The fecal and mucosal microbiome in acute appendicitis patients: an observational study.

AIM: We aimed to study the mucosal microbiota of the appendix in a prospective appendicitis cohort and to compare the fecal microbiota of patients and controls. We hypothesized that the microbiota may be associated with susceptibility to appendicitis. PATIENTS & METHODS: The fecal microbiota of 99 patients and 106 controls were characterized using 16S-23S intergenic spacer profiling. Richness, diversity and community structure were compared. The appendiceal microbiota from 90 patients was analyzed according to the severity of appendicitis. RESULTS: Overall fecal microbial richness and diversity were similar in patients and controls, yet richness and diversity within the group of Firmicutes, Actinobacteria, Fusobacteria and Verrucomicrobia phyla were lower in patients. Discriminant analyses could correctly classify patients and controls with fair accuracy. No differences were found according to severity in appendiceal or fecal microbiota. CONCLUSION: This study demonstrates differences in the composition of intestinal microbiota of appendicitis patients and healthy individuals.

Comparative analysis of four methods to extract DNA from paraffin-embedded tissues: effect on downstream molecular applications.

BACKGROUND: A large portion of tissues stored worldwide for diagnostic purposes is formalin-fixed and paraffin-embedded (FFPE). These FFPE-archived tissues are an extremely valuable source for retrospective (genetic) studies. These include mutation screening in cancer-critical genes as well as pathogen detection. In this study we evaluated the impact of several widely used DNA extraction methods on the quality of molecular diagnostics on FFPE tissues. FINDINGS: We compared 4 DNA extraction methods from 4 identically processed FFPE mammary-, prostate-, colon- and lung tissues with regard to PCR inhibition, real time SNP detection and amplifiable fragment size. The extraction methods, with and without proteinase K pre-treatment, tested were: 1) heat-treatment, 2) QIAamp DNA-blood-mini-kit, 3) EasyMAG NucliSens and 4) Gentra Capture-Column-kit.Amplifiable DNA fragment size was assessed by multiplexed 200-400-600 bp PCR and appeared highly influenced by the extraction method used. Proteinase K pre-treatment was a prerequisite for proper purification of DNA from FFPE. Extractions with QIAamp, EasyMAG and heat-treatment were found suitable for amplification of fragments up to 400 bp from all tissues, 600 bp amplification was marginally successful (best was QIAamp). QIAamp and EasyMAG extracts were found suitable for downstream real time SNP detection. Gentra extraction was unsuitable. Hands-on time was lowest for heat-treatment, followed by EasyMAG. CONCLUSIONS: We conclude that the extraction method plays an important role with regard to performance in downstream molecular applications.