Onward transmission of HIV among MSM is significantly associated with recent infection, sexually transmitted diseases and higher viral load, and reduced by effective HAART. The majority of new infections appear to occur from individuals whose infection was undiagnosed at the time of transmission.

BackgroundMolecular genetic testing is recommended for diagnosis of inherited cardiac disease, to guide prognosis and treatment, but access is often limited by cost and availability. Recently introduced high-throughput bench-top DNA sequencing platforms have the potential to overcome these limitations.Methodology/Principal FindingsWe evaluated two next-generation sequencing (NGS) platforms for molecular diagnostics. The protein-coding regions of six genes associated with inherited arrhythmia syndromes were amplified from 15 human samples using parallelised multiplex PCR (Access Array, Fluidigm), and sequenced on the MiSeq (Illumina) and Ion Torrent PGM (Life Technologies). Overall, 97.9% of the target was sequenced adequately for variant calling on the MiSeq, and 96.8% on the Ion Torrent PGM. Regions missed tended to be of high GC-content, and most were problematic for both platforms. Variant calling was assessed using 107 variants detected using Sanger sequencing: within adequately sequenced regions, variant calling on both platforms was highly accurate (Sensitivity: MiSeq 100%, PGM 99.1%. Positive predictive value: MiSeq 95.9%, PGM 95.5%). At the time of the study the Ion Torrent PGM had a lower capital cost and individual runs were cheaper and faster. The MiSeq had a higher capacity (requiring fewer runs), with reduced hands-on time and simpler laboratory workflows. Both provide significant cost and time savings over conventional methods, even allowing for adjunct Sanger sequencing to validate findings and sequence exons missed by NGS.Conclusions/SignificanceMiSeq and Ion Torrent PGM both provide accurate variant detection as part of a PCR-based molecular diagnostic workflow, and provide alternative platforms for molecular diagnosis of inherited cardiac conditions. Though there were performance differences at this throughput, platforms differed primarily in terms of cost, scalability, protocol stability and ease of use. Compared with current molecular genetic diagnostic tests for inherited cardiac arrhythmias, these NGS approaches are faster, less expensive, and yet more comprehensive.

Our results demonstrate that the technique is appropriate for surveillance of drug resistance in untreated individuals and those with virological failure on therapy.

We report the evaluation of a new real-time PCR assay for hepatitis C virus (HCV) genotyping. The assay design is such that genotype 1 isolates are typed by amplification targeting the nonstructural 5b (NS5b) subgenomic region. Non-genotype 1 isolates are typed by type-specific amplicon detection in the 5 noncoding region (5NC) (method 1; HCV genotyping analyte-specific reagent assay). This method was compared with 5NC reverse hybridization (method 2; InnoLiPA HCV II) and 5NC sequencing (method 3; Trugene HCV 5NC). Two hundred ninety-five sera were tested by method 1; 223 of them were also typed by method 2 and 89 by method 3. Sequencing and phylogenetic analysis of an NS5b fragment were used to resolve discrepant results. Suspected multiple-genotype infections were confirmed by PCR cloning and pyrosequencing. Even though a 2% rate of indeterminates was obtained with method 1, concordance at the genotype level with results with methods 2 and 3 was high. Among eight discordant results, five mixed infections were confirmed. Genotype 1 subtyping efficiencies were 100%, 77%, and 74% for methods 1, 2, and 3, respectively; there were 11/101 discordants between methods 1 and 2 (method 1 was predominantly correct) and 2/34 between methods 2 and 3. Regarding genotype 2, subtyping efficiencies were 100%, 45%, and 92% by methods 1, 2, and 3, respectively; NS5b sequencing of discordants (16/17) revealed a putative new subtype within genotype 2 and that most subtype calls were not correct. Although only sequencing-based methods provide the possibility of identifying new variants, the real-time PCR method is rapid, straightforward, and simple to interpret, thus providing a good single-step alternative to more-time-consuming assays.Hepatitis C virus (HCV) is the most important cause of chronic liver disease and is the leading indication for liver transplantation (2). It is estimated that HCV infects 3% (170 million people) of the world's population (26). HCV possesses a positive-sense single-stranded RNA genome of approximately 9.5 kb, which is flanked by noncoding (NC) regions. The HCV genome encodes at least 11 proteins, which include both structural and nonstructural (NS) proteins (5, 7).HCV is known to have a high rate of genetic heterogeneity. This has allowed HCV strains to be classified into a number of genetically distinct groups, known as genotypes, subtypes, isolates, and quasispecies (5). Genome sequence heterogeneity arises due to poor fidelity of the viral polymerase during replication. Sequence variability is not evenly distributed throughout the genome. The lowest sequence variability is found in the 5ЈNC region, which is the target of choice for many molecular diagnostics assays, including genotyping tests. Nevertheless, nucleotide sequencing coupled with phylogenetic analysis of more-variable genomic regions has been recommended for HCV genotyping in consensus proposals (27). As patients infected with different genotypes respond differently to antiviral drug therapy, identification of the infecting genotype ha...