DNA extraction and amplicon production strategies deeply inf luence the outcome of gut mycobiome studies.

Microbial ecology studies are often performed through extraction of metagenomic DNA followed by amplification and sequencing of a marker. It is known that each step may bias the results. These biases have been explored for the study of bacterial communities, but rarely for fungi. Our aim was therefore to evaluate methods for the study of the gut mycobiome. We first evaluated DNA extraction methods in fungal cultures relevant to the gut. Afterwards, to assess how these methods would behave with an actual sample, stool from a donor was spiked with cells from the same cultures. We found that different extraction kits favour some species and bias against others. In terms of amplicon sequencing, we evaluated five primer sets, two for ITS2 and one for ITS1, 18S and 28S rRNA. Results showed that 18S rRNA outperformed the other markers: it was able to amplify all the species in the mock community and to discriminate among them. ITS primers showed both amplification and sequencing biases, the latter related to the variable length of the product. We identified several biases in the characterisation of the gut mycobiome and showed how crucial it is to be aware of these before drawing conclusions from the results of these studies.

Community-acquired pneumonia: doctors do not follow national guidelines.

OBJECTIVES: Appropriate assessment of community-acquired pneumonia (CAP) allows accurate severity scoring and hence optimal management, leading to reduced morbidity and mortality. British Thoracic Society (BTS) guidelines provide an appropriate score. Adherence to BTS guidelines was assessed in our medical assessment unit (MAU) in 2001/2 and again in 2005/6, 3 years after introducing an educational programme. METHODS: A retrospective case-note study, comparing diagnosis, documentation of severity, management and outcome of CAP during admission to MAU during 3 months of each winter in 2001/2 and 2005/6. RESULTS: In 2001/2, 65/165 patients were wrongly coded as CAP and 100 were included in the study. In 2005/6 43/130 were excluded and 87 enrolled. In 2005/6, 87% did not receive a severity score, a significant increase from 48% in 2001/2 (p<0.0001). Parenteral antibiotics were given to 79% of patients in 2001/2 and 77% in 2005/6, and third generation cephalosporins were given to 63% in 2001/2 and 54% in 2005/6 (p = NS). In 2001, 15 different antibiotic regimens were prescribed, increasing to 19 in 2005/6. CONCLUSIONS: Coding remains poor. Adherence to CAP management guidelines was poor and has significantly worsened. Educational programmes, alone, do not improve adherence. Restriction of antibiotic prescribing should be considered.