Comparison of multiplexed-tandem real-time PCR panel with reference real-time PCR molecular diagnostic assays for detection of Giardia intestinalis and Tritrichomonas foetus in cats.

Giardia intestinalis and Tritrichomonas foetus are frequent enteric protozoan parasites of the gastrointestinal track of domestic cats. Because of different treatment options for the parasites, confirmation of presence of one or both pathogens is necessary. The PCR based assays are suitable for differential diagnosis. We evaluated the performance of Small Animal Diarrhoea panel, a multiplexed-tandem real-time PCR (MT-PCR) assay, that detects DNA of both G. intestinalis and T. foetus. The sensitivity and specificity were compared to reference real-time PCR assays using 105 faecal samples, 39.05% (n = 41) positive for G. intestinalis and 30.48% (n = 32) were positive for T. foetus. The faecal samples positive for T. foetus had a high proportion of late amplifiers, determined by an arbitrary threshold of Ct-values > 35. On the other hand, only one G. intestinalis positive sample was considered a late amplifier. For G. intestinalis DNA, the MT-PCR assay had 95.1% sensitivity and 92.1% specificity. For T. foetus DNA, the MT-PCR assay had 41.9% sensitivity and 100.0% specificity. To evaluate the interlaboratory reproducibility of the MT-PCR assay, results were compared in two different laboratories and found to be in a very good agreement (Kappa = 0.9). Further analysis of the DNA using conventional PCR determined presence of G. intestinalis Assemblage F and T. foetus genotype 'feline'. In conclusion, the MT-PCR Small Animal Diarrhoea panel had a good and poor performance against reference assays for G. intestinalis and T. foetus, respectively. The assay is suitable for detection and differential diagnosis of G. intestinalis and moderate to high burdens of T. foetus in small animal clinical practice.

Author Correction: Seasonal total methane depletion in limestone caves.

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Seasonal total methane depletion in limestone caves.

Methane concentration in caves is commonly much lower than the external atmosphere, yet the cave CH4 depletion causal mechanism is contested and dynamic links to external diurnal and seasonal temperature cycles unknown. Here, we report a continuous 3-year record of cave methane and other trace gases in Jenolan Caves, Australia which shows a seasonal cycle of extreme CH4 depletion, from ambient ~1,775 ppb to near zero during summer and to ~800 ppb in winter. Methanotrophic bacteria, some newly-discovered, rapidly consume methane on cave surfaces and in external karst soils with lifetimes in the cave of a few hours. Extreme bacterial selection due to the absence of alternate carbon sources for growth in the cave environment has resulted in an extremely high proportion 2-12% of methanotrophs in the total bacteria present. Unexpected seasonal bias in our cave CH4 depletion record is explained by a three-step process involving methanotrophy in aerobic karst soil above the cave, summer transport of soil-gas into the cave through epikarst, followed by further cave CH4 depletion. Disentangling cause and effect of cave gas variations by tracing sources and sinks has identified seasonal speleothem growth bias, with implied palaeo-climate record bias.