The emergence of rifampin-resistant strains of pathogenic mycobacteria has threatened the usefulness of this drug in treating mycobacterial diseases. Critical to the treatment of individuals infected with resistant strains is the rapid identification of these strains directly from clinical specimens. It Rifampin is an important component of effective multidrug therapies for tuberculosis and leprosy; however, widespread use has led to the emergence of rifampin-resistant (Rif) strains, threatening its usefulness in treating mycobacterial diseases (4-6, 8, 26, 27). Rapid information about drug susceptibility patterns is critical to the treatment of individuals with mycobacterial disease for which rifampin is indicated. Since conventional drug susceptibility testing can require 2 to 4 weeks after growth detection (Mycobacterium tuberculosis) or up to a year (Mycobacterium leprae) in mouse footpads, improvements are needed to yield accurate analysis in a shorter time. DNA diagnostic assays have the potential to provide rapid analysis of rifampin resistance in mycobacteria because of their high degree of sensitivity and specificity and the fact that they do not rely on in vitro growth for results. Shortening the time between diagnosis and the onset of effective therapy should improve patients' survival (tuberculosis) or decrease physical deformities and ocular manifestations resulting in disabilities and blindness (leprosy).Developing such assays requires knowledge of the molecular basis of Rif' in pathogenic mycobacteria. Mutations resulting in the Rif' phenotype in prokaryotes have been mapped to the gene encoding the 1-subunit of the DNA-dependent RNA polymerase (rpoB gene) (10, 11). Recently, the entire rpoB genes of M. leprae (7) resistance have been identified in both species (8,12,28,29). To further characterize mutations associated with the Rif phenotype in M. tuberculosis, M. leprae, and other pathogenic mycobacteria, we developed a rapid PCR-based, DNA sequencing protocol targeted to a 305-bp region of rpoB. By direct DNA sequencing of PCR products, the nucleic acid sequence within this region was determined in 4 rifampinsusceptible (Rifs) and 4 Rif' strains of M. leprae and in 12 Rif' and 110 Rif' strains of M. tuberculosis. In addition, mutations were identified in this region of Rif' strains of Mycobacterium africanum and Mycobacterium avium, the latter causing frequent opportunistic infections in immunocompromised hosts. On the basis of these results we have established conditions for a PCR-heteroduplex formation assay (PCR-HDF) for the rapid detection of the Rif' phenotype in pathogenic mycobacteria. MATERUILS AND METHODSMycobacterial strains. Rifampin-susceptible and -resistant strains of M. leprae were isolated initially from homogenates of skin biopsy samples from lepromatous leprosy patients not responding to antileprosy therapy, which included rifampin, and were subsequently defined as resistant to rifampin by the standard mouse footpad drug susceptibility assay (23). These strains were amplifie...

A sample of 124 isoniazid (INH)-resistant and 88 susceptible strains of Mycobacterium tuberculosis complex from south, central, and west Africa was analyzed by direct sequence analysis and PCR-restriction fragment length polymorphism analysis of their catalase-peroxidase (katG) genes. Point mutations at codon 315 were found in the genomes of 64% of INH-resistant strains, but no complete deletions were identified. Mutations at codon 463 were independent of INH resistance and were linked to the geographic origins of the strains.

In 3 patients with Lyme radiculomyelitis, cellular immune reactions of cerebrospinal fluid (CSF) lymphocytes were analyzed. Phenotypic analysis of CSF cells demonstrated that the majority were T cells (CD3+) of the helper/inducer subset (CD4+). These T cells were directly expanded from the CSF by limiting dilution. A total of 505 T-cell lines were tested for Borrelia burgdorferi (Bb)-specific proliferation and also partly tested for reactivity to a panel of central and peripheral nervous system antigens. Proliferative assays revealed 33 of them to be Bb specific, 16 to be specific for myelin basic protein, 16 to be specific for peripheral myelin, 1 to be specific for cardiolipin, and 2 to be specific for galactocerebrosides. The antigen-specific proliferation was restricted by autologous human leukocyte antigen (HLA) class II molecules. The majority of CSF-derived T-cell lines stained positively for CD3, CD4, and HLA class II antigens and negatively for CD8 (cytotoxic/suppressor subset). One T-cell line provided help for the production of specific IgG by autologous B cells and secreted gamma-interferon upon stimulation with Bb antigen in the presence of autologous antigen-presenting cells. These data show that in patients with severe neurological manifestations of late Lyme disease, not only Bb-specific T-cell lines but also T cells reactive to central or peripheral nervous system autoantigens can be found.

Mycobacterium africanum is a pathogen found in tuberculosis patients in certain parts of Africa and is a member of the Mycobacterium tuberculosis complex. Biochemically, strains of M. africanum exhibit a high degree of variability, with some tendency to cluster according to their geographical origin. To investigate whether this phenotypic variability is reflected at the genetic level, we performed DNA fingerprint analysis of strains isolated from patients with pulmonary tuberculosis in Uganda and Sierra Leone. IS6110 DNA fingerprinting was carried out by the mixed-linker PCR method. A total of 138 strains of M. africanum were analyzed: 42 isolates from Uganda and 96 isolates from Sierra Leone. With few exceptions, the resulting DNA fingerprint patterns grouped together according to their country of origin. A striking lack of variability of DNA fingerprints was found for strains from Sierra Leone, where 70 of 96 isolates (61.5%) fell into clusters. The two largest clusters accounted for 41.7% of all isolates and differed by only one band, as confirmed by standard DNA fingerprinting. In contrast, only two clusters (7.1%) with two and three isolates, respectively, were found for M. africanum isolates collected in Uganda, and three of the DNA fingerprints contained fewer than seven bands. Strains of M. tuberculosis collected and processed during the same time period were highly variable in both countries. Our results support the concept of geographically defined subtypes of M. africanum. In addition, they demonstrate that natural geographic differences in the variability of IS6110 DNA fingerprints within the M. tuberculosis complex must be considered if this technique is used for epidemiologic studies.