SwabExpress: An End-to-End Protocol for Extraction-Free COVID-19 Testing.

BACKGROUND: The urgent need for massively scaled clinical testing for SARS-CoV-2, along with global shortages of critical reagents and supplies, has necessitated development of streamlined laboratory testing protocols. Conventional nucleic acid testing for SARS-CoV-2 involves collection of a clinical specimen with a nasopharyngeal swab in transport medium, nucleic acid extraction, and quantitative reverse-transcription PCR (RT-qPCR). As testing has scaled across the world, the global supply chain has buckled, rendering testing reagents and materials scarce. To address shortages, we developed SwabExpress, an end-to-end protocol developed to employ mass produced anterior nares swabs and bypass the requirement for transport media and nucleic acid extraction. METHODS: We evaluated anterior nares swabs, transported dry and eluted in low-TE buffer as a direct-to-RT-qPCR alternative to extraction-dependent viral transport media. We validated our protocol of using heat treatment for viral inactivation and added a proteinase K digestion step to reduce amplification interference. We tested this protocol across archived and prospectively collected swab specimens to fine-tune test performance. RESULTS: After optimization, SwabExpress has a low limit of detection at 2-4 molecules/µL, 100% sensitivity, and 99.4% specificity when compared side by side with a traditional RT-qPCR protocol employing extraction. On real-world specimens, SwabExpress outperforms an automated extraction system while simultaneously reducing cost and hands-on time. CONCLUSION: SwabExpress is a simplified workflow that facilitates scaled testing for COVID-19 without sacrificing test performance. It may serve as a template for the simplification of PCR-based clinical laboratory tests, particularly in times of critical shortages during pandemics.

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.

[Morphological aspects of diaphragm dysfunction in COVID-19-associated pneumonia]. / Morfologicheskie aspekty disfunktsii diafragmy pri COVID-19-assotsiirovannoi pnevmonii.

OBJECTIVE: To assess morphological changes in the diaphragm and phrenic nerve in patients who died from COVID-19. MATERIAL AND METHODS: In a case-control study, an analysis was made of autopsy material of the diaphragm and phrenic nerve of those who died from COVID-19 infection complicated by SARS-CoV-2-associated pneumonia, confirmed in vivo by the presence of SARS-CoV-2 RNA (Group 1, n=12), and those who died with a diagnosis of acute cerebrovascular accident of the ischemic type without parenchymal respiratory failure (Group 2, n=3). RESULTS: The main histopathological features in the diaphragm of the 1st group were the edema of the pericellular spaces of muscle fibers, edema of perivascular spaces, diapedese hemorrhages, plethora in arteriolas, in most veins and capillaries, red blood clots were revealed; in the diaphragmatic nerve - swelling of the perineral space, severe edema around the nerve fibers inside the nerve trunk. In the diaphragm of group 2, edema of pericellular spaces of muscle fibers and edema of perivascular spaces were less pronounced (p<0.001), hemorrhages were not determined; in the diaphragmatic nerve, moderate edema of the perineral space, mild swelling inside the nerve trunk around the nerve fibers was revealed (p<0.001). The glycogen content in the muscle cells of group 1 is significantly lower compared to group 2 (p<0.001). CONCLUSION: The study confirms the characteristic pathological picture of organ damage in COVID-19. However, the leading pathological mechanism of organ damage requires further investigation.

Development of giant cell arteritis after vaccination against SARS-CoV2: A case report and literature review.

RATIONALE: Giant cell arteritis (GCA) is an autoimmune vasculitis that affects large and medium-sized blood vessels. The mRNA vaccine against severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) has been associated with the development of immune-mediated diseases. In this article, we present a case of GCA that developed after vaccination against SARS-CoV2. PATIENT CONCERNS: A 77-year-old man developed fever, general fatigue, and headache 1 day after the third dose of vaccination against SARS-CoV2. Nodular swelling and tenderness of the bilateral temporal arteries were observed. DIAGNOSES: Although right temporal artery biopsies were negative, the patient was diagnosed with GCA based on criteria established by the American College of Rheumatology for the classification of GCA. INTERVENTIONS: The patient received methylprednisolone 1000 mg for 3 days. This was followed by prednisolone 1 mg/kg/d, which was decreased by 10 mg every week to 30 mg. From day 16 of hospitalization, the patient received tocilizumab 162 mg/wk every other week. OUTCOMES: There was no occurrence of acute side effects. After 38 days of treatment, the condition improved and the patient was discharged from the hospital; as stated above, the dose of prednisolone was tapered to 30 mg/d. LESSONS: We experienced a case of GCA that occurred immediately after vaccination against SARS-CoV2 with an mRNA vaccine. Early signs of GCA include fever, fatigue, and headache, and often resemble those noted after vaccination against SARS-CoV2. The potential presence of GCA should be determined in individuals with persistent fever and headache after vaccination against SARS-CoV2.

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.

Wastewater early warning system for SARS-CoV-2 outbreaks and variants in a Coruña, Spain.

Wastewater-based epidemiology has been widely used as a cost-effective method for tracking the COVID-19 pandemic at the community level. Here we describe COVIDBENS, a wastewater surveillance program running from June 2020 to March 2022 in the wastewater treatment plant of Bens in A Coruña (Spain). The main goal of this work was to provide an effective early warning tool based in wastewater epidemiology to help in decision-making at both the social and public health levels. RT-qPCR procedures and Illumina sequencing were used to weekly monitor the viral load and to detect SARS-CoV-2 mutations in wastewater, respectively. In addition, own statistical models were applied to estimate the real number of infected people and the frequency of each emerging variant circulating in the community, which considerable improved the surveillance strategy. Our analysis detected 6 viral load waves in A Coruña with concentrations between 103 and 106 SARS-CoV-2 RNA copies/L. Our system was able to anticipate community outbreaks during the pandemic with 8-36 days in advance with respect to clinical reports and, to detect the emergence of new SARS-CoV-2 variants in A Coruña such as Alpha (B.1.1.7), Delta (B.1.617.2), and Omicron (B.1.1.529 and BA.2) in wastewater with 42, 30, and 27 days, respectively, before the health system did. Data generated here helped local authorities and health managers to give a faster and more efficient response to the pandemic situation, and also allowed important industrial companies to adapt their production to each situation. The wastewater-based epidemiology program developed in our metropolitan area of A Coruña (Spain) during the SARS-CoV-2 pandemic served as a powerful early warning system combining statistical models with mutations and viral load monitoring in wastewater over time.

Identification of Flavonoids from Scutellaria barbata D. Don as Inhibitors of HIV-1 and Cathepsin L Proteases and Their Structure-Activity Relationships.

Scutellaria barbata D. Don (SB, Chinese: Ban Zhi Lian), a well-known medicinal plant used in traditional Chinese medicine, is rich in flavonoids. It possesses antitumor, anti-inflammatory, and antiviral activities. In this study, we evaluated the inhibitory activities of SB extracts and its active components against HIV-1 protease (HIV-1 PR) and SARS-CoV2 viral cathepsin L protease (Cat L PR). UPLC/HRMS was used to identify and quantify the major active flavonoids in different SB extracts, and fluorescence resonance energy transfer (FRET) assays were used to determine HIV-1 PR and Cat L PR inhibitions and identify structure-activity relationships. Molecular docking was also performed, to explore the diversification in bonding patterns of the active flavonoids upon binding to the two PRs. Three SB extracts (SBW, SB30, and SB60) and nine flavonoids inhibited HIV-1 PR with an IC50 range from 0.006 to 0.83 mg/mL. Six of the flavonoids showed 10~37.6% inhibition of Cat L PR at a concentration of 0.1 mg/mL. The results showed that the introduction of the 4'-hydroxyl and 6-hydroxyl/methoxy groups was essential in the 5,6,7-trihydroxyl and 5,7,4'-trihydroxyl flavones, respectively, to enhance their dual anti-PR activities. Hence, the 5,6,7,4'-tetrahydroxyl flavone scutellarein (HIV-1 PR, IC50 = 0.068 mg/mL; Cat L PR, IC50 = 0.43 mg/mL) may serve as a lead compound to develop more effective dual protease inhibitors. The 5,7,3',4'-tetrahydroxyl flavone luteolin also showed a potent and selective inhibition of HIV-1 PR (IC50 = 0.039 mg/mL).

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.

Optimizing the Cas13 antiviral train: cargo and delivery.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic in 2020 highlighted the need for rapid, widespread responses against infectious disease. One such innovation uses CRISPR-Cas13 technology to directly target and cleave viral RNA, thereby inhibiting replication. Due to their programmability, Cas13-based antiviral therapies can be rapidly deployed to target emerging viruses, in comparison with traditional therapeutic development that takes at least 12-18 months, and often many years. Moreover, similar to the programmability of mRNA vaccines, Cas13 antivirals can be developed to target mutations as the virus evolves.

Results of the 2021 French National Perinatal Survey and trends in perinatal health in metropolitan France since 1995.

OBJECTIVE: To report results of the 2021 French National Perinatal Survey (ENP) in metropolitan France and assess trends in the main indicators of perinatal health, medical practices, and risk factors in France since 1995. POPULATION AND METHOD: All the samples included all women giving birth at a gestational age of at least 22 weeks of gestation and/or to an infant weighing at least 500 grams in all maternity units in metropolitan France during one week in 1995 (N=13 048), 2003 (N=14 324), 2010 (N=14 546), 2016 (N=12 553), and 2021 (N=12 088). The data came from postpartum interviews of the women at the hospital and their medical records. Comparisons between surveys showed trends over time. RESULTS: Between 1995 and 2021, maternal characteristics changed. Maternal age and the frequency of women with obesity rose: in 2021, 24.6% of women were 35 years or older (21.1% in 2016, 19.2% in 2010, 15.9% in 2003 and 12.4% in 1995) and 14.4% were obese (11.8% in 2016, 9.9% in 2010 and 7.4% in 2003). Some antenatal prevention behaviors that improved in 2021 were not smoking during the third trimester, acid folic administration before pregnancy, and vaccination against influenza. The percentage of women with an early prenatal appointment ("4th month appointment"), implemented to facilitate screening of maternal vulnerability during pregnancy, has continued to rise. The percentage of women receiving prenatal care by midwives has risen markedly (39.0% in 2021 versus 11.7% in 2016). Serum screening for Down syndrome continues to increase (91.8% of women in 2021). The rate of induction of labor has risen significantly (20.2% in 1995 and 25.8% in 2021). The mode of delivery has not varied significantly since 2003; in 2021, the cesarean rate was 21.4% and the instrumental vaginal delivery rate 12.4%. Episiotomy was increasingly rare, among both primiparous and multiparous women (16.5% and 2.9% in 2021, respectively). The prevalence of coronavirus (SARS-CoV2) infection during pregnancy was 5.7%. Preterm live births increased regularly, slightly but significantly over the 1995-2016 period and then remained stable between 2016 and 2021 (7.0%). In 2021, 56.3% of women exclusively breastfed during their hospital stay, a modest increase in comparison with 2016 (54.6%). CONCLUSION: Routine national perinatal surveys highlight positive trends over time in some preventive practices, decreases in some medical interventions consistent with national guidelines, and the increasing role of midwives in prenatal care. Nonetheless, some indicators remain less than optimal and require more detailed analyses.

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.

COVID-19-associated pulmonary aspergillosis in immunocompetent patients: a virtual patient cohort study.

The opportunistic fungus Aspergillus fumigatus infects the lungs of immunocompromised hosts, including patients undergoing chemotherapy or organ transplantation. More recently however, immunocompetent patients with severe SARS-CoV2 have been reported to be affected by COVID-19 Associated Pulmonary Aspergillosis (CAPA), in the absence of the conventional risk factors for invasive aspergillosis. This paper explores the hypothesis that contributing causes are the destruction of the lung epithelium permitting colonization by opportunistic pathogens. At the same time, the exhaustion of the immune system, characterized by cytokine storms, apoptosis, and depletion of leukocytes may hinder the response to A. fumigatus infection. The combination of these factors may explain the onset of invasive aspergillosis in immunocompetent patients. We used a previously published computational model of the innate immune response to infection with Aspergillus fumigatus. Variation of model parameters was used to create a virtual patient population. A simulation study of this virtual patient population to test potential causes for co-infection in immunocompetent patients. The two most important factors determining the likelihood of CAPA were the inherent virulence of the fungus and the effectiveness of the neutrophil population, as measured by granule half-life and ability to kill fungal cells. Varying these parameters across the virtual patient population generated a realistic distribution of CAPA phenotypes observed in the literature. Computational models are an effective tool for hypothesis generation. Varying model parameters can be used to create a virtual patient population for identifying candidate mechanisms for phenomena observed in actual patient populations.

6†The impact of COVID-19 on corneal transplantation in England.

INTRODUCTION/BACKGROUND: At the beginning of the COVID-19 pandemic, eye banks around the world had to assess the impact of SARS-CoV-2 infection in potential ocular tissue donors and decide how to characterise donors to meet ongoing demand for tissue for transplantation.NHSBT eye banks normally issue cornea grafts for over 4000 transplants per annum (pre-pandemic). SARS-CoV2 RNA screening is not a requirement for eye donor characterisation. Donor authorisation is based on review of donor medical and contact history and any available COVID test results (e.g. from hospital testing or as part of organ donor characterisation). After retrieval, globes are disinfected with PVP-iodine, and corneas stored in organ culture.This presentation explores the impact of COVID-19 on corneal donation and transplantation in England. METHODS: UK Transplant Registry data were analysed on all corneal donors and transplants in England from 1 January 2020 to 2 July 2021. All laboratory confirmed SARS CoV-2 infections were collected by Public Health England from 16 March 2020. Information was available until mid-November 2021.To assess the possibility of transmission through a transplanted graft, cases with a diagnosis of infection within 14 days post transplant were identified for further review. RESULTS: 4130 corneal grafts were performed in England. We are aware of 222 recipients who tested positive for SARS-CoV2. 2 of these have been reported to have died within 28 days of testing positive. The diagnosis of SARS-CoV2 infection in these 2 recipients had been made beyond 30 days post transplant.In 3 of the 222 infected recipients, the interval between transplant and infection was within 14 days (all 3 recipients alive). 2 of the 3 donors were fully characterised organ donors (universally screened for SARS-CoV-2 RNA in upper and lower respiratory tract samples), and one was an eye only donor who had tested negative in hospital 2 days prior to death. CONCLUSIONS: The linkage of large registries allows collection of useful data in a large cohort of patients transplanted during the COVID-19 pandemic. The incidence of COVID-19 and characteristics of corneal transplant recipients who tested positive for SARS-CoV2 were found to be similar to those for the general population of England.These data have not identified any epidemiological evidence for transmission of COVID-19 through corneal transplantation, and offer reassurance about the safety and quality systems that are in place to allow ongoing corneal transplantation during the pandemic.

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.

Case Report: Favorable outcome of allogeneic hematopoietic stem cell transplantation in SARSCoV2 positive recipient, risk-benefit balance between infection and leukemia.

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) in SARS-CoV-2 positive candidates is usually delayed until the clinical resolution of the infection's symptoms and a negative nasopharyngeal molecular test. However, prolonged SARS-CoV-2 positivity has been frequently observed in haematological malignancies, thus representing a challenge for the timing of transplant procedures. Here, we report on the case of a 34-year-old patient with recent pauci-symptomatic COVID-19 undergoing transplant for high-risk acute B-lymphoblastic leukemia before achieving viral clearance. Shortly before their scheduled allogeneic HSCT from a matched unrelated donor, the patient developed mild Omicron BA.5 infection receiving nirmatrelvir/ritonavir with fever resolution within 72 hours. Twenty-three days after COVID-19 diagnosis, because of increasing minimal residual disease values in the context of high-risk refractory leukemia and clinical resolution of SARS-2-CoV infection with reduction of viral load at surveillance nasopharyngeal swabs, it was decided not to delay further allo-HSCT. During myelo-ablative conditioning, the nasopharyngeal SARS-CoV-2 viral load increased while the patient remained asymptomatic. Consequently, two days before the transplant, intra-muscular tixagevimab/cilgavimab 300/300 mg and a 3-day course of intravenous remdesivir were administered. During the pre-engraftment phase, veno-occlusive disease (VOD) occurred at day +13, requiring defibrotide treatment to obtain a slow but complete recovery. The post-engraftment phase was characterized by mild COVID-19 at day +23 (cough, rhino-conjunctivitis, fever) that spontaneously resolved, achieving viral clearance at day +28. At day +32, she experienced grade I acute graft-versus host disease (a-GVHD, skin grade II) treated with steroids and photo-apheresis, without further complications during follow-up until day +180. Addressing the issue of allo-HSCT timing in patients recovering from SARS-CoV-2 infection with high-risk malignant diseases is challenging because of 1] the high risk of COVID-19 clinical progression, 2] the impact of transplant delay on leukemia prognosis and 3] the occurrence of endothelial complications such as VOD, a-GVHD, and transplant associated thrombotic micro-angiopathy. Our report describes the favourable outcome of allo-HSCT in a recipient with active SARS-CoV2 infection and high-risk leukemia thanks to timely anti-SARS-CoV-2 preventive therapies and prompt management of transplant-related complications.

Performances of Dried Blood Spots and Point-of-Care Devices to Identify Virological Failure in HIV-Infected Patients: A Systematic Review and Meta-Analysis.

To broaden access to HIV viral load monitoring (VLM), the use of blood samples from dried blood spots (DBS) or point-of-care (POC) devices, could be of great help in settings where plasma is not easily accessible. The variety of assays available makes the choice complex. This systematic review and meta-analysis aims to estimate the sensitivity and specificity of DBS and POC devices to identify patients in virological failure using World Health Organization (WHO) recommendations (viral load ≥1000 copies/mL), compared with plasma, for the assays currently available. Four databases were searched for articles, and two reviewers independently identified articles reporting sensitivity and specificity of DBS and/or POC to identify patients in virological failure. We excluded articles that used other thresholds as well as articles with a total number of participants below 50 to avoid reporting bias. Heterogeneity and factors associated with assays' performances were assessed by I2 statistics and metaregression. The protocol of this review follows the PRISMA guidelines. Out of 941 articles, 47 were included: 32 DBS evaluations and 16 POC evaluations. Overall, when using DBS, the Abbott RT HIV-1, Roche CAP-CTM, NucliSENS BioMerieux and Aptima assays presented sensitivity and specificity exceeding 85%, but reported results were highly heterogeneous. Factors associated with better performances were high volume of blood and the use of the same assay for DBS and plasma VLM. Regarding the POC devices, SAMBA I, SAMBA II, and GeneXpert devices presented high sensitivity and specificity exceeding 90%, with less heterogeneity. DBS is suitable VLM, but performances can vary greatly depending on the protocols, and should be performed in trained centers. POC is suitable for VLM with less risk of heterogeneity but is more intensive in costs and logistics.

Risk factors for prolonged viral RNA shedding in patients with COVID-19; a nested case-control study.

INTRODUCTION: The coronavirus disease 2019 (COVID-19) pandemic continues to have a global impact. The behavior and viral course of severe acute respiratory syndrome coronavirus (SARS-CoV-2) remains unpredictable. We aimed to investigate the prediction factors associated with prolonged viral shedding in COVID-19 patients. METHODOLOGY: This is a retrospective, nested, case-control study with 155 confirmed COVID-19 infected patients divided into two groups based on nucleic acid conversion time (NCT), a prolonged group (viral RNA shedding >14 days, n = 31) and a non-prolonged group (n = 124). RESULTS: The mean age of participants was 57.16 years, and 54.8% were male. Inpatient numbers were 67.7% across both groups. No statistically significant differences between the two groups were observed in terms of clinical manifestation, comorbidities, computer tomography, severity index, antiviral treatment, and vaccination. However, C-reactive protein and D-dimer levels were significantly higher in the prolonged group (p = 0.01; p = 0.01). Using conditional logistic regression analysis, D-dimer and bacterial co-infection were found to be independent factors associated with the prolonged NCT (OR: 1.001, 95% CI: 1.000-1.001, p = 0.043; OR: 12.479, 95% CI: 2.701-57.654, p = 0.001 respectively). We evaluated the diagnostic value of the conditional logistic regression model by using receiver operating characteristic curve analysis. The area under the curve was 0.7 (95% Cl: 0.574-0.802; p < 0.001). CONCLUSIONS: Our study design included controlling confounders. We showed a clear result associating predicting factors with prolonged NCT of SARS-CoV-2. D-dimer level and bacterial co-infection were considered as independent predictors of prolonged NCT.

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.