SARS-CoV-2 RNA detection on environmental surfaces in COVID-19 wards.

This study monitored the presence of SARS-Cov-2 RNA on environmental surfaces in hospital wards housing patients with mild, severe, and convalescent Coronavirus Disease 2019 (COVID-19), respectively. From 29 October to 4 December 2021, a total of 787 surface samples were randomly collected from a General Ward, Intensive Care Unit, and Convalescent Ward at a designated hospital for COVID-19 patients in China. All of the samples were used for SARS-Cov-2 detection. Descriptive statistics were generated and differences in the positivity rates between the wards were analyzed using Fisher's exact tests, Yates chi-squared tests, and Pearson's chi-squared tests. During the study period, 787 surface samples were collected, among which, 46 were positive for SARS-Cov-2 RNA (5.8%). The positivity rate of the contaminated area in the Intensive Care Unit was higher than that of the General Ward (23.5% vs. 10.4%, P<0.05). The positivity rate of the semi-contaminated area in the Intensive Care Unit (4.5%) was higher than that of the General Ward (1.5%), but this difference was not statistically significant (P>0.05). In the clean area, only one sample was positive in the Intensive Care Unit (0.5%). None of the samples were positive in the Convalescent Ward. These findings reveal that the SARS-Cov-2 RNA environmental pollution in the Intensive Care Unit was more serious than that in the General Ward, while the pollution in the Convalescent Ward was the lowest. Strict disinfection measures, personal protection, and hand hygiene are necessary to limit the spread of SARS-Cov-2.

Purification/Amplification-Free RNA Detection Platform for Rapid and Multiplex Diagnosis of Plant Viral Infections.

Genetic tests are highly sensitive, and quantitative methods for diagnosing human viral infections, including COVID-19, are also being used to diagnose plant diseases in various agricultural settings. Conventional genetic tests for plant viruses are mostly based on methods that require purification and amplification of viral genomes from plant samples, which generally take several hours in total, making it difficult to use them in rapid detection at point-of-care testing (POCT). In this study, we developed Direct-SATORI, a rapid and robust genetic test that eliminates the purification and amplification processes of viral genomes by extending the recently developed amplification-free digital RNA detection platform called SATORI, allowing the detection of various plant viral genes in a total of less than 15 min with a limit of detection (LoD) of 98 ∼ copies/µL using tomato viruses as an example. In addition, the platform can simultaneously detect eight plant viruses directly from ∼1 mg of tomato leaves with a sensitivity of 96% and a specificity of 99%. Direct-SATORI can be applied to various infections related to RNA viruses, and its practical use is highly anticipated as a versatile platform for plant disease diagnostics in the future.

Structure-Based Discovery of Inhibitors of the SARS-CoV-2 Nsp14 N7-Methyltransferase.

An under-explored target for SARS-CoV-2 is the S-adenosyl methionine (SAM)-dependent methyltransferase Nsp14, which methylates the N7-guanosine of viral RNA at the 5'-end, allowing the virus to evade host immune response. We sought new Nsp14 inhibitors with three large library docking strategies. First, up to 1.1 billion lead-like molecules were docked against the enzyme's SAM site, leading to three inhibitors with IC50 values from 6 to 50 µM. Second, docking a library of 16 million fragments revealed 9 new inhibitors with IC50 values from 12 to 341 µM. Third, docking a library of 25 million electrophiles to covalently modify Cys387 revealed 7 inhibitors with IC50 values from 3.5 to 39 µM. Overall, 32 inhibitors encompassing 11 chemotypes had IC50 values < 50 µM and 5 inhibitors in 4 chemotypes had IC50 values < 10 µM. These molecules are among the first non-SAM-like inhibitors of Nsp14, providing starting points for future optimization.

Infectious SARS-CoV-2 Particles from Rectal Swab Samples from COVID-19 Patients in Brazil.

The main objective of this study was to investigate the dynamic of SARS-CoV-2 viral excretion in rectal swab (RS), saliva, and nasopharyngeal swab (NS) samples from symptomatic patients and asymptomatic contacts. In addition, in order to evaluate the replication potential of SARS-CoV-2 in the gastrointestinal (GI) tract and the excretion of infectious SARS-CoV-2 from feces, we investigated the presence of subgenomic nucleoprotein gene (N) mRNA (sgN) in RS samples and cytopathic effects in Vero cell culture. A prospective cohort study was performed to collect samples from symptomatic patients and contacts in Rio de Janeiro, Brazil, from May to October 2020. One hundred and seventy-six patients had samples collected at home visits and/or during the follow up, resulting in a total of 1633 RS, saliva, or NS samples. SARS-CoV-2 RNA was detected in 130 (73.9%) patients who had at least one sample that tested positive for SARS-CoV-2. The presence of replicating SARS-CoV-2 in RS samples, measured by the detection of sgN mRNA, was successfully achieved in 19.4% (6/31) of samples, whilst infectious SARS-CoV-2, measured by the generation of cytopathic effects in cell culture, was identified in only one RS sample. Although rare, our results demonstrated the replication capacity of SARS-CoV-2 in the GI tract, and infectious viruses in one RS sample. There is still a gap in the knowledge regarding SARS-CoV-2 fecal-oral transmission. Additional studies are warranted to investigate fecal or wastewater exposure as a risk factor for transmission in human populations.

SARS-CoV-2 N-Antigen Quantification in Respiratory Tract, Plasma and Urine: Kinetics and Association with RT-qPCR Results.

Qualitative SARS-CoV-2 antigen assays based on immunochromatography are useful for mass diagnosis of COVID-19, even though their sensitivity is poor in comparison with RT-PCR assays. In addition, quantitative assays could improve antigenic test performance and allow testing with different specimens. Using quantitative assays, we tested 26 patients for viral RNA and N-antigen in respiratory samples, plasma and urine. This allowed us to compare the kinetics between the three compartments and to compare RNA and antigen concentrations in each. Our results showed the presence of N-antigen in respiratory (15/15, 100%), plasma (26/59, 44%) and urine (14/54, 28.9%) samples, whereas RNA was only detected in respiratory (15/15, 100%) and plasma (12/60, 20%) samples. We detected N-antigen in urine and plasma samples until the day 9 and day 13 post-inclusion, respectively. The antigen concentration was found to correlate with RNA levels in respiratory (p < 0.001) and plasma samples (p < 0.001). Finally, urinary antigen levels correlated with plasma levels (p < 0.001). Urine N-antigen detection could be part of the strategy for the late diagnosis and prognostic evaluation of COVID-19, given the ease and painlessness of sampling and the duration of antigen excretion in this biological compartment.

Evaluation of Analytical and Clinical Performance and Usefulness in a Real-Life Hospital Setting of Two in-House Real-Time RT-PCR Assays to Track SARS-CoV-2 Variants of Concern.

Since the end of 2020, multiple severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) have emerged and spread worldwide. Tracking their evolution has been a challenge due to the huge number of positive samples and limited capacities of whole-genome sequencing. Two in-house variant-screening RT-PCR assays were successively designed in our laboratory in order to detect specific known mutations in the spike region and to rapidly detect successively emerging VOCs. The first one (RT-PCR#1) targeted the 69-70 deletion and the N501Y substitution simultaneously, whereas the second one (RT-PCR#2) targeted the E484K, E484Q, and L452R substitutions simultaneously. To evaluate the analytical performance of these two RT-PCRs, 90 negative and 30 positive thawed nasopharyngeal swabs were retrospectively analyzed, and no discordant results were observed. Concerning the sensitivity, for RT-PCR#1, serial dilutions of the WHO international standard SARS-CoV-2 RNA, corresponding to the genome of an Alpha variant, were all detected up to 500 IU/mL. For RT-PCR#2, dilutions of a sample harboring the E484K substitution and of a sample harboring the L452R and E484Q substitutions were all detected up to 1000 IU/mL and 2000 IU/mL, respectively. To evaluate the performance in a real-life hospital setting, 1308 and 915 profiles of mutations, obtained with RT-PCR#1 and RT-PCR#2, respectively, were prospectively compared to next-generation sequencing (NGS) data. The two RT-PCR assays showed an excellent concordance with the NGS data, with 99.8% for RT-PCR#1 and 99.2% for RT-PCR#2. Finally, for each mutation targeted, the clinical sensitivity, the clinical specificity and the positive and negative predictive values showed excellent clinical performance. Since the beginning of the SARS-CoV-2 pandemic, the emergence of variants-impacting the disease's severity and the efficacy of vaccines and therapies-has forced medical analysis laboratories to constantly adapt to the strong demand for screening them. Our data showed that in-house RT-PCRs are useful and adaptable tools for monitoring such rapid evolution and spread of SARS-CoV-2 VOCs.

Optimization of the molecular diagnosis of the acute hepatitis E virus infection.

To evaluate the diagnostic value of the combination of two broad-range PCR assays targeting two different and conserved regions of the viral genome for the diagnosis of acute Hepatitis E virus (HEV) infection. Patients with acute hepatitis were prospectively recruited. In all, HEV-IgM antibodies were tested together with evaluation of HEV viraemia by two PCR assays (ORF3 and ORF1). The number of individuals exhibiting negative IgM antibody results but carrying viral RNA was calculated by each PCR assay. Four-hundred and seventy individuals were included, of whom 145 (30.8%) were diagnosed as having acute HEV. Of them, 122 (84.1%) exhibited HEV-IgM antibodies, and 81 (55.8%) had detectable viral RNA for at least one PCR. Using the ORF3 molecular assay, 70 (48.3%) individuals were identified with HEV infection. When the ORF1 molecular assay was applied, 49 (33.8%) individuals were identified. The ORF3 assay detected viral RNA in 32 patients not detected by the ORF1 assay. In contrast, the ORF1 assay could amplify viral RNA in 11 patients who were not detected by the ORF3 assay. The parallel use of two broad-range PCR assays significantly increased the performance of the molecular diagnosis of HEV.

[Evaluation of the dynamics of detection of viable SARS-CoV-2 (Coronaviridae: Betacoronavirus: Sarbecovirus) in biological samples obtained from patients with COVID-19 in a health care setting, as one of the indicators of the infectivity of the virus].

INTRODUCTION: The study of the mechanisms of transmission of the SARS-CoV-2 virus is the basis for building a strategy for anti-epidemic measures in the context of the COVID-19 pandemic. Understanding in what time frame a patient can spread SARS-CoV-2 is just as important as knowing the transmission mechanisms themselves. This information is necessary to develop effective measures to prevent infection by breaking the chains of transmission of the virus. The aim of the work is to identify the infectious SARS-CoV-2 virus in patient samples in the course of the disease and to determine the duration of virus shedding in patients with varying severity of COVID-19. MATERIALS AND METHODS: In patients included in the study, biomaterial (nasopharyngeal swabs) was subjected to analysis by quantitative RT-PCR and virological determination of infectivity of the virus. RESULTS: We have determined the timeframe of maintaining the infectivity of the virus in patients hospitalized with severe and moderate COVID-19. Based on the results of the study, we made an analysis of the relationship between the amount of detected SARS-CoV-2 RNA and the infectivity of the virus in vitro in patients with COVID-19. The median time of the infectious virus shedding was 8 days. In addition, a comparative analysis of different protocols for the detection of the viral RNA in relation to the identification of the infectious virus was carried out. CONCLUSION: The obtained data make it possible to assess the dynamics of SARS-CoV-2 detection and viral load in patients with COVID-19 and indicate the significance of these parameters for the subsequent spread of the virus and the organization of preventive measures.

A viral pan-end RNA element and host complex define a SARS-CoV-2 regulon.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19, generates multiple protein-coding, subgenomic RNAs (sgRNAs) from a longer genomic RNA, all bearing identical termini with poorly understood roles in regulating viral gene expression. Insulin and interferon-gamma, two host-derived, stress-related agents, and virus spike protein, induce binding of glutamyl-prolyl-tRNA synthetase (EPRS1), within an unconventional, tetra-aminoacyl-tRNA synthetase complex, to the sgRNA 3'-end thereby enhancing sgRNA expression. We identify an EPRS1-binding sarbecoviral pan-end activating RNA (SPEAR) element in the 3'-end of viral RNAs driving agonist-induction. Translation of another co-terminal 3'-end feature, ORF10, is necessary for SPEAR-mediated induction, independent of Orf10 protein expression. The SPEAR element enhances viral programmed ribosomal frameshifting, thereby expanding its functionality. By co-opting noncanonical activities of a family of essential host proteins, the virus establishes a post-transcriptional regulon stimulating global viral RNA translation. A SPEAR-targeting strategy markedly reduces SARS-CoV-2 titer, suggesting a pan-sarbecoviral therapeutic modality.

Detection of SARS-CoV-2 in Terrestrial Animals in Southern Nigeria: Potential Cases of Reverse Zoonosis.

Since SARS-CoV-2 caused the COVID-19 pandemic, records have suggested the occurrence of reverse zoonosis of pets and farm animals in contact with SARS-CoV-2-positive humans in the Occident. However, there is little information on the spread of the virus among animals in contact with humans in Africa. Therefore, this study aimed to investigate the occurrence of SARS-CoV-2 in various animals in Nigeria. Overall, 791 animals from Ebonyi, Ogun, Ondo, and Oyo States, Nigeria were screened for SARS-CoV-2 using RT-qPCR (n = 364) and IgG ELISA (n = 654). SARS-CoV-2 positivity rates were 45.9% (RT-qPCR) and 1.4% (ELISA). SARS-CoV-2 RNA was detected in almost all animal taxa and sampling locations except Oyo State. SARS-CoV-2 IgGs were detected only in goats from Ebonyi and pigs from Ogun States. Overall, SARS-CoV-2 infectivity rates were higher in 2021 than in 2022. Our study highlights the ability of the virus to infect various animals. It presents the first report of natural SARS-CoV-2 infection in poultry, pigs, domestic ruminants, and lizards. The close human-animal interactions in these settings suggest ongoing reverse zoonosis, highlighting the role of behavioral factors of transmission and the potential for SARS-CoV-2 to spread among animals. These underscore the importance of continuous monitoring to detect and intervene in any eventual upsurge.

[Detection of SARS-CoV-2 RNA in the mucosa of the appendices of children with COVID-19]. / Vyyavlenie RNK SARS-CoV-2 v slizistoi obolochke cherveobraznogo otrostka u detei s COVID-19.

Background. The novel coronavirus infection (COVID-19) often manifests in children as diarrhea, vomiting, abdominal pain, and some children develop acute appendicitis. To elucidate the role of SARS-CoV-2 in the development of acute appendicitis, a more detailed study of the presence of its genetic material in the tissue of the appendix. OBJECTIVE: Determination of SARS-CoV-2 RNA in appendices of children with COVID-19 by fluorescence in situ hybridization (FISH). MATERIAL AND METHODS: A retrospective analysis of case histories and morphological analysis using FISH of appendices of pediatric patients with established clinical diagnosis of acute appendicitis and confirmed infection with SARS-CoV-2 was performed. The material was divided into 3 groups: 1st -appendices obtained during appendectomy in children with established clinical diagnosis of «coronavirus infection¼ (COVID-19, PCR+) (n=42; mean age 10.8 years); 2nd - appendices of children (n=55; mean age 9.7 years) with acute appendicitis obtained before the onset of the COVID-19 pandemic; 3rd (control) group (n=38; mean age 10.3 years) - autopsy material of the appendices (intact). RESULTS: In all samples of the appendices of the 1st group, a positive SARS-CoV-2 viral RNA signal was noted in the cytoplasm of most epithelial cells and single immunocompetent cells. The signal intensity remained the same in all slides, regardless of age. In all samples obtained from patients without COVID-19 (groups 2 and 3), confocal microscopy did not reveal a signal, which indicates successful adaptation of the FISH method in this study and excludes the false positive results. CONCLUSION: In the epithelium of the appendices of children of different age with COVID-19, the FISH method revealed SARS-CoV-2 RNA, which does not exclude the association between viral invasion and the development of acute appendicitis.

Establishing severe acute respiratory infection (SARI) surveillance in a sentinel hospital, Ireland, 2021 to 2022.

BackgroundIn 2020, due to the COVID-19 pandemic, the European Centre for Disease Prevention and Control (ECDC) accelerated development of European-level severe acute respiratory infection (SARI) surveillance.AimWe aimed to establish SARI surveillance in one Irish hospital as part of a European network E-SARI-NET.MethodsWe used routine emergency department records to identify cases in one adult acute hospital. The SARI case definition was adapted from the ECDC clinical criteria for a possible COVID-19 case. Clinical data were collected using an online questionnaire. Cases were tested for SARS-CoV-2, influenza and respiratory syncytial virus (RSV), including whole genome sequencing (WGS) on SARS-CoV-2 RNA-positive samples and viral characterisation/sequencing on influenza RNA-positive samples. Descriptive analysis was conducted for SARI cases hospitalised between July 2021 and April 2022.ResultsOverall, we identified 437 SARI cases, the incidence ranged from two to 28 cases per week (0.7-9.2/100,000 hospital catchment population). Of 431 cases tested for SARS-CoV-2 RNA, 226 (52%) were positive. Of 349 (80%) cases tested for influenza and RSV RNA, 15 (4.3%) were positive for influenza and eight (2.3%) for RSV. Using WGS, we identified Delta- and Omicron-dominant periods. The resource-intensive nature of manual clinical data collection, specimen management and laboratory supply shortages for influenza and RSV testing were challenging.ConclusionWe successfully established SARI surveillance as part of E-SARI-NET. Expansion to additional sentinel sites is planned following formal evaluation of the existing system. SARI surveillance requires multidisciplinary collaboration, automated data collection where possible, and dedicated personnel resources, including for specimen management.

SERS Signature of SARS-CoV-2 in Saliva and Nasopharyngeal Swabs: Towards Perspective COVID-19 Point-of-Care Diagnostics.

In this study, the intrinsic surface-enhanced Raman spectroscopy (SERS)-based approach coupled with chemometric analysis was adopted to establish the biochemical fingerprint of SARS-CoV-2 infected human fluids: saliva and nasopharyngeal swabs. The numerical methods, partial least squares discriminant analysis (PLS-DA) and support vector machine classification (SVMC), facilitated the spectroscopic identification of the viral-specific molecules, molecular changes, and distinct physiological signatures of pathetically altered fluids. Next, we developed the reliable classification model for fast identification and differentiation of negative CoV(-) and positive CoV(+) groups. The PLS-DA calibration model was described by a great statistical value-RMSEC and RMSECV below 0.3 and R2cal at the level of ~0.7 for both type of body fluids. The calculated diagnostic parameters for SVMC and PLS-DA at the stage of preparation of calibration model and classification of external samples simulating real diagnostic conditions evinced high accuracy, sensitivity, and specificity for saliva specimens. Here, we outlined the significant role of neopterin as the biomarker in the prediction of COVID-19 infection from nasopharyngeal swab. We also observed the increased content of nucleic acids of DNA/RNA and proteins such as ferritin as well as specific immunoglobulins. The developed SERS for SARS-CoV-2 approach allows: (i) fast, simple and non-invasive collection of analyzed specimens; (ii) fast response with the time of analysis below 15 min, and (iii) sensitive and reliable SERS-based screening of COVID-19 disease.

CBPH assay for the highest sensitive detection of SARS-COV-2 in the semen.

BACKGROUND: Great concerns have been raised on SARS-CoV-2 impact on men's andrological well-being, and many studies have attempted to determine whether SARS-CoV-2 is present in the semen and till now the data are unclear and somehow ambiguous. However, these studies used quantitative real-time (qRT) PCR, which is not sufficiently sensitive to detect nucleic acids in clinical samples with a low viral load. METHODS: The clinical performance of various nucleic acid detection methods (qRT-PCR, OSN-qRT-PCR, cd-PCR, and CBPH) was assessed for SARS-CoV-2 using 236 clinical samples from laboratory-confirmed COVID-19 cases. Then, the presence of SARS-CoV-2 in the semen of 12 recovering patients was investigated using qRT-PCR, OSN-qRT-PCR, cd-PCR, and CBPH in parallel using 24 paired semen, blood, throat swab, and urine samples. RESULTS: The sensitivity and specificity along with AUC of CBPH was markedly higher than the other 3methods. Although qRT-PCR, OSN-qRT-PCR and cdPCR detected no SARS-CoV-2 RNA in throat swab, blood, urine, and semen samples of the 12 patients, CBPH detected the presence of SARS-CoV-2 genome fragments in semen samples, but not in paired urine samples, of 3 of 12 patients. The existing SARS-CoV-2 genome fragments were metabolized over time. CONCLUSIONS: Both OSN-qRT-PCR and cdPCR had better performance than qRT-PCR, and CBPH had the highest diagnostic performance in detecting SARS-CoV-2, which contributed the most improvement to the determination of the critical value in gray area samples with low vrial load, which then provides a rational screening strategy for studying the clearance of coronavirus in the semen over time in patients recovering from COVID-19. Although the presence of SARS-CoV-2 fragments in the semen was demonstrated by CBPH, COVID-19 is unlikely to be sexually transmitted from male partners for at least 3 months after hospital discharge.

Comparison of the Ct-values for genomic and subgenomic SARS-CoV-2 RNA reveals limited predictive value for the presence of replication competent virus.

SARS-CoV-2 is the causative agent of the acute respiratory disease COVID-19. In addition to the full length positive-sensed, single-stranded genomic RNA (gRNA), viral subgenomic RNAs (sgRNAs) that are required for expression of the 3' region of the genome are synthesized in virus-infected cells. However, whether these sgRNA-species might be used as a measure of active virus replication and to predict infectivity is still under debate. The commonly used methods to monitor and quantitate SARS-CoV-2 infections are based on RT-qPCR analysis and the detection of gRNA. The infectivity of a sample obtained from nasopharyngeal or throat swabs is associated with the viral load and inversely correlates with Ct-values, however, a cut-off value predicting the infectivity highly depends on the performance of the assay. Furthermore, gRNA derived Ct-values result from nucleic acid detection and do not necessarily correspond to active replicating virus. We established a multiplex RT-qPCR assay on the cobas 6800 omni utility channel concomitantly detecting SARS-CoV-2 gRNAOrf1a/b, sgRNAE,7a,N, and human RNaseP-mRNA used as human input control. We compared the target specific Ct-values with the viral culture frequency and performed ROC curve analysis to determine the assay sensitivity and specificity. We found no advantage in the prediction of viral culture when using sgRNA detection compared to gRNA only, since Ct-values for gRNA and sgRNA were highly correlated and gRNA offered a slightly more reliable predictive value. Single Ct-values alone only provide a very limited prediction for the presence of replication competent virus. Hence, careful consideration of the medical history including symptom onset has to be considered for risk stratification.

First Round of External Quality Assessment Scheme for SARS-CoV-2 Laboratories During the COVID-19 Pandemic in Thailand.

The emergence of SARS-CoV-2 necessitated the rapid deployment of tests to diagnose COVID-19. To monitor the accuracy of testing across the COVID-19 laboratory network in Thailand, the Department of Medical Sciences under the Ministry of Public Health launched a national external quality assessment (EQA) scheme using samples containing inactivated SARS-CoV-2 culture supernatant from a predominant strain in the early phase of the Thailand outbreak. All 197 laboratories in the network participated; 93% (n=183) of which reported correct results for all 6 EQA samples. Ten laboratories reported false-negative results, mostly for samples with low viral concentrations, and 5 laboratories reported false-positive results (1 laboratory reported false positives and false negatives). An intralaboratory investigation of 14 laboratories reporting incorrect results revealed 2 main causes of error: (1) RNA contamination of the rRT-PCR reaction and (2) poor-quality RNA extraction. Specific reagent combinations were significantly associated with false-negative reports. Thailand's approach to national EQA for SARS-CoV-2 can serve as a roadmap for other countries interested in implementing a national EQA program to ensure laboratories provide accurate testing results, which is crucial in diagnosis, prevention, and control strategies. A national EQA program can be less costly and thus more sustainable than commercial EQA programs. National EQA is recommended to detect and correct testing errors and provide postmarket surveillance for diagnostic test performance.

Clinical utility of SARS-CoV-2 subgenomic RT-PCR in a pediatric quaternary care setting.

BACKGROUND: During active transcription, SARS-CoV-2 generates subgenomic regions of viral RNA. While standard SARS-CoV-2 RT-PCR amplifies region(s) of genomic RNA, it cannot distinguish active infection from remnant viral genomic material. However, screening for subgenomic RNA (sgRNA) by RT-PCR may aid in the determination of actively transcribing virus. OBJECTIVES: To evaluate the clinical utility of SARS-CoV-2 sgRNA RT-PCR testing in a pediatric population. STUDY DESIGN: Retrospective analysis was performed on inpatients from February-September 2022 positive for SARS-CoV-2 by RT-PCR with a concomitant order for sgRNA RT-PCR. Chart abstractions were conducted to determine clinical outcomes, management, and infection prevention and control (IPC) practices. RESULTS: Of 95 SARS-CoV-2 positive samples from 75 unique patients, 27 (28.4%) were positive by sgRNA RT-PCR. A negative sgRNA RT-PCR test allowed for de-isolation in 68 (71.6%) patient episodes. Regardless of age or sex, a positive sgRNA RT-PCR result significantly correlated with disease severity (P = 0.007), generalized COVID-19 symptoms (P = 0.012), hospitalization for COVID-19 (P = 0.019), and immune status (P = 0.024). Moreover, sgRNA RT-PCR results prompted changes in management in 28 patients (37.3%); specifically, therapeutic escalation in 13/27 (48.1%) positives and de-escalation in 15/68 (22.1%) negatives. CONCLUSIONS: Taken together, these findings underscore the clinical utility of sgRNA RT-PCR testing in a pediatric population as we report significant associations between sgRNA RT-PCR results and clinical parameters related to COVID-19. These findings align with the proposed use of sgRNA RT-PCR testing to guide patient management and IPC practices in the hospital setting.

Metagenomics characterization of respiratory viral RNA pathogens in children under five years with severe acute respiratory infection in the Free State, South Africa.

Prompt detection of viral respiratory pathogens is crucial in managing respiratory infection including severe acute respiratory infection (SARI). Metagenomics next-generation sequencing (mNGS) and bioinformatics analyses remain reliable strategies for diagnostic and surveillance purposes. This study evaluated the diagnostic utility of mNGS using multiple analysis tools compared with multiplex real-time PCR for the detection of viral respiratory pathogens in children under 5 years with SARI. Nasopharyngeal swabs collected in viral transport media from 84 children admitted with SARI as per the World Health Organization definition between December 2020 and August 2021 in the Free State Province, South Africa, were used in this study. The obtained specimens were subjected to mNGS using the Illumina MiSeq system, and bioinformatics analysis was performed using three web-based analysis tools; Genome Detective, One Codex and Twist Respiratory Viral Research Panel. With average reads of 211323, mNGS detected viral pathogens in 82 (97.6%) of the 84 patients. Viral aetiologies were established in nine previously undetected/missed cases with an additional bacterial aetiology (Neisseria meningitidis) detected in one patient. Furthermore, mNGS enabled the much needed viral genotypic and subtype differentiation and provided significant information on bacterial co-infection despite enrichment for RNA viruses. Sequences of nonhuman viruses, bacteriophages, and endogenous retrovirus K113 (constituting the respiratory virome) were also uncovered. Notably, mNGS had lower detectability rate for severe acute respiratory syndrome coronavirus 2 (missing 18/32 cases). This study suggests that mNGS, combined with multiple/improved bioinformatics tools, is practically feasible for increased viral and bacterial pathogen detection in SARI, especially in cases where no aetiological agent could be identified by available traditional methods.

Persistence of SARS-CoV-2 RNA shedding and infectivity in immunized population: Prospective study along different epidemiological periods in Argentina.

During the pandemic of COVID-19, numerous waves of infections affected the two hemispheres with different impacts on each country. Throughout these waves, and with the emergence of new variants, health systems and scientists have tried to provide real-time responses to the complex biology of SARS-CoV-2, dealing with different clinical presentations, biological characteristics, and clinical impact of these variants. In this context, knowing the extent period in which an infected individual releases infectious viral particles has important implications for public health. This work aimed to investigate viral RNA shedding and infectivity of SARS-CoV-2 beyond 10 days after symptom onset (SO). A prospective multicenter study was performed between July/2021 and February/2022 on 116 immunized strategic personnel with COVID-19 diagnosed by RT-qPCR, with asymptomatic (7%), mild (91%) or moderate disease (2%). At the time of diagnosis, 70% had 2 doses of vaccines, 26% had 2 plus a booster, and 4% had one dose. After day 10 from SO, sequential nasopharyngeal swabs were taken to perform RT-qPCR, viral isolation, and S gene sequencing when possible. Viral sequences were obtained in 98 samples: 43% were Delta, 16% Lambda, 15% Gamma, 25% Omicron (BA.1) and 1% Non-VOC/VOI, in accordance with the main circulating variants at each moment. SARS-CoV-2 RNA was detected 10 days post SO in 57% of the subjects. Omicron was significantly less persistent. Noteworthy, infective viruses could not be isolated in any of the samples. In conclusion, a 10-days isolation period was useful to prevent further infections, and proved valid for the variants studied. Recently, even shorter periods have been applied, as the Omicron variant is prevalent, and worldwide population is largely vaccinated. In the future, facing the possible emergence of new variants and considering immunological status, a return to 10 days may be necessary.

Modelling the structures of frameshift-stimulatory pseudoknots from representative bat coronaviruses.

Coronaviruses (CoVs) use -1 programmed ribosomal frameshifting stimulated by RNA pseudoknots in the viral genome to control expression of enzymes essential for replication, making CoV pseudoknots a promising target for anti-coronaviral drugs. Bats represent one of the largest reservoirs of CoVs and are the ultimate source of most CoVs infecting humans, including those causing SARS, MERS, and COVID-19. However, the structures of bat-CoV frameshift-stimulatory pseudoknots remain largely unexplored. Here we use a combination of blind structure prediction followed by all-atom molecular dynamics simulations to model the structures of eight pseudoknots that, together with the SARS-CoV-2 pseudoknot, are representative of the range of pseudoknot sequences in bat CoVs. We find that they all share some key qualitative features with the pseudoknot from SARS-CoV-2, notably the presence of conformers with two distinct fold topologies differing in whether or not the 5' end of the RNA is threaded through a junction, and similar conformations for stem 1. However, they differed in the number of helices present, with half sharing the 3-helix architecture of the SARS-CoV-2 pseudoknot but two containing 4 helices and two others only 2. These structure models should be helpful for future work studying bat-CoV pseudoknots as potential therapeutic targets.