Clinical impact of rapid molecular detection of respiratory pathogens in patients with acute respiratory infection.

BACKGROUND: Acute respiratory infections (ARI) are a leading cause of morbidity and mortality worldwide. There is a need to demonstrate the clinical impact of using the new, rapid and sensitive molecular assays in prospectively designed studies. OBJECTIVES: To study the impact on medical management of a rapid molecular assay in patients with respiratory infections. STUDY DESIGN: A prospective, randomized, non-blinded study was performed in patients presenting to the Emergency Department during two respiratory seasons (2016-2017). Diagnosis was performed by FilmArray Respiratory Panel (FilmArray-RP) or by immunofluorescence assay (IFA). RESULTS: A total of 432 patients (156 children and 276 adults) were analyzed. Diagnosis with FilmArray-RP was associated with significant changes in medical management including withholding antibiotic prescriptions (OR:15.52, 95%CI:1.99-120.83 in adults and OR:12.23, 95%CI:1.56-96.09 in children), and reduction in complementary studies in children (OR:9.64, 95%CI:2.13-43.63) compared to IFA. Decrease in oseltamivir prescriptions was significantly higher in adults in the FilmArray-RP group (p = 0.042; OR:1.19, 95%CI:0.51-2.79) compared to adults managed with IFA. Diagnostic yield was significantly higher by FilmArray-RP (81%) than by IFA (31%)(p < 0.001). The median time from sample collection to reporting was 1 h 52 min by FilmArray-RP and 26 h by IFA (p < 0.001). CONCLUSIONS: The high respiratory viruses' detection rate and availability of results within two hours when using FilmArray-RP were associated with decreases in antibiotic prescriptions and complementary studies and more accurate use of oseltamivir.

Measuring Digital PCR Quality: Performance Parameters and Their Optimization.

Digital PCR is rapidly being adopted in the field of DNA-based food analysis. The direct, absolute quantification it offers makes it an attractive technology for routine analysis of food and feed samples for their composition, possible GMO content, and compliance with labelling requirements. However, assessing the performance of dPCR assays is not yet well established. This article introduces three straightforward parameters based on statistical principles that allow users to evaluate if their assays are robust. In addition, we present post-run evaluation criteria to check if quantification was accurate. Finally, we evaluate the usefulness of Poisson confidence intervals and present an alternative strategy to better capture the variability in the analytical chain.

Development of an innovative immunoassay for CP4EPSPS and Cry1AB genetically modified protein detection and quantification.

An innovative immunoassay, called enzyme-linked immunoabsorbant assay (ELISA) Reverse, based on a new conformation of the solid phase, was developed. The solid support was expressly designed to be immersed directly in liquid samples to detect the presence of protein targets. Its application is proposed in those cases where a large number of samples have to be screened simultaneously or when the simultaneous detection of different proteins is required. As a first application, a quantitative immunoassay for Cry1AB protein in genetically modified maize was optimized. The method was tested using genetically modified organism concentrations from 0.1 to 2.0%. The limit of detection and limit of quantitation of the method were determined as 0.0056 and 0.0168 (expressed as the percentage of genetically modified organisms content), respectively. A qualitative multiplex assay to assess the presence of two genetically modified proteins simultaneously was also established for the case of the Cry1AB and the CP4EPSPS (5-enolpyruvylshikimate-3-phosphate synthase) present in genetically modified maize and soy, respectively.

Agrobacterium transient expression system as a tool for the isolation of disease resistance genes: application to the Rx2 locus in potato.

Rx2 confers resistance against potato virus X (PVX). To clone Rx2, we developed a system based on Agrobacterium-mediated transient expression of candidate R genes in transgenic tobacco leaves expressing the PVX coat protein elicitor of Rx2-mediated resistance. Using this system, a potato gene eliciting HR specifically in the presence of the elicitor was identified. Based on genetical and functional analysis, it is concluded that the cloned gene is Rx2. The transient expression system is potentially adaptable to cloning of any other resistance gene. The Rx2 locus is on chromosome V of potato and the encoded protein is highly similar to the products of Rx1 and Rxh1 encoded on potato chromosome XII. Rxh1 has been shown elsewhere to encode a potato cyst nematode resistance gene Gpa2. All three proteins are in the leucine zipper-nucleotide binding site-leucine rich repeat class of resistance gene products. Rx1 and Rx2 are functionally identical and are almost identical in the C terminal region consistent with a role of the leucine rich repeats in recognition of the PVX coat protein. In the N terminal, half there are some regions where the Rx1 and Rx2 proteins are more similar to each other than to the Rxh1 protein. However, in other regions these proteins are more similar to Rxh1 than to each other. Based on this mosaic pattern of sequence similarity, we conclude that sequence exchange occurs repeatedly between genetically unlinked disease resistance genes through a process of gene conversion.

Other Natural Hosts of Potato virus T.

Potato virus T (PVT), a member of the genus Trichovirus, was isolated from leaves of naturally infected ulluco (Ullucus tuberosus), oca (Oxalis tuberosa), and mashua (Tropaeolum tuberosum). These Andean tuber crops are often grown in small plots in association with potato (Solanum tuberosum) in the Peruvian highlands. PVT isolates from ulluco, oca, mashua, and potato infected virus-free ulluco, oca, and potato genotypes by mechanical inoculation. The incidence of PVT in mashua, oca, and ulluco accessions from the International Potato Center (CIP) in vitro germplasm bank was less than 10%. A polymerase chain reaction (PCR) product of approximately 330 bp was obtained from each of the four isolates using primers designed from the published PVT sequence. Restriction enzyme digestions of the PCR product did not demonstrate variability.

Evidence for heterologous encapsidation of potato spindle tuber viroid in particles of potato leafroll virus.

The aphid Myzus persicae (Sulz.) was shown to transmit potato spindle tuber viroid (PSTVd) to potato clone DTO-33 from source plants doubly infected with potato leafroll virus (PLRV) and PSTVd. Transmission was of the persistent type and did not occur when the insects were allowed to feed on singly infected plants. Only low levels of PSTVd were associated with purified PLRV virions, but its resistance to digestion with micrococcal nuclease indicates that the viroid RNA is encapsidated within the PLRV particles. Epidemiological surveys carried out at three locations in China revealed a strong correlation between PSTVd infection and the presence of PLRV, suggesting that PLRV can facilitate PSTVd spread under field conditions.

RNA sequence of potato virus X strain HB.

The genomic RNA of the potato virus X (PVX) strain HB, isolated in Bolivia and able to overcome all known resistance genes, has been cloned and sequenced. The PVXHB RNA sequence is 6432 nucleotides long and contains, similarly to the RNAs of other PVX strains, five open reading frames encoding proteins of M(r)s 165.1K, 24.5K, 12.4K, 7.6K and 25.1K (coat protein), respectively. Multiple amino acid sequence alignments of the coat proteins of four PVX strains identified eight amino acid residues unique for PVXHB. Structural prediction comparisons of the coat proteins of PVXHB and of the other strains suggest a general structural similarity. However, two of the eight amino acid residues unique for strain HB gave rise to a change in the predicted coat protein structure, suggesting a possible involvement in the resistance-breaking activity of PVXHB.