Explore the vast range of titles available.

Objective To investigate the validity of recommendations in treatment guidelines to use higher than approved doses of oseltamivir in patients with severe influenza.Design Double blind randomised trial.Setting Thirteen hospitals in Indonesia, Singapore, Thailand, and Vietnam.Participants Patients aged ≥1 year admitted to hospital with confirmed severe influenza.Interventions Oral oseltamivir at double dose (150 mg twice a day/paediatric equivalent) versus standard dose (75 mg twice a day/paediatric equivalent).Main outcome measure Viral status according to reverse transcriptase polymerase chain reaction (RT-PCR) for influenza RNA in nasal and throat swabs on day five.Results Of 326 patients (including 246 (75.5%) children aged <15), 165 and 161 were randomised to double or standard dose oseltamivir, respectively. Of these, 260 (79.8%) were infected with influenza virus A (133 (40.8%) with A/H3N2, 72 (22.1%) with A/H1N1-pdm09, 38 (11.7%) with seasonal A/H1N1, 17 (5.2%) with A/H5N1) and 53 (16.2%) with influenza virus B. A further 3.9% (13) were false positive by rapid antigen test (negative by RT-PCR and no rise in convalescent haemagglutination inhibition titers). Similar proportions of patients were negative for RT-PCR on day five of treatment: 115/159 (72.3%, 95% confidence interval 64.9% to 78.7%) double dose recipients versus 105/154 (68.2%, 60.5% to 75.0%) standard dose recipients; difference 4.2% (−5.9 to 14.2); P=0.42. No differences were found in clearance of virus in subgroup analyses by virus type/subtype, age, and duration of illness before randomisation. Mortality was similar: 12/165 (7.3%, 4.2% to 12.3%) in double dose recipients versus 9/161 (5.6%, 3.0% to 10.3%) in standard dose recipients. No differences were found between double and standard dose arms in median days on supplemental oxygen (3 (interquartile range 2-5) v 3.5 (2-7)), in intensive care (4.5 (3-6) v 5 (2-11), and on mechanical ventilation (2.5 (1-16) v 8 (1-16)), respectively. No important differences in tolerability were found.Conclusions There were no virological or clinical advantages with double dose oseltamivir compared with standard dose in patients with severe influenza admitted to hospital.Registration Clinical Trials NCT00298233

Reverse transcription-quantitative PCR (RT-qPCR) has been widely adopted to measure differences in mRNA levels; however, biological and technical variation strongly affects the accuracy of the reported differences. RT-qPCR specialists have warned that, unless researchers minimize this variability, they may report inaccurate differences and draw incorrect biological conclusions. The Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines describe procedures for conducting and reporting RT-qPCR experiments. The MIQE guidelines enable others to judge the reliability of reported results; however, a recent literature survey found low adherence to these guidelines. Additionally, even experiments that use appropriate procedures remain subject to individual variation that statistical methods cannot correct. For example, since ideal reference genes do not exist, the widely used method of normalizing RT-qPCR data to reference genes generates background noise that affects the accuracy of measured changes in mRNA levels. However, current RT-qPCR data reporting styles ignore this source of variation. In this commentary, we direct researchers to appropriate procedures, outline a method to present the remaining uncertainty in data accuracy, and propose an intuitive way to select reference genes to minimize uncertainty. Reporting the uncertainty in data accuracy also serves for quality assessment, enabling researchers and peer reviewers to confidently evaluate the reliability of gene expression data.

Background and aims:Infliximab is an effective treatment for ulcerative colitis with over 60% of patients responding to treatment and up to 30% reaching remission. The mechanism of resistance to anti-tumour necrosis factor α (anti-TNFα) is unknown. This study used colonic mucosal gene expression to provide a predictive response signature for infliximab treatment in ulcerative colitis.Methods:Two cohorts of patients who received their first treatment with infliximab for refractory ulcerative colitis were studied. Response to infliximab was defined as endoscopic and histological healing. Total RNA from pre-treatment colonic mucosal biopsies was analysed with Affymetrix Human Genome U133 Plus 2.0 Arrays. Quantitative RT-PCR was used to confirm microarray data.Results:For predicting response to infliximab treatment, pre-treatment colonic mucosal expression profiles were compared for responders and non-responders. Comparative analysis identified 179 differentially expressed probe sets in cohort A and 361 in cohort B with an overlap of 74 probe sets, representing 53 known genes, between both analyses. Comparative analysis of both cohorts combined, yielded 212 differentially expressed probe sets. The top five differentially expressed genes in a combined analysis of both cohorts were osteoprotegerin, stanniocalcin-1, prostaglandin-endoperoxide synthase 2, interleukin 13 receptor alpha 2 and interleukin 11. All proteins encoded by these genes are involved in the adaptive immune response. These markers separated responders from non-responders with 95% sensitivity and 85% specificity.Conclusion:Gene array studies of ulcerative colitis mucosal biopsies identified predictive panels of genes for (non-)response to infliximab. Further study of the pathways involved should allow a better understanding of the mechanisms of resistance to infliximab therapy in ulcerative colitis.ClinicalTrials.gov number, NCT00639821.

The COVID-19 pandemic provides an urgent example where a gap exists between availability of state-of-the-art diagnostics and current needs. As assay protocols and primer sequences become widely known, many laboratories perform diagnostic tests using methods such as RT-PCR or reverse transcription loop mediated isothermal amplification (RT-LAMP). Here, we report an RT-LAMP isothermal assay for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus and demonstrate the assay on clinical samples using a simple and accessible point-of-care (POC) instrument. We characterized the assay by dipping swabs into synthetic nasal fluid spiked with the virus, moving the swab to viral transport medium (VTM), and sampling a volume of the VTM to perform the RT-LAMP assay without an RNA extraction kit. The assay has a limit of detection (LOD) of 50 RNA copies per μL in the VTM solution within 30 min. We further demonstrate our assay by detecting SARS-CoV-2 viruses from 20 clinical samples. Finally, we demonstrate a portable and real-time POC device to detect SARS-CoV-2 from VTM samples using an additively manufactured three-dimensional cartridge and a smartphone-based reader. The POC system was tested using 10 clinical samples, and was able to detect SARS-CoV-2 from these clinical samples by distinguishing positive samples from negative samples after 30 min. The POC tests are in complete agreement with RT-PCR controls. This work demonstrates an alternative pathway for SARS-CoV-2 diagnostics that does not require conventional laboratory infrastructure, in settings where diagnosis is required at the point of sample collection.

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is commonly diagnosed by reverse transcription polymerase chain reaction (RT-PCR) to detect viral RNA in patient samples, but RNA extraction constitutes a major bottleneck in current testing. Methodological simplification could increase diagnostic availability and efficiency, benefitting patient care and infection control. Here, we describe methods circumventing RNA extraction in COVID-19 testing by performing RT-PCR directly on heat-inactivated or lysed samples. Our data, including benchmarking using 597 clinical patient samples and a standardised diagnostic system, demonstrate that direct RT-PCR is viable option to extraction-based tests. Using controlled amounts of active SARS-CoV-2, we confirm effectiveness of heat inactivation by plaque assay and evaluate various generic buffers as transport medium for direct RT-PCR. Significant savings in time and cost are achieved through RNA-extraction-free protocols that are directly compatible with established PCR-based testing pipelines. This could aid expansion of COVID-19 testing. SARS-CoV-2 infection is widely diagnosed by RT-PCR, but RNA extraction is a bottleneck for fast and cheap diagnosis. Here, the authors develop protocols to perform RT-PCR directly on heat-inactivated subject samples or samples lysed with readily available detergents and benchmark performance against 597 clinically diagnosed patient samples.

Intramammary infection (IMI), also known as mastitis, is the most frequently occurring and economically the most important infectious disease in dairy cattle. This study provides a validation of the analytical specificity and sensitivity of a real-time PCR-based assay that identifies 11 major pathogen species or species groups responsible for IMI, and a gene coding for staphylococcal β-lactamase production (penicillin resistance). Altogether, 643 culture isolates originating from clinical bovine mastitis, human, and companion animal samples were analyzed using the assay. The isolates represented 83 different species, groups, or families, and originated from 6 countries in Europe and North America. The analytical specificity and sensitivity of the assay was 100% in bacterial and β-lactamase identification across all isolates originating from bovine mastitis (n=454). When considering the entire culture collection (including also the isolates originating from human and companion animal samples), 4 Streptococcus pyogenes, 1 Streptococcus salivarius, and 1 Streptococcus sanguis strain of human origin were identified as Streptococcus uberis, and 3 Shigella spp. strains were identified as Escherichia coli, decreasing specificity to 99% in Strep. uberis and to 99.5% in E. coli. These false-positive results were confirmed by sequencing of the 16S rRNA gene. Specificity and sensitivity remained at 100% for all other bacterial targets across the entire culture collection. In conclusion, the real-time PCR assay shows excellent analytical accuracy and holds much promise for use in routine bovine IMI testing programs. This study provides the basis for evaluating the assay's diagnostic performance against the conventional bacterial culture method in clinical field trials using mastitis milk samples.

With the ongoing COVID-19 (Coronavirus Disease 2019) pandemic, caused by the novel coronavirus SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2), there is a need for sensitive, specific, and affordable diagnostic tests to identify infected individuals, not all of whom are symptomatic. The most sensitive test involves the detection of viral RNA using RT-qPCR (quantitative reverse transcription PCR), with many commercial kits now available for this purpose. However, these are expensive, and supply of such kits in sufficient numbers cannot always be guaranteed. We therefore developed a multiplex assay using well-established SARS-CoV-2 targets alongside a human cellular control ( RPP30 ) and a viral spike-in control (Phocine Herpes Virus 1 [PhHV-1]), which monitor sample quality and nucleic acid extraction efficiency, respectively. Here, we establish that this test performs as well as widely used commercial assays, but at substantially reduced cost. Furthermore, we demonstrate >1,000-fold variability in material routinely collected by combined nose and throat swabbing and establish a statistically significant correlation between the detected level of human and SARS-CoV-2 nucleic acids. The inclusion of the human control probe in our assay therefore provides a quantitative measure of sample quality that could help reduce false-negative rates. We demonstrate the feasibility of establishing a robust RT-qPCR assay at approximately 10% of the cost of equivalent commercial assays, which could benefit low-resource environments and make high-volume testing affordable.

Salivary microRNAs (miRNAs) have been recently revealed as the next generation of non-invasive biomarkers for the diagnostics of diverse diseases. However, their short and highly homologous sequences make their quantification by RT-qPCR technique highly heterogeneous and study dependent, thus limiting their implementation for clinical applications. In this study, we evaluated the use of a widely used commercial RT-qPCR kit for quantification of salivary miRNAs for clinical diagnostics. Saliva from ten healthy volunteers were sampled four times within a three month time course and submitted for small RNA extraction followed by RT-qPCR analysed. Six miRNAs with different sequence homologies were analysed. Sensitivity and specificity of the tested miRNA assays were corroborated using synthetic miRNAs to evaluate the reliability of all tested assays. Significant variabilities in expression profiles of six miRNAs from ten healthy participants were revealed, yet the poor specificity of the assays offered insufficient performance to associate these differences to biological context. Indeed, as the limit of quantification (LOQ) concentrations are from 2–4 logs higher than that of the limit of detection (LOD) ones, the majority of the analysis for salivary miRNAs felt outside the quantification region. Most importantly, a remarkable number of crosstalk reactions exhibiting considerable OFF target signal intensities was detected, indicating their poor specificity and limited reliability. However, the spike-in of synthetic target miRNA increased the capacity to discriminate endogenous salivary miRNA at the LOQ concentrations from those that were significantly lower. Our results demonstrate that comparative analyses for salivary miRNA expression profiles by this commercial RT-qPCR kit are most likely associated to technical limitations rather than to biological differences. While further technological breakthroughs are still required to overcome discrepancies, standardization of rigorous sample handling and experimental design according to technical parameters of each assay plays a crucial role in reducing data inconsistencies across studies.

Since 1998, avian reovirus (ARV) infection has been detected in broiler and breeding chicken flocks in Tunisia. The genotype of avian reoviruses was established using simple and rapid approaches. Reverse transcription PCR (RT-PCR) on both sigma C (σC) and sigma B (σB)-encoding genes followed by restriction fragment length polymorphism (RFLP) analyses were used to better characterize Tunisian isolated strains. The RT-PCR amplified fragments of 738 and 540 bp for σC- and σB-encoding genes, respectively, of 15 ARV Tunisian strains. DNA fragments amplified from S1133 vaccine and isolated strains were digested with different restrictions enzymes. RFLP on the σC gene indicated that the field isolates and the S1133 vaccine strain have identical profiles when separately digested with TaqI, PstI, DdeI, and HincII. Considering the σB gene, RFLP profiles were identical with RsaI, BclI, DpnII, and NciI restriction enzymes for all the strains. However, using MseI and AciI enzymes, it was shown that all tested isolates could be clearly distinguished from the vaccine strain. ARV strains could be classified in groups with strong relatedness. Strain-typing based on cleavage site results are in agreement with ARV clustering based on nucleotide sequences of both the σC and σB genes. RT-PCR–RFLP provides a simple and a rapid approach for genotyping ARV isolates, especially when a large number of isolates are being studied. Additionally, this approach may also determine whether a new variant strain has been introduced into a flock or if a given virus strain is being spread from one flock to another. Genotipificación y clasificación utilizando RT-PCR y análisis RFLP de cepas de reovirus aviar originarias de Túnez. Desde 1998, la infección por reovirus aviar (ARV) se ha detectado en pollos de engorde y en parvadas de reproductoras en Túnez. El genotipo de los reovirus aviares se estableció utilizando métodos sencillos y rápidos. Se llevó a cabo la transcripción inversa y PCR (RT-PCR) de los genes C (σC) y sigma B (σB) seguido del análisis del polimorfismo de la longitud de los fragmentos de restricción (RFLP). Los análisis se utilizaron para caracterizar mejor las cepas aisladas de Túnez. El método de RT-PCR amplificó fragmentos de 738 pares de bases (pb) y de 540 pb que codificaban para los genes σC y σB, respectivamente, amplificados de quince cepas de reovirus aviar de Túnez. Los fragmentos amplificados de ADN de la vacuna S1133 y de las cepas aisladas se sometieron a la digestión con diferentes enzimas de restricción. Los patrones RFLP del gene σC indicaron que los aislamientos de campo y la cepa vacunal S1133 tienen perfiles idénticos cuando se digirieron por separado con la enzimas TaqI, PstI, DdeI y HincII. Teniendo en cuenta que los perfiles de RFLP del gen σB fueron idénticos con las enzimas de restricción RsaI, BclI, DpnII y NciI para todas las cepas. Sin embargo, mediante el uso de las enzimas MseI y AciI, se demostró que todos los aislamientos ensayados podían distinguirse claramente de la cepa vacunal. Las cepas de reovirus aviares pudieron clasificarse en grupos con una fuerte relación. Los resultados basados en los sitios de restricción estuvieron de acuerdo con la agrupación basada en las secuencias de nucleótidos de los genes σC y σB. El método de RT-PCR y seguido de RFLP proporciona un enfoque sencillo y rápido para la genotipificación de los aislamientos, especialmente cuando se analizan un gran número de aislamientos. Además, este enfoque también puede determinar si una nueva cepa variante se ha introducido en una parvada o si una cepa de un determinado virus está siendo propagada de una parvada a otra.