Performance monitoring of mycobacterium tuberculosis dried culture spots for use with the GeneXpert system within a national program in South Africa.

The use of dried culture spots (DCSs) has been reported in the verification of GeneXpert instruments as being "fit for purpose" for the South African National implementation program. We investigated and compared the performance of the DCSs for verification across different bulk batches, testing the settings and cadre of staff, and the Xpert MTB/RIF assay version. Four bulk batches (V005 to V008) were used to prepare (i) 619 DCS panels for laboratory testing on G3 or G4 cartridges by a technologist, (ii) 13 DCS panels (batch V005) used for clinic verification on G3 cartridges by a nurse or lay counselor, and (iii) 20 DCS panels (batch V005) used for the verification of 10 GeneXpert 16 module instruments in mobile vehicles on the G3 cartridge performed by a scientist. The stabilities of the DCSs over 6 months at 4°C, room temperature, and 37°C were investigated. The mean cycle threshold (CT) and standard deviation (SD) for probe A were calculated. The proportions of variability in the CT values across bulk batches, assay versions, and settings and cadre of staff were determined using regression analysis. Overall, the DCSs demonstrated SDs of 3.3 (n = 660) for the G3 cartridges and 3.8 (n = 1,888) for the G4 cartridges, with an overall error rate of 1.5% and false rifampin resistance rate of 0.1%. The proportions of variability (R(2)) in the CT values explained by batch were 14%, by setting and cadre of staff, 5.6%, and by assay version, 4.2%. The most stable temperature in a period of up to 6 months was 37°C (SD, 2.7). The DCS is a robust product suitable for storage, transport, and use at room temperature for the verification of the GeneXpert instrument, and the testing can be performed by non-laboratory-trained personnel in nonlaboratory settings.

Dried culture spots for Xpert MTB/RIF external quality assessment: results of a phase 1 pilot study in South Africa.

Implementation of Xpert MTB/RIF requires quality assessment. A pilot program using dried culture spots (DCSs) of inactivated Mycobacterium tuberculosis is described. Of 274 DCS results received, 2.19% generated errors; the remainder yielded 100% correct Mycobacterium tuberculosis detection. The probe A cycle threshold (C(T)) variability of three DCS batches was ≤ 3.47. The study of longer-term DCS stability is ongoing.

Characterization of the cydAB-encoded cytochrome bd oxidase from Mycobacterium smegmatis.

The cydAB genes from Mycobacterium smegmatis have been cloned and characterized. The cydA and cydB genes encode the two subunits of a cytochrome bd oxidase belonging to the widely distributed family of quinol oxidases found in prokaryotes. The cydD and cydC genes located immediately downstream of cydB encode a putative ATP-binding cassette-type transporter. At room temperature, reduced minus oxidized difference spectra of membranes purified from wild-type M. smegmatis displayed spectral features that are characteristic of the gamma-proteobacterial type cytochrome bd oxidase. Inactivation of cydA or cydB by insertion of a kanamycin resistance marker resulted in loss of d-heme absorbance at 631 nm. The d-heme could be restored by transformation of the M. smegmatis cyd mutants with a replicating plasmid carrying the highly homologous cydABDC gene cluster from Mycobacterium tuberculosis. Inactivation of cydA had no effect on the ability of M. smegmatis to exit from stationary phase at 37 or 42 degrees C. The growth rate of the cydA mutant was tested under oxystatic conditions. Although no discernible growth defect was observed under moderately aerobic conditions (9.2 to 37.5 x 10(2) Pa of pO(2) or 5 to 21% air saturation), the mutant displayed a significant growth disadvantage when cocultured with the wild type under extreme microaerophilia (0.8 to 1.7 x 10(2) Pa of pO(2) or 0.5 to 1% air saturation). These observations were in accordance with the two- to threefold increase in cydAB gene expression observed upon reduction of the pO(2) of the growth medium from 21 to 0.5% air saturation and with the concomitant increase in d-heme absorbance in spectra of membranes isolated from wild-type M. smegmatis cultured at 1% air saturation. Finally, the cydA mutant displayed a competitive growth disadvantage in the presence of the terminal oxidase inhibitor, cyanide, when cocultured with wild type at 21% air saturation in an oxystat. In conjunction with these findings, our results suggest that cytochrome bd is an important terminal oxidase in M. smegmatis.

Heat shock cognate protein 70 chaperone-binding site in the co-chaperone murine stress-inducible protein 1 maps to within three consecutive tetratricopeptide repeat motifs.

Murine stress-inducible protein 1 (mSTI1) is a co-chaperone homologous with the human heat shock cognate protein 70 (hsc70)/heat shock protein 90 (hsp90)-organizing protein (Hop). The concomitant interaction of mSTI1 with hsp70 and hsp90 at its N- and C-termini respectively is mediated by the tetratricopeptide repeat (TPR) motifs in these regions. With the use of co-precipitation assays, we show here that the N-terminal TPR domain of mSTI1 without extensive flanking regions is both necessary and sufficient to mediate a specific interaction with hsc70. In contrast, other TPR-containing co-chaperones require TPR flanking regions for target substrate recognition, suggesting different mechanisms of TPR-mediated chaperone-co-chaperone interactions. Furthermore, the interaction between mSTI1 and hsc70 was analysed to ascertain the effect of replacing or deleting conserved amino acid residues and sequences within the three TPR motifs constituting the N-terminal TPR domain of full-length mSTI1. Replacement of a bulky hydrophobic residue in TPR1 disrupted the interaction of mSTI1 with hsc70. A highly conserved sequence in TPR2 was altered by deletion or single amino acid replacement. These derivatives retained a specific interaction with hsc70. These results are consistent with a model in which conserved residues within the N-terminal TPR region of mSTI1 contribute differentially to the interaction with hsc70, and in which TPR1 has a significant role in targeting mSTI1 to hsc70. The contribution of the TPR domain mutations and deletions are discussed with respect to their effect on target substrate interactions.

Fossil mammals and artefacts from the Middle Awash Valley, Ethiopia.

A review of fossil mammalian faunas from the Middle Awash indicates they span most of the later Neogene and document evolutionary change in several mammalian groups, especially Primates, Proboscidea and Artiodactyla. Oldowan artefacts first appear in the late Pliocene, while Acheulian and later industries and apparent occupation sites occur in Pleistocene beds.