Boroujeni, Azar Dokht Khosravi; (1996) Variation within Mycobacterium scrofulaceum and its relevance to the aetiology of disease. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Mycobacterial strains were isolated from patients with diagnoses including cervical lymphadenitis, HIV, and Crohn's disease. These were collected from different centres and were examined. Of them, 54 were identified as Mycobacterium scrofulaceum based on: a.) preliminary cultural and biochemical tests, b.) thin-layer chromatography of lipids and pigments, and c.) double diffusion. Results from these techniques, identified M. scrofulaceum as a clearcut separate species and the techniques enabled me to differentiate M. scrofulaceum from closely related species such as MAI complex and the other scotochromogens. The next step was to find a reliable, rapid molecular technique for the identification of M. scrofulaceum at the species level. A two-step assay combining PCR and RFLP analysis was applied to differentiate M. scrofulaceum strains. PCR with genus specific primers (Tb11 and Tb12) was done and all the strains yielded a DNA product between positions 564 and 125 of EcoR1, Hind III, Lambda Marker. RFLP was performed on the PCR products. For restriction enzyme analysis two enzymes Bst EII and Hae III were used. The restriction patterns enabled us to classify M. scrofulaceum into 7 different variants. Most of the tested strains had 1 to 2 fragments less compared to a previous report based on a single strain. The data obtained showed the method to be a rapid and reliable tool for studying variation within M. scrofulaceum, but it was unable to discriminate between this organism and genetically close species except when combined with other complementary techniques. It is not applicable to the identification of unknown strains from clinical samples suspected to be M. scrofulaceum. In the last step, the IgG level against sonicated antigens of three different reference strains of M. scrofulaceum were measured by ELISA. The sera come from HIV negative, asymptomatic HIV seropositive, and Crohn's disease patients. There was no significant response to sonicated strain159 in any of the 3 groups. The Crohn's group showed a higher response to sonicated strains 22 and 1281 and the asymptomatic HIV seropositive group showed a very low titre of antibodies to all sonicated antigens. If the very high response of the HIV negative group to sonicated strain 1281 is ignored, the data show that the Crohn's disease patients had significantly raised antibody levels to all the M. scrofulaceum sonicates.

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