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As one of the major approaches in combating the COVID-19 pandemics, the availability of specific and reliable assays for the SARS-CoV-2 viral genome and its proteins is essential to identify the infection in suspected populations, make diagnoses in symptomatic or asymptomatic individuals, and determine clearance of the virus after the infection. For these purposes, use of the quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) for detection of the viral nucleic acid remains the most valuable in terms of its specificity, fast turn-around, high-throughput capacity, and reliability. It is critical to update the sequences of primers and probes to ensure the detection of newly emerged variants. Various assays for increased levels of IgG or IgM antibodies are available for detecting ongoing or past infection, vaccination responses, and persistence and for identifying high titers of neutralizing antibodies in recovered individuals. Viral genome sequencing is increasingly used for tracing infectious sources, monitoring mutations, and subtype classification and is less valuable in diagnosis because of its capacity and high cost. Nanopore target sequencing with portable options is available for a quick process for sequencing data. Emerging CRISPR-Cas-based assays, such as SHERLOCK and AIOD-CRISPR, for viral genome detection may offer options for prompt and point-of-care detection. Moreover, aptamer-based probes may be multifaceted for developing portable and high-throughput assays with fluorescent or chemiluminescent probes for viral proteins. In conclusion, assays are available for viral genome and protein detection, and the selection of specific assays depends on the purposes of prevention, diagnosis and pandemic control, or monitoring of vaccination efficacy. During the COVID-19 pandemics, sensitive and reliable assays for SARS-CoV-2 detection are essential for screening the population, identifying asymptomatic individuals, making diagnoses, monitoring treatment responses, and determining viral clearance. This review summarizes the principles, advantages, disadvantages, and specific applications of currently available assays for detection of the viral nucleotide, genome or proteins, as well as host antibody responses, and provide overall guidelines for selection of optimal assays for specific usage.

Novel diagnostic approaches for Zika and dengue are on the rise but may not make it to the market because of bottlenecks in access to samples for validation. An international reference laboratory response is needed to address these challenges, which include networks of in-country laboratories, with well-characterized samples to facilitate assay validation and ensure quality control. Abstract Epidemics of dengue, Zika, and other arboviral diseases are increasing in frequency and severity. Current efforts to rapidly identify and manage these epidemics are limited by the short diagnostic window in acute infection, the extensive serologic cross-reactivity among flaviviruses, and the lack of point-of-care diagnostic tools to detect these viral species in primary care settings. The Partnership for Dengue Control organized a workshop to review the current landscape of Flavivirus diagnostic tools, identified current gaps, and developed strategies to accelerate the adoption of promising novel technologies into national programs. The rate-limiting step to bringing new diagnostic tools to the market is access to reference materials and well-characterized clinical samples to facilitate performance evaluation. We suggest the creation of an international laboratory-response consortium for flaviviruses with a decentralized biobank of well-characterized samples to facilitate assay validation. Access to proficiency panels are needed to ensure quality control, in additional to in-country capacity building.

Inappropriate and inaccurate antimicrobial therapy can lead to adverse patient outcomes and also the development of antimicrobial resistance. Peptide nucleic acid (PNA) fluorescence in situ hybridization (FISH) gives rapid reporting with highly sensitive and specific results to clinicians within 3 h after blood cultures turn positive, thereby offering targeted therapeutics where necessary. It is simple to establish compared with real-time PCR and has resulted in significant cost savings for hospitals. PNA FISH is a promising future technology for the microbiology laboratory that will impact on patient management and clinical guidelines. This article will review the clinical data supporting these new technologies.

The ability to rapidly detect viable pathogens in food is important for public health and food safety reasons. Culture-based detection methods, the traditional means of demonstrating microbial viability, tend to be laborious, time consuming and slow to provide results. Several culture-independent methods to detect viable pathogens have been reported in recent years, including both nucleic acid–based (PCR combined with use of cell viability dyes or reverse-transcriptase PCR to detect messenger RNA) and phage-based (plaque assay or phage amplification and lysis plus PCR/qPCR, immunoassay or enzymatic assay to detect host DNA, progeny phages or intracellular components) methods. Some of these newer methods, particularly phage-based methods, show promise in terms of speed, sensitivity of detection and cost compared with culture for food testing. This review provides an overview of these new approaches and their food testing applications, and discusses their current limitations and future prospects in relation to detection of viable pathogens in food.Key points• Cultural methods may be ‘gold standard’ for assessing viability of pathogens, but they are too slow.• Nucleic acid–based methods offer speed of detection but not consistently proof of cell viability.• Phage-based methods appear to offer best alternative to culture for detecting viable pathogens.

Prognostic factors of coronavirus disease 2019 (COVID-19) patients among European population are lacking. Our objective was to identify early prognostic factors upon admission to optimize the management of COVID-19 patients hospitalized in a medical ward. This French single-center prospective cohort study evaluated 152 patients with positive severe acute respiratory syndrome coronavirus 2 real-time reverse transcriptase-polymerase chain reaction assay, hospitalized in the Internal Medicine and Clinical Immunology Department, at Pitié-Salpêtrière's Hospital, in Paris, France, a tertiary care university hospital. Predictive factors of intensive care unit (ICU) transfer or death at day 14 (D14), of being discharge alive and severe status at D14 (remaining with ventilation, or death) were evaluated in multivariable logistic regression models; models' performances, including discrimination and calibration, were assessed (C-index, calibration curve, R2, Brier score). A validation was performed on an external sample of 132 patients hospitalized in a French hospital close to Paris, in Aulnay-sous-Bois, Île-de-France. The probability of ICU transfer or death was 32% (47/147) (95% CI 25-40). Older age (OR 2.61, 95% CI 0.96-7.10), poorer respiratory presentation (OR 4.04 per 1-point increment on World Health Organization (WHO) clinical scale, 95% CI 1.76-9.25), higher CRP-level (OR 1.63 per 100mg/L increment, 95% CI 0.98-2.71) and lower lymphocytes count (OR 0.36 per 1000/mm3 increment, 95% CI 0.13-0.99) were associated with an increased risk of ICU requirement or death. A 9-point ordinal scale scoring system defined low (score 0-2), moderate (score 3-5), and high (score 6-8) risk patients, with predicted respectively 2%, 25% and 81% risk of ICU transfer or death at D14. Therefore, in this prospective cohort study of laboratory-confirmed COVID-19 patients hospitalized in a medical ward in France, a simplified scoring system at admission predicted the outcome at D14.

There is an increasing interest in using microRNAs (miRNA) as biomarkers in autoimmune diseases. They are easily accessible in many body fluids but it is controversial if they are circulating freely or are encapsulated in microvesicles, particularly exosomes. We investigated if the majority of miRNas in serum and saliva are free-circulating or concentrated in exosomes. Exosomes were isolated by ultracentrifugation from fresh and frozen human serum and saliva. The amount of selected miRNAs extracted from the exosomal pellet and the exosome-depleted serum and saliva was compared by quantitative RT-PCR. Some miRNAs tested are ubiquitously expressed, others were previously reported as biomarkers. We included miRNAs previously reported to be free circulating and some thought to be exosome specific. The purity of exosome fraction was confirmed by electronmicroscopy and western blot. The concentration of miRNAs was consistently higher in the exosome pellet compared to the exosome-depleted supernatant. We obtained the same results using an equal volume or equal amount of total RNA as input of the RT-qPCR. The concentration of miRNA in whole, unfractionated serum, was between the exosomal pellet and the exosome-depleted supernatant. Selected miRNAs, which were detectable in exosomes, were undetectable in whole serum and the exosome-depleted supernantant. Exosome isolation improves the sensitivity of miRNA amplification from human biologic fluids. Exosomal miRNA should be the starting point for early biomarker studies to reduce the probability of false negative results involving low abundance miRNAs that may be missed by using unfractionated serum or saliva.

In April 2020, Congress appropriated $1.5 billion to the NIH to increase national testing capacity for SARS-CoV-2. The NIH established the Rapid Acceleration of Diagnostics program to support the development and deployment of accurate, rapid tests. Among the goals of RADx is to increase capacity to enable testing of approximately 2% of the U.S. population (6 million persons) per day by December 2020.

Background Shading includes low light intensity and varying quality. However, a low red/far-red (R/Fr) ratio of light is a signal that affects plant growth in intercropping and close- planting systems. Thus, the low R/Fr ratio uncoupling from shading conditions was assessed to identify the effect of light quality on photosynthesis and CO 2 assimilation. Soybean plants were grown in a growth chamber with natural solar radiation under four treatments, that is, normal (N, sunlight), N + Fr, Low (L) + Fr, and L light. Results Low R/Fr ratio significantly increased the total biomass, leaf area, starch and sucrose contents, chlorophyll content, net photosynthetic rate, and quantum efficiency of the photosystem II compared with normal R/Fr ratio under the same light level ( P  < 0.05). Proteomic analysis of soybean leaves under different treatments was performed to quantify the changes in photosynthesis and CO 2 assimilation in the chloroplast. Among the 7834 proteins quantified, 12 showed a > 1.3-fold change in abundance, of which 1 was related to porphyrin and chlorophyll metabolism, 2 were involved in photosystem I (PS I), 4 were associated with PS II, 3 proteins participated in photosynthetic electron transport, and 2 were involved in starch and sucrose metabolism. The dynamic change in these proteins indicates that photosynthesis and CO 2 assimilation were maintained in the L treatment by up-regulating the component protein levels compared with those in N treatment. Although low R/Fr ratio increased the photosynthetic CO 2 assimilation parameters, the differences in most protein expression levels in N + Fr and L + Fr treatments compared with those in N treatment were insignificant. Similar trends were found in gene expression through quantitative reverse transcription polymerase chain reaction excluding the gene expression of sucrose synthase possible because light environment is one of the factors affecting carbon assimilation. Conclusions Low R/Fr ratio (high Fr light) can increase the photosynthetic CO 2 assimilation in the same light intensity by improving the photosynthetic efficiency of the photosystems.

RNA interference (RNAi) has previously been shown to be effective in western corn rootworm (WCR, Diabrotica virgifera virgifera LeConte) larvae via oral delivery of synthetic double-stranded RNA (dsRNA) in an artificial diet bioassay, as well as by ingestion of transgenic corn plant tissues engineered to express dsRNA. Although the RNAi machinery components appear to be conserved in Coleopteran insects, the key steps in this process have not been reported for WCR. Here we characterized the sequence of events that result in mortality after ingestion of a dsRNA designed against WCR larvae. We selected the Snf7 ortholog (DvSnf7) as the target mRNA, which encodes an essential protein involved in intracellular trafficking. Our results showed that dsRNAs greater than or equal to approximately 60 base-pairs (bp) are required for biological activity in artificial diet bioassays. Additionally, 240 bp dsRNAs containing a single 21 bp match to the target sequence were also efficacious, whereas 21 bp short interfering (si) RNAs matching the target sequence were not. This result was further investigated in WCR midgut tissues: uptake of 240 bp dsRNA was evident in WCR midgut cells while a 21 bp siRNA was not, supporting the size-activity relationship established in diet bioassays. DvSnf7 suppression was observed in a time-dependent manner with suppression at the mRNA level preceding suppression at the protein level when a 240 bp dsRNA was fed to WCR larvae. DvSnf7 suppression was shown to spread to tissues beyond the midgut within 24 h after dsRNA ingestion. These events (dsRNA uptake, target mRNA and protein suppression, systemic spreading, growth inhibition and eventual mortality) comprise the overall mechanism of action by which DvSnf7 dsRNA affects WCR via oral delivery and provides insights as to how targeted dsRNAs in general are active against insects.