Minimizing errors in RT-PCR detection and quantification of SARS-CoV-2 RNA for wastewater surveillance

Wastewater surveillance for pathogens using reverse transcription-polymerase chain reaction (RT-PCR) is an effective and resource-efficient tool for gathering community-level public health information, including the incidence of coronavirus disease-19 (COVID-19). Surveillance of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) in wastewater can potentially provide an early warning signal of COVID-19 infections in a community. The capacity of the world's environmental microbiology and virology laboratories for SARS-CoV-2 RNA characterization in wastewater is increasing rapidly. However, there are no standardized protocols or harmonized quality assurance and quality control (QA/QC) procedures for SARS-CoV-2 wastewater surveillance. This paper is a technical review of factors that can cause false-positive and false-negative errors in the surveillance of SARS-CoV-2 RNA in wastewater, culminating in recommended strategies that can be implemented to identify and mitigate some of these errors. Recommendations include stringent QA/QC measures, representative sampling approaches, effective virus concentration and efficient RNA extraction, PCR inhibition assessment, inclusion of sample processing controls, and considerations for RT-PCR assay selection and data interpretation. Clear data interpretation guidelines (e.g., determination of positive and negative samples) are critical, particularly when the incidence of SARS-CoV-2 in wastewater is low. Corrective and confirmatory actions must be in place for inconclusive results or results diverging from current trends (e.g., initial onset or reemergence of COVID-19 in a community). It is also prudent to perform interlaboratory comparisons to ensure results' reliability and interpretability for prospective and retrospective analyses. The strategies that are recommended in this review aim to improve SARS-CoV-2 characterization and detection for wastewater surveillance applications. A silver lining of the COVID-19 pandemic is that the efficacy of wastewater surveillance continues to be demonstrated during this global crisis. In the future, wastewater should also play an important role in the surveillance of a range of other communicable diseases

Oxidation contributes to low glutathione in the airways of children with cystic fibrosis

Glutathione is an important antioxidant in the lungs but its concentration is low in the airways of patients with cystic fibrosis. Whether this deficit occurs from an early age or how oxidative stress contributes to lowering glutathione is unknown. We measured glutathione, its oxidation products, myeloperoxidase, and biomarkers of hypochlorous acid in bronchoalveolar lavage from children with cystic fibrosis and disease controls using mass spectrometry and immunological techniques. The concentration of glutathione was lower in bronchoalveolar lavage from children with cystic fibrosis, whereas glutathione sulfonamide, a specific oxidation product of hypochlorous acid, was higher. Oxidised glutathione and glutathione sulfonamide correlated with myeloperoxidase and a biomarker of hypochlorous acid. The percentage of glutathione attached to proteins was higher in children with cystic fibrosis than controls. Pulmonary infections in cystic fibrosis resulted in lower levels of glutathione but higher levels of oxidised glutathione and glutathione sulfonamide in bronchoalveolar lavage. The concentration of glutathione is low in the airways of patients with cystic fibrosis from an early age. Increased oxidation of glutathione by hypochlorous acid and its attachment to proteins contribute to this deficiency. Therapies targeted against myeloperoxidase may boost antioxidant defence and slow the onset and progression of lung disease in cystic fibrosis

Subarcsecond view on the high-redshift blazar GB 1508+5714 by the International LOFAR Telescope

Context. Studies of the most distant active galactic nuclei (AGNs) allow us to test our current understanding of the physics present in radio-jetted AGNs across a range of environments, and probe their interactions with these environments. The decrease in apparent luminosity with distance is the primary difficulty to overcome in the study of these distant AGNs, which requires highly sensitive instruments. Aims. Our goal is to employ new long wavelength radio data to better parametrise the broad-band spectral energy distribution (SED) of GB 1508+5714, a high-redshift (z = 4.30) AGN. Its high redshift, high intrinsic luminosity and classification as a blazar allow us to test emission models that consider the efficient cooling of jet electrons via inverse Compton losses in interactions with the dense cosmic microwave background (CMB) photon field at high redshifts. A significant detection of this effect in GB 1508+5714 may partly explain the apparent sparsity of high-redshift radio galaxies in wide-field surveys, detections of this kind are only becoming possible with the current generation of Square Kilometre Array (SKA) precursors. Methods. We used the LOw-Frequency ARray (LOFAR) to image the long wavelength radio emission around the high-redshift blazar GB 1508+5714 on arcsecond scales at frequencies between 128 and 160 MHz. This allowed us to compare the spatially resolved structure with higher frequency observations, and construct spectral index maps to study the spectral properties of the different components. Results. The LOFAR image shows a compact unresolved core and two resolved emission regions around 2 arcsec to the east and to the west of the radio core. We find structure consistent with previous Very Large Array (VLA) observations, as well as a previously unreported emission region to the east. The region in the west shows a spectral index of −1.2−0.2+0.4 while the region in the east indicates a spectral index of ≲−1.1. The radio core features aflat spectral index of 0.02 ± 0.01. Conclusions. We interpret the arcsecond-scale radio structure of GB 1508+5714 as a FR II-like radio galaxy at a small viewing angle, and the western component as the region containing the approaching jet’s terminal hot spot while the eastern diffuse component near the core can be interpreted as the counter-hot spot region. Our SED modelling shows that a scenario featuring significant quenching effects caused by interaction with the CMB provides a good description of the data, and notably explains the suppressed radio emission