Rapid identification of fungi in culture-negative clinical blood and respiratory samples by DNA sequence analyses.

BACKGROUND: Clinical diagnoses of fungal infections often rely upon culture techniques followed by microscopic examination of positive cultures and histopathological specimens. Culturing of microorganisms is prone to false negatives, while microscopy methods can be complicated by atypical phenotypes and organisms that are morphologically indistinguishable in tissues. Delays in diagnoses (or the lack thereof) and inaccurate identification of infectious organisms contribute to increased morbidity and mortality in patients. METHODS: Two-hundred randomized, heterogeneous patient blood and respiratory samples that were culture-negative were tested using polymerase chain reaction (PCR) amplification of internal transcribed spacer regions of ribosomal RNA genes utilizing panfungal primers. Amplicons were sequenced, subjected to sequence similarity searches, and compared using phylogenetic analyses. RESULTS: Thirteen fungal sequences were detected in three whole-blood samples and nine respiratory samples. Bioinformatic analyses were performed which indicated the presence of multiple pathogens and potential pathogens. CONCLUSIONS: The results from this pilot study demonstrate the utility of PCR assays and sequence analyses in clinical tests for fungi to facilitate rapid diagnosis and appropriate treatments to deal with the false negatives from culture results.

Seasonal shifts in bacteria associated with Jersey cows on a small dairy farm and the potential for bedding choice and low levels of iodine use to inhibit mastitic pathogens.

Milk products from small dairies are increasingly in demand, as access to pasture provides benefits to the cow, consumer, and environment. The productivity and profitability of small dairy farms particularly rely on the prevention of infectious diseases. Cattle on seasonal grazing dairies live primarily outdoors until inclement weather warrants relocation indoors. While shifts in the amounts of bacteria associated with livestock may be expected from this transition, potentially increasing risks for infectious diseases, changes in bacteria levels on cows relocated to indoor facilities have not been well-studied. In addition, the optimal use of bedding materials and iodine are critical in bovine infectious diseases prevention. However, the antibacterial potential of bedding material with high polyphenol content or low concentrations of iodine, are poorly understood. Cow teats were swabbed and total bacteria and coliform counts, as well as extracellular enzyme activities (EEA) were utilized to assess shifts in bacterial levels on cows at pasture and then housed indoors. To test the antibacterial efficacy of bedding materials, as well as low concentrations of povidone-iodine, growth curves with laboratory strains of Staphylococcus aureus and Klebsiella pneumoniae, as well as S. aureus isolated from a dairy farm, were performed with three concentrations of red cedar shavings or iodine. Post hoc multiple comparisons indicated that total bacteria, coliform, and ß-galactosidase activities were significantly greater among cows housed indoors compared to bacterial samples from cows at pasture. Laboratory strains of S. aureus, but not K. pneumoniae, were significantly inhibited by moderate and high treatments of red cedar shavings, while S. aureus isolated from a dairy were inhibited by the high treatment only. All low iodine concentrations significantly inhibited each bacterial strain investigated. These results may help optimize strategies for the prevention of infectious diseases of bovine udders critical to the productivity and profitability of small dairies.