mRNA from 47 leprosy patients using paraffin-embedded biopsy samples. The assay used was specific, sensitive and reproducible. The applicability of this approach in monitoring the chemotherapy of leprosy was examined. A reduction in DNA and mRNA during chemotherapy was observed and hsp18 mRNA could not be detected in patients who underwent 2 years of multidrug therapy (MDT). However, a considerable amount of M. leprae DNA could be detected even after 2 years of MDT. A significant amount of hsp18 mRNA was found in reactional cases as well. This raises important questions regarding the role of bacterial antigens in leprosy reactions and the rationale of omitting antibiotics in the treatment of reactional cases. Results in this study show that real-time PCR could be a better tool for the careful monitoring of bacillary DNA and mRNA in lesions, which will help to improve diagnosis, disease progression and the treatment regimen.
Lini
Nirmala
N
Department of Genetic Engineering, School of Biotechnology, Madurai Kamaraj University, Madurai 625 021, Tamil Nadu, India.
Shankernarayan
Nallakandy Panangadan
NP
Voluntary Health Services, Leprosy Project, Shaktinagar, Periyar District, Tamil Nadu, India.
Dharmalingam
Kuppamuthu
K
Department of Genetic Engineering, School of Biotechnology, Madurai Kamaraj University, Madurai 625 021, Tamil Nadu, India.
D001426:Bacterial Proteins / Q000235:genetics; D001706:Biopsy; D004269:DNA, Bacterial / Q000032:analysis* / Q000235:genetics / Q000302:isolation & purification; D006360:Heat-Shock Proteins / Q000235:genetics; D006801:Humans; D007917:Leprostatic Agents / Q000627:therapeutic use; D007918:Leprosy / Q000175:diagnosis / Q000188:drug therapy / Q000382:microbiology / Q000503:physiopathology; D009166:Mycobacterium leprae / Q000145:classification / Q000187:drug effects / Q000235:genetics / Q000302:isolation & purification*; D010232:Paraffin; D016133:Polymerase Chain Reaction / Q000379:methods*; D012333:RNA, Messenger / Q000032:analysis* / Q000235:genetics / Q000302:isolation & purification; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D012867:Skin / Q000382:microbiology*; D016610:Tissue Embedding / Q000379:methods; D016896:Treatment Outcome
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D006360:Heat-Shock Proteins; D007917:Leprostatic Agents; D012333:RNA, Messenger; C081475:hsp18 protein, bacteria; D010232:Paraffin