Rafi, Abdolnasser; (1995) Molecular and immunological studies on "fully treated" long-term leprosy patients. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

In a preceding visit, 279 leprosy patients in the Baba Baghi Leprosy Sanatorium in Iran with long histories of treatment with dapsone, were subjected to skin-testing with 4 new tuberculins, and randomised to receive an injection of saline as placebo or killed M. vaccae as immunotherapy. A year later, in the first sampling visit, the study began on selected groups of these patients, each of whom provided sputum and skin-tissue fluid specimens for DNA studies, and serum samples for antibody estimation. Eighteen months later, in the second sampling visit, further samples were obtained from 50 of the same patients. The aim was to search for leprosy and tubercle bacilli by PCR in appropriate specimens, and relate the findings to the skin test, immunotherapy data, and immunoassays. Three different DNA extraction methods for preparation of template DNA were evaluated. The chosen DNA extraction method was based on the lysing and nuclease-inactivating properties of guanidine thiocyanate (GuSCN) together with the nucleic acid-binding properties of diatoms. Species-specific primers for a 530-bp fragment of the gene encoding the 36 kDa antigen of M. leprae and for a 123-bp fragment of the repetitive DNA sequence (IS6110) of M. tuberculosis were used for the study and PCR results were compared with conventional methods. Using the chosen techniques, 25% of the selected "fully treated" long-term leprosy patients were found to be PCR-positive for M. leprae and 13.6% of the same patients were found to be PCR-positive for M. tuberculosis. Comparison of PCR results showed no concurrent positivity for both organisms. The serum samples from the same selected patients were tested by indirect ELISA to determine the level of IgG against seven mycobacterial antigen preparations. A modified technique was used to measure the level of agalactosyl IgG (G₀). The serological results correlated very well with the molecular findings, 81.1% of the leprosy PCR-positive patients could be confirmed with PGL-I ELISA and 83.3% of the tuberculosis PCR-positive patients were confirmed by the G₀ assay. Additionally, 29 CSF specimens from patients thought to have tuberculous meningitis (TBM) were tested by the same PCR technique for tuberculosis and PCR results showed much more positivity than did microscopy or culture. For the first time, ancient DNA of M. leprae was extracted and amplified from a bone dating from 600 AD. The results illustrate the importance of tuberculin testing as a marker of M. tuberculosis infection and the value of leprosin A as a marker for M. leprae in long-treated leprosy patients. PCR results from M. leprae demonstrated the efficacy of immunotherapy for leprosy. The ELISA studies could differentiate between leprosy patients positive or negative by PCR for M. leprae. A ratio of antibodies to antigens of tuberculosis and leprosy, and G₀ were found to be valuable serological markers for tuberculosis in long-treated leprosy patients, and antibody to 65 kDa heat shock protein (hsp) was negatively associated with past immunotherapy with M. vaccae.

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