Detection of viral respiratory pathogens in mild and severe acute respiratory infections in Singapore.

To investigate the performance of laboratory methods and clinical case definitions in detecting the viral pathogens for acute respiratory infections (ARIs) from a prospective community cohort and hospital inpatients, nasopharyngeal swabs from cohort members reporting ARIs (community-ARI) and inpatients admitted with ARIs (inpatient-ARI) were tested by Singleplex Real Time-Polymerase Chain Reaction (SRT-PCR), multiplex RT-PCR (MRT-PCR) and pathogen-chip system (PathChip) between April 2012 and December 2013. Community-ARI and inpatient-ARI was also combined with mild and severe cases of influenza from a historical prospective study as mild-ARI and severe-ARI respectively to evaluate the performance of clinical case definitions. We analysed 130 community-ARI and 140 inpatient-ARI episodes (5 inpatient-ARI excluded because multiple pathogens were detected), involving 138 and 207 samples respectively. Detection by PCR declined with days post-onset for influenza virus; decrease was faster for community-ARI than for inpatient-ARI. No such patterns were observed for non-influenza respiratory virus infections. PathChip added substantially to viruses detected for community-ARI only. Clinical case definitions discriminated influenza from other mild-ARI but performed poorly for severe-ARI and for older participants. Rational strategies for diagnosis and surveillance of influenza and other respiratory virus must acknowledge the differences between ARIs presenting in community and hospital settings.

Species identification and profiling of complex microbial communities using shotgun Illumina sequencing of 16S rRNA amplicon sequences.

The high throughput and cost-effectiveness afforded by short-read sequencing technologies, in principle, enable researchers to perform 16S rRNA profiling of complex microbial communities at unprecedented depth and resolution. Existing Illumina sequencing protocols are, however, limited by the fraction of the 16S rRNA gene that is interrogated and therefore limit the resolution and quality of the profiling. To address this, we present the design of a novel protocol for shotgun Illumina sequencing of the bacterial 16S rRNA gene, optimized to amplify more than 90% of sequences in the Greengenes database and with the ability to distinguish nearly twice as many species-level OTUs compared to existing protocols. Using several in silico and experimental datasets, we demonstrate that despite the presence of multiple variable and conserved regions, the resulting shotgun sequences can be used to accurately quantify the constituents of complex microbial communities. The reconstruction of a significant fraction of the 16S rRNA gene also enabled high precision (>90%) in species-level identification thereby opening up potential application of this approach for clinical microbial characterization.

How are "Atypical" Sulfite Dehydrogenases Linked to Cell Metabolism? Interactions between the SorT Sulfite Dehydrogenase and Small Redox Proteins.

Sulfite dehydrogenases (SDHs) are enzymes that catalyze the oxidation of the toxic and mutagenic compound sulfite to sulfate, thereby protecting cells from adverse effects associated with sulfite exposure. While some bacterial SDHs that have been characterized to date are able to use cytochrome c as an electron acceptor, the majority of these enzymes prefer ferricyanide as an electron acceptor and have therefore been termed "atypical" SDHs. Identifying the natural electron acceptor of these enzymes, however, is crucial for understanding how the "atypical" SDHs are integrated into cell metabolism. The SorT sulfite dehydrogenase from Sinorhizobium meliloti is a representative of this enzyme type and we have investigated the interactions of SorT with two small redox proteins, a cytochrome c and a Cu containing pseudoazurin, that are encoded in the same operon and are co-transcribed with the sorT gene. Both potential acceptor proteins have been purified and characterized in terms of their biochemical and electrochemical properties, and interactions and enzymatic studies with both the purified SorT sulfite dehydrogenase and components of the respiratory chain have been carried out. We were able to show for the first time that an "atypical" sulfite dehydrogenase can couple efficiently to a cytochrome c isolated from the same organism despite being unable to efficiently reduce horse heart cytochrome c, however, at present the role of the pseudoazurin in SorT electron transfer is unclear, but it is possible that it acts as an intermediate electron shuttle between. The SorT system appears to couple directly to the respiratory chain, most likely to a cytochrome oxidase.