Amplicon identification using SparsE representation of multiplex PYROsequencing signal (AdvISER-M-PYRO): application to bacterial resistance genotyping

MOTIVATION: Pyrosequencing is a cost-effective DNA sequencing technology that has many applications, including rapid genotyping of a broad spectrum of bacteria. When molecular typing requires to genotype multiple DNA stretches, several pyrosequencing primers could be used simultaneously but this would create overlapping primer-specific signals, which are visually uninterpretable. Accordingly, the objective was to develop a new method for signal processing (AdvISER-M-PYRO) to automatically analyze and interpret multiplex pyrosequencing signals. In parallel, the nucleotide dispensation order was improved by developing the SENATOR ('SElecting the Nucleotide dispensATion Order') algorithm. RESULTS: In this proof-of-concept study, quintuplex pyrosequencing was applied on eight bacterial DNA and targeted genetic alterations underlying resistance to β-lactam antibiotics. Using SENATOR-driven dispensation order, all genetic variants (31 of 31; 100%) were correctly identified with AdvISER-M-PYRO. Among nine expected negative results, there was only one false positive that was tagged with an 'unsafe' label

Simple technique for in field samples collection in the cases of skin rash illness and subsequent PCR detection of orthopoxviruses and varicella zoster virus.

In case of outbreak of rash illness in remote areas, clinically discriminating monkeypox (MPX) from severe form of chickenpox and from smallpox remains a concern for first responders. OBJECTIVE: The goal of the study was therefore to use MPX and chickenpox outbreaks in Democratic Republic of Congo (DRC) as a test case for establishing a rapid and specific diagnosis in affected remote areas. METHODS: In 2008 and 2009, successive outbreaks of presumed MPX skin rash were reported in Bena Tshiadi, Yangala and Ndesha healthcare districts of the West Kasai province (DRC). Specimens consisting of liquid vesicle dried on filter papers or crusted scabs from healing patients were sampled by first responders. A field analytical facility was deployed nearby in order to carry out a real-time PCR (qPCR) assay using genus consensus primers, consensus orthopoxvirus (OPV) and smallpox-specific probes spanning over the 14 kD fusion protein encoding gene. A PCR-restriction fragment length polymorphism was used on-site as backup method to confirm the presence of monkeypox virus (MPXV) in samples. To complete the differential diagnosis of skin rash, chickenpox was tested in parallel using a commercial qPCR assay. In a post-deployment step, a MPXV-specific pyrosequencing was carried out on all biotinylated amplicons generated on-site in order to confirm the on-site results. RESULTS: Whereas MPXV proved to be the agent causing the rash illness outbreak in the Bena Tshiadi, VZV was the causative agent of the disease in Yangala and Ndesha districts. In addition, each on-site result was later confirmed by MPXV-specific pyrosequencing analysis without any discrepancy. CONCLUSION: This experience of rapid on-site dual use DNA-based differential diagnosis of rash illnesses demonstrates the potential of combining tests specifically identifying bioterrorism agents and agents causing natural outbreaks. This opens the way to rapid on-site DNA-based identification of a broad spectrum of causative agents in remote areas

A Novel Splice-Site Mutation in Angiotensin I-Converting Enzyme (ACE) Gene, c.3691+1G > A (IVS25+1G > A), Causes a Dramatic Increase in Circulating ACE through Deletion of the Transmembrane Anchor

<p>Background: Angiotensin-converting enzyme (ACE) (EC 4.15.1) metabolizes many biologically active peptides and plays a key role in blood pressure regulation and vascular remodeling. Elevated ACE levels are associated with different cardiovascular and respiratory diseases.</p><p>Methods and Results: Two Belgian families with a 8-16-fold increase in blood ACE level were incidentally identified. A novel heterozygous splice site mutation of intron 25 - IVS25+1G>A (c.3691+1G>A) - cosegregating with elevated plasma ACE was identified in both pedigrees. Messenger RNA analysis revealed that the mutation led to the retention of intron 25 and Premature Termination Codon generation. Subjects harboring the mutation were mostly normotensive, had no left ventricular hypertrophy or cardiovascular disease. The levels of renin-angiotensin-aldosterone system components in the mutated cases and wild-type controls were similar, both at baseline and after 50 mg captopril. Compared with non-affected members, quantification of ACE surface expression and shedding using flow cytometry assay of dendritic cells derived from peripheral blood monocytes of affected members, demonstrated a 50% decrease and 3-fold increase, respectively. Together with a dramatic increase in circulating ACE levels, these findings argue in favor of deletion of transmembrane anchor, leading to direct secretion of ACE out of cells.</p><p>Conclusions: We describe a novel mutation of the ACE gene associated with a major familial elevation of circulating ACE, without evidence of activation of the renin-angiotensin system, target organ damage or cardiovascular complications. These data are consistent with the hypothesis that membrane-bound ACE, rather than circulating ACE, is responsible for Angiotensin II generation and its cardiovascular consequences.</p>