Comparison of quantitative real time PCR with Sequencing and ribosomal RNA-FISH for the identification of fungi in Formalin fixed, paraffin-embedded tissue specimens

Background: Identification of the causative agents of invasive fungal infections (IFI) is critical for guiding antifungal therapy. Cultures remain negative in a substantial number of IFI cases. Accordingly, species identification from formalin fixed, paraffin embedded (FFPE) tissue specimens by molecular methods such as fluorescence in situ hybridisation (FISH) and PCR provides an appealing approach to improve management of patients. Methods: We designed FISH probes targeting the 28S rRNA of Aspergillus and Candida and evaluated them with type strains. Fluorescence microscopy (FM), using FISH probes and quantitative broadrange fungal PCR targeting the rRNA gene were applied to FFPE tissue specimens from patients with proven IFI in order to explore benefits and limitations of each approach. Results: PCR followed by sequencing identified a broad spectrum of pathogenic fungi in 28 of 40 evaluable samples (70%). Hybridisation of FISH probes to fungal rRNA was documented in 19 of 40 tissue samples (47.5%), including 3 PCR negative samples with low fungal burden. The use of FISH was highly sensitive in invasive yeast infections, but less sensitive for moulds. In samples with hyphal elements, the evaluation of hybridisation was impaired due to autofluorescence of hyphae and necrotic tissue background. Conclusions: While PCR appears to be more sensitive in identifying the causative agents of IFI, some PCR negative and FISH positive samples suggest that FISH has some potential in the rapid identification of fungi from FFPE tissue samples

In Search of an Uncultured Human-Associated TM7 Bacterium in the Environment

We have identified an environmental bacterium in the Candidate Division TM7 with ≥98.5% 16S rDNA gene homology to a group of TM7 bacteria associated with the human oral cavity and skin. The environmental TM7 bacterium (referred to as TM7a-like) was readily detectable in wastewater with molecular techniques over two years of sampling. We present the first images of TM7a-like cells through FISH technique and the first images of any TM7 as viable cells through the STARFISH technique. In situ quantification showed TM7 concentration in wastewater up to five times greater than in human oral sites. We speculate that upon further characterization of the physiology and genetics of the TM7a-like bacterium from environmental sources and confirmation of its genomic identity to human-associated counterparts it will serve as model organisms to better understand its role in human health. The approach proposed circumvents difficulties imposed by sampling humans, provides an alternative strategy to characterizing some diseases of unknown etiology, and renders a much needed understanding of the ecophysiological role hundreds of unique Bacteria and Archaea strains play in mixed microbial communities

What is the relative contribution of biological and psychosocial factors to the generation of hypoxia headache?

BACKGROUND: The biopsychosocial model claims that illness is generated by both biological and psychosocial factors. Accordingly, several studies have shown that both factors contribute to the generation of pain. AIMS: The aim of the present study is to manipulate biological, psychological, and social factors in hypobaric hypoxia headache in order to understand their relative contribution to the generation of headache pain. METHODS: Healthy subjects were subdivided into three groups and brought to our high-altitude labs for the assessment of hypoxia-induced headache, blood oxygen saturation (SO(2)), prostaglandins, and cortisol during the first 24 h after arrival. The first group did not undergo any manipulation. The second group (negative expectation) was told that severe headache would occur if SO(2) dropped to less than 80% and their oximeters were set to display a saturation of 75%, even though real SO(2) was much higher. The third group (negative expectation and social interaction) underwent the same manipulation as the second group, but these subjects spent the night together with people experiencing headache and insomnia. RESULTS: Although none of the three groups differed significantly for SO(2), the second group, compared to the first, experienced more severe headache and showed an increase in prostaglandins and cortisol. The third group, compared to the second group, showed a further increase of headache as well as of prostaglandin (PG) E2 and cortisol. CONCLUSIONS: These findings indicate that biological, psychological, and social factors are additive not only in the generation of headache but also for the biochemical changes related to hypoxia