No abstract

Mycobacterium tuberculosis resistance to rifampin results from nucleotide changes in the gene encoding the beta-subunit of the RNA polymerase (rpoB). We developed a reverse hybridization-based line probe assay (LiPA; the INNO-LiPA Rif. TB) carrying one oligonucleotide probe for the detection of M. tuberculosis complex strains and nine probes designed to detect nucleotide changes in the relevant part of rpoB. This assay was evaluated with 107 M. tuberculosis isolates with known rpoB sequences, 52 non-M. tuberculosis complex strains, and 61 and 203 clinical isolates found to be sensitive and resistant, respectively, by in vitro testing. The results indicated that (i) the M. tuberculosis complex probe was 100% specific, (ii) when compared to the results of nucleotide sequencing, no discrepancies with the results of INNO-LiPA Rif. TB were observed, (iii) all strains sensitive by in vitro susceptibility testing were correctly identified, and (iv) among the strains resistant by in vitro susceptibility testing, only 4 (2%) yielded conflicting results. The INNO-LiPA Rif. TB is therefore a reliable and widely applicable assay and a valuable tool for routine diagnostic use, given its simplicity and rapid performance.

Two strains of simian immunodeficiency viruses (SIV) isolated from chimpanzees (SIVCPZ-GAB and SIVCPZ-GAB2) originating from Gabon have previously been genetically characterized and shown to belong phylogenetically to the same lineage as the human immunodeficiency virus type 1 (HIV-1). We describe the sequence analysis of a third HIV-1-related virus, SIVCPZ-ANT, isolated from a wild captured chimpanzee originating from Zaire. This virus displayed the same genetic organization as HIV-1 and was found to fall on the same lineage as HIV-1 and SIVCPZ-GAB. Protein sequence identity with SIVCPZ-GAB ranged from 72% (Pol) to 48% (Env) for the structural proteins, while a particularly divergent Vpu was found (only 25% identity to SIVCPZ-GAB). The V3 regions of the SIVCPZ isolates were exceptionally conserved in contrast to the high divergence of V3 among HIV-1 isolates. However, SIVCPZ-ANT did not show a greater degree of sequence similarity with SIVCPZ-GAB than with HIV-1 isolates and represents a quite divergent outgroup of the HIV-1 lineage. Our data suggest multiple introductions of HIV-1 in the human population and shed new light on the origin of the HIV-1 pandemic.

The 16S-23S rRNA spacer regions of Brucella abortus, B. melitensis, and B. suis were cloned and subcloned after PCR amplification. Sequence analysis of the inserts revealed a spacer of about 800 bp with very high ( > 99%) homology among the three species examined. Two genus-specific primer pairs, BRU-P5-BRU-P8 and BRU-P6-BRU-P7, that could be used in a nested PCR format and three genus-specific DNA probes, BRU-ICG2, BRU-ICG3, and BRU-ICG4, were deduced from this spacer. The specificity and sensitivity of both primer sets and probes were examined by testing them against a collection of 18 Brucella strains and 56 strains from other relevant taxa by using PCR and the Line Probe Assay (LiPA), respectively. A method for direct detection of Brucella spp. in 1 ml of raw milk was developed on the basis of enzymatic treatment of the milk components and subsequent PCR and LiPA hybridization. After a single PCR, sensitivities of 2.8 x 10(5) and 2.8 x 10(4) CFU/ml were obtained for detection by agarose gel electrophoresis and LiPA, respectively. Nested PCR yielded a sensitivity of 2.8 x 10(2) CFU/ml for both methods.