Comparison of two in-house real-time PCR assays with MTB Q-PCR Alert and GenoType MTBDRplus for the rapid detection of mycobacteria in clinical specimens.

An in-house IS6110 real-time PCR (IH IS6110), MTB Q-PCR Alert (Q-PCR) and GenoType MTBDRplus (MTBDR; Hain Lifescience) were compared for the direct detection of Mycobacterium tuberculosis complex (MTBC) in 87 specimens following automated NucliSENS easyMAG DNA extraction. This included 82 first smear-positive specimens and three smear-negative specimens. Another in-house real-time PCR with a Mycobacterium genus-specific probe for the internal transcribed spacer (ITS) region (IH ITS) was used to allow a full comparison with culture results. The sensitivities of IH IS6110, Q-PCR, MTBDR and IH ITS for MTBC detection were 100, 92, 87 and 87 %, respectively, compared with culture. Both IS6110-based real-time PCRs (in-house and Q-PCR) were similar in performance, with 91.2 % concordant results for MTBC detection. Inhibition rates were low, with zero to three specimens producing uninterpretable results. However, the Q-PCR failed to detect MTBC in five samples that were smear negative or had few acid-fast bacilli (one to 10 bacilli in 10 microscopic fields) detected by IH IS6110. IH ITS was the least sensitive assay but may be useful when used in conjunction with IS6110 PCR results to determine the presence of non-tuberculous mycobacteria in smear-negative specimens. None of the real-time PCR assays tested provided drug-resistance data. It was concluded that an IH IS6110 assay could easily be incorporated into the workflow of a diagnostic laboratory for rapid and accurate identification of MTBC from clinical specimens. The inclusion of an internal control and amplification of an ITS target enhance the diagnostic utility of the test.

Similarity of microbial and meiofaunal community analyses for mapping ecological effects of heavy-metal contamination in soil.

The effect of long-term heavy-metal contamination on soil microbial and meiofaunal communities was assessed with a view to determining whether analysis of these communities could be used for ecological assessment of contaminated sites. Thirty soil cores were taken from an industrial site formerly used for the burning of waste from an explosives factory. The predominant contaminants in the soil were a range of heavy metals, including lead, copper and zinc. The numbers of culturable bacteria (especially those grown on Pseudomonas selective media) and the microbial community response to a suite of 95 carbon sources were suppressed in samples with high heavy-metal contamination. This corresponded to a reduction in the density and evenness of the nematode communities in the samples with high metal concentrations. Conversely the bacterial counts and responses to the 95 carbon sources were greater at sites with higher and more diverse populations of nematodes. However, epifluorescence counts of bacteria and the profiles of extracted fatty acids were not consistently altered by the heavy-metal contamination. These results suggest that culturable bacteria are effective indicators of pollution in soil, and reflect the perturbations seen in other components of the soil biota. Furthermore, this is the first study to show that both meiofaunal communities and microbial communities give similar indications of the ecological impact of heavy-metal contamination in soil.