Immortalized hepatocyte-like cells: A competent hepatocyte model for studying clinical HCV isolate infection.

More than 58 million individuals worldwide are inflicted with chronic HCV. The disease carries a high risk of end stage liver disease, i.e., cirrhosis and hepatocellular carcinoma. Although direct-acting antiviral agents (DAAs) have revolutionized therapy, the emergence of drug-resistant strains has become a growing concern. Conventional cellular models, Huh7 and its derivatives were very permissive to only HCVcc (JFH-1), but not HCV clinical isolates. The lack of suitable host cells had hindered comprehensive research on patient-derived HCV. Here, we established a novel hepatocyte model for HCV culture to host clinically pan-genotype HCV strains. The immortalized hepatocyte-like cell line (imHC) derived from human mesenchymal stem cell carries HCV receptors and essential host factors. The imHC outperformed Huh7 as a host for HCV (JFH-1) and sustained the entire HCV life cycle of pan-genotypic clinical isolates. We analyzed the alteration of host markers (i.e., hepatic markers, cellular innate immune response, and cell apoptosis) in response to HCV infection. The imHC model uncovered the underlying mechanisms governing the action of IFN-α and the activation of sofosbuvir. The insights from HCV-cell culture model hold promise for understanding disease pathogenesis and novel anti-HCV development.

CRISPR/Cas9 genome editing of CCR5 combined with C46 HIV-1 fusion inhibitor for cellular resistant to R5 and X4 tropic HIV-1.

Hematopoietic stem-cell (HSC) transplantation using a donor with a homozygous mutation in the HIV co-receptor CCR5 (CCR5Δ32/Δ32) holds great promise as a cure for HIV-1. Previously, there were three patients that had been reported to be completely cured from HIV infection by this approach. However, finding a naturally suitable Human Leukocyte Antigen (HLA)-matched homozygous CCR5Δ32 donor is very difficult. The prevalence of this allele is only 1% in the Caucasian population. Therefore, additional sources of CCR5Δ32/Δ32 HSCs are required. The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated (Cas) system is one method to mediate CCR5 knockout in HSCs that has been successfully employed as a gene editing tool in clinical trials. Additional anti-HIV-1 strategies are still required for broad-spectrum inhibition of HIV-1 replication. Here in this study, we combined an additional anti-HIV-1 therapy, which is C46, a cell membrane-anchored HIV-1 fusion inhibitor with the CRISPR/Cas9 mediated knockout CCR5. The combined HIV-1 therapeutic genes were investigated for the potential prevention of both CCR5 (R5)- and CXCR4 (X4)-tropic HIV-1 infections in the MT4CCR5 cell line. The combinatorial CRISPR/Cas9 therapies were superior compared to single method therapy for achieving the HIV-1 cure strategy and shows potential for future applications.

Immune reconstitution in children after haploidentical haematopoietic stem cell transplantation.

INTRODUCTION: Immune reconstitution (IR) kinetics of paediatric patients underwent haploidentical haematopoietic stem cell transplantation (HSCT) with post-transplant cyclophosphamide (PTCy) have not been extensively studied. We compared IR patterns of children receiving HSCT from haploidentical (n = 92) and HLA-matched donors (n = 36), and analysed risk factors for viral infection in these patients. METHODS: We prospectively measured lymphocyte subset numbers before HSCT and at 1, 3, 6 and 12 months after HSCT. Blood cytomegalovirus (CMV), Epstein-Barr virus, adenovirus, BK virus (BKV) and urine adenovirus and BKV viral loads were measured at designated time points. RESULTS: The median numbers of total T and T helper cells at 1 month were significantly lower in the haploidentical group compared with the HLA-matched group. Haploidentical HSCT recipients had significantly lower median numbers of several T cell subsets and B cells for 1 year after HSCT. The median NK cell count of the haploidentical group was lower at 1 month. BKV haemorrhagic cystitis, blood CMV and urine adenovirus reactivation were more frequently found in the haploidentical group. Post-haploidentical HSCT patients receiving anti-T lymphocyte globulin (ATG) had significantly lower median numbers of total T cells (at 1 month) and T helper cells (at 6 and 12 months) and higher rate of blood BKV reactivation compared with those without ATG. CONCLUSION: Paediatric patients who undergo haploidentical HSCT with PTCy are likely to have delayed IR and an increased risk of viral reactivation/infection compared with HLA-matched HSCT. The addition of ATG to PTCy delayed T cell recovery and increased risk of BKV reactivation.

Characterizing a visual lateral flow device for rapid SARS-CoV-2 virus protein detection: pre-clinical and system assessment.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infections have affected more than 769 million individuals worldwide over the last few years. Although the pandemic is transitioning into an endemic, the COVID-19 outbreak is still a global concern. A rapid screening platform is needed for effective preventive and control measures. Herein, a visual rapid lateral flow platform for SARS-CoV-2 nucleocapsid protein detection is developed. Under optimal conditions, the system demonstrated good detection sensitivity and selectivity against tested respiratory viruses. The system provides direct visual detection with a limit of 0.7 ng of the nucleocapsid protein per mL of a sample (0.7 ng mL-1) within 15 minutes. Further, a correlation between direct visual detection and semi-quantitative analysis using a reader showed a similar detection limit (R2 = 0.9571). The repeatability and reproducibility studies highlighted the potential of the system for the rapid screening of SARS-CoV-2 infection, with variations within 5% and 10% at high and low protein concentrations, respectively. Subsequent pre-clinical validation to correlate the performance with the standard molecular approach (RT-PCR) using 170 nasopharyngeal swabs demonstrated 98% estimated sensitivity (95% CI, 89.35-99.95%) and 100% specificity (95% CI, 96.38-100%). The positive and negative predictive values were reported to be 100% and 99%, respectively, with an accuracy of 99.3%. With high viral load samples (Ct value ≤25, n = 47), the system demonstrated 100% detection sensitivity and specificity. The proposed technique provides a valuable platform for potential use in rapid screening, particularly during pandemics, where diagnostic capacity and mass screening are crucial.

HIV-1 proviral DNA in purified peripheral blood CD34+ stem and progenitor cells in individuals with long-term HAART; paving the way to HIV gene therapy.

Human immunodeficiency virus (HIV)-1 infection is an important public health problem worldwide. After primary HIV-1 infection, transcribed HIV-1 DNA is integrated into the host genome, serving as a reservoir of the virus and hindering a definite cure. Although highly active antiretroviral therapy suppresses active viral replication, resulting in undetectable levels of HIV RNA in the blood, a viral rebound can be detected after a few weeks of treatment interruption. This supports the concept that there is a stable HIV-1 reservoir in people living with HIV-1. Recently, a few individuals with HIV infection were reported to be probably cured by hematopoietic stem transplantation (HSCT). The underlying mechanism for this success involved transfusion of uninfected hematopoietic stem and progenitor cells (HSPCs) from CCR5-mutated donors who were naturally resistant to HIV infection. Thus, gene editing technology to provide HIV-resistant HSPC has promise in the treatment of HIV infections by HSCT. In this study, we aimed to find HIV-infected individuals likely to achieve a definite cure via gene editing HSCT. We screened for total HIV proviral DNA by Alu PCR in peripheral blood mononuclear cells (PBMCs) of 20 HIV-infected individuals with prolonged viral suppression. We assessed the amount of intact proviral DNA via a modified intact proviral DNA assay (IPDA) in purified peripheral CD34+ HSPCs. PBMCs from all 20 individuals were positive for the gag gene in Alu PCR, and peripheral CD34+ HSPCs were IPDA-negative for six individuals. Our results suggested that these six HIV-infected individuals could be candidates for further studies into the ability of gene editing HSCT to lead to a definite HIV cure.

Sequential Flow Controllable Microfluidic Device for G-Quadruplex DNAzyme-Based Electrochemical Detection of SARS-CoV-2 Using a Pyrrolidinyl Peptide Nucleic Acid.

The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been a significant health issue globally. Point-of-care (POC) testing that can offer a rapid and accurate diagnosis of SARS-CoV-2 at the early stage of infection is highly desirable to constrain this outbreak, especially in resource-limited settings. Herein, we present a G-quadruplex DNAzyme-based electrochemical assay that is integrated with a sequential flow controllable microfluidic device for the detection of SARS-CoV-2 cDNA. According to the detection principle, a pyrrolidinyl peptide nucleic acid probe is immobilized on a screen-printed graphene electrode for capturing SARS-CoV-2 DNA. The captured DNA subsequently hybridizes with another DNA probe that carries a G-quadruplex DNAzyme as the signaling unit. The G-quadruplex DNAzyme catalyzes the H2O2-mediated oxidation of hydroquinone to benzoquinone that can be detected using square-wave voltammetry to give a signal that corresponds to the target DNA concentration. The assay exhibited high selectivity for SARS-CoV-2 DNA and showed a good experimental detection limit at 30 pM. To enable automation, the DNAzyme-based assay was combined with a capillary-driven microfluidic device featuring a burst valve technology to allow sequential sample and reagent delivery as well as the DNA target hybridization and enzymatic reaction to be operated in a precisely controlled fashion. The developed microfluidic device was successfully applied for the detection of SARS-CoV-2 from nasopharyngeal swab samples. The results were in good agreement with the standard RT-PCR method and could be performed within 20 min. Thus, this platform offers desirable characteristics that make it an alternative POC tool for COVID-19 diagnosis.

Diagnostic stewardship to limit repeat plasma cytomegalovirus viral load testing.

BACKGROUND: Frequent serial monitoring of plasma cytomegalovirus (CMV) viral load caused unnecessary budgets for laboratory testing without changes in treatment. We aimed to implement diagnostic stewardship to limit CMV viral load testing at appropriate intervals. METHODS: A quasi-experimental study was performed. To avoid unnecessary plasma CMV viral load testing, the inpatient electronic pop-up reminder was launched in 2021. In cases with plasma CMV viral load testing was ordered in intervals of less than five days, telephone interview and feedback were performed. Pre-post intervention data was compared in terms of clinical and monetary outcomes. The rate of plasma CMV viral load testing performed in intervals of less than five days was compared between 2021 and 2019 using the Poisson regression model. RESULTS: After the protocol implementation, there was a significant decrease in the rate of plasma CMV viral load test orders in intervals of less than five days from 17.5% to 8.0% [incidence rate ratio 0.40, p < 0.001]. There was no statistically significant difference in the incidence of CMV DNAemia and CMV disease (p = 0.407 and 0.602, respectively). As a result, the hospital could save the costs of plasma CMV viral load testing per 1,000 patients performed with intervals of less than five days from 2,646,048.11 to 1,360,062.89 Thai Baht. CONCLUSIONS: The diagnostic stewardship program is safe and helpful in reducing unnecessary plasma CMV viral load testing and costs.

Self-enhancement lateral flow immunoassay for COVID-19 diagnosis.

Equipment-free colorimetric-based lateral flow immunoassay (LFIA) is the most convenient and popular tool for various applications, including diagnostic tools requiring high sensitivity for the detection of pathogens. Thus, improvements and developments of LFIA are constantly being reported. Herein, we enriched the sensitivity of LFIA using the gold enhancement principle, emphasizing needlessly complicated apparatus, only one step for the strip test operation, and typical time incubation (15 min) process. Self-enhanced LFIA was then executed for subsequent flows by overlapping the additionally enhanced pad composed of gold ions and reducing agent on the conjugate pad and the sample pad. Self-enhanced LFIA was performed to detect SARS-CoV-2 antigens in saliva. The obtained result depicted that the achieved sensitivity was up to tenfold compared with that of conventional LFIA by visual measurements. The detection limits of self-enhanced LFIA detecting nucleocapsid protein antigens in the saliva sample was 0.50 and 0.10 ng/mL employed by naked eye detection and calibration curve-based calculation, respectively. When the proposed device was applied to 207 human saliva samples, the diagnostic performance presented a 96.10 % sensitivity and 99.23 % specificity. This self-enhanced LFIA could be implemented in large-scale production and demonstrates higher sensitivity with effortless use, which meets the requirements for point-of-care testing and on-field mass screening.

Electrochemical lateral-flow device for rapid COVID-19 antigen-diagnostic testing.

Antigen test kits (ATK) are extensively utilized for screening and diagnosing COVID-19 because they are easy to operate. However, ATKs exhibit poor sensitivity and cannot detect low concentrations of SARS-CoV-2. Herein, we present a new, highly sensitive, and selective device obtained by combining the principle of ATKs with electrochemical detection for COVID-19 diagnosis, which can be quantitatively assessed using a smartphone. An electrochemical test strip (E-test strip) was constructed by attaching a screen-printed electrode inside a lateral-flow device to exploit the remarkable binding affinity of SARS-CoV-2 antigen to ACE2. The ferrocene carboxylic acid attached to SARS-CoV-2 antibody acts as an electroactive species when it binds to SARS-CoV-2 antigen in the sample before it flows continuously to the ACE2-immobilization region on the electrode. Electrochemical-assay signal intensity on smartphones increased proportionally to the concentration of SARS-CoV-2 antigen (LOD = 2.98 pg/mL, under 12 min). Additionally, the application of the single-step E-test strip for COVID-19 screening was demonstrated using nasopharyngeal samples, and the results were consistent with those obtained using the gold standard (RT-PCR). Therefore, the sensor demonstrated excellent performance in assessing and screening COVID-19, and it can be used professionally to accurately verify diagnostic data while remaining rapid, simple, and inexpensive.

Saliva and wastewater surveillance for SARS-CoV-2 during school reopening amid COVID-19 pandemic in Thailand.

Objectives: School closure during the coronavirus disease 2019 (COVID-19) pandemic resulted in a negative impact on children. Serial testing of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been proposed as a measure for safety school reopening. We aimed to study the usefulness of SARS-CoV-2 surveillance by saliva testing and performing wastewater surveillance for SARS-CoV-2 in a day school in a resource-limited setting. Methods: We conducted a cluster randomized study to investigate the potential use of saliva antigen testing compared to saliva pooling for nucleic acid detection in a primary school in Thailand from December 2021 to March 2022. Wastewater surveillance in the school was also performed. Results: A total of 484 participants attended the study. SARS-CoV-2 was detected in two participants from the tests provided by the study (one in the pool nucleic acid test arm, and another in the quantitative antigen test arm). Additional ten participants reported positive results on an additional rapid antigen test (RAT) performed by nasal swab when they had symptoms or household contact. There was no difference among arms in viral detection by intention-to-treat and per protocol analysis (p = 0.304 and 0.894, respectively). We also investigated the feasibility of wastewater surveillance to detect the virus in this setting. However, wastewater surveillance could not detect the virus. Conclusions: In a low COVID-19 prevalence, serial saliva testing and wastewater surveillance for SARS-CoV-2 rarely detected the virus in a day school setting. Performing RAT on nasal swabs when students, teachers or staff have symptoms or household contact might be more reasonable.

Seroprevalence of hepatitis E virus after pediatric liver transplantation.

BACKGROUND: Southeast Asia is the endemic area of hepatitis E virus (HEV) infection. We aimed to determine the seroprevalence of the virus, its association, and the prevalence of chronic infection after pediatric liver transplantation (LT). METHODS: A cross-sectional study was performed in Bangkok, Thailand. Patients aged <18 years who had LT for >2 years underwent serologic and real-time polymerase chain reaction (rt-PCR) tests. Acute HEV infection was defined by the presence of positive anti-HEV immunoglobulin (Ig)M and HEV viremia from the rt-PCR. If the viremia persisted for >6 months, chronic HEV infection was diagnosed. RESULTS: A total of 101 patients had a median age of 8.4 years [interqartile range (IQR): 5.8-11.7]. The seroprevalence of anti-HEV IgG and IgM was 15% and 4%, respectively. Positive IgM and/or IgG were associated with a history of elevated transaminases with an unknown cause after LT (p = 0.04 and p = 0.01, respectively). The presence of HEV IgM was associated with a history of elevated transaminases with an unknown cause within 6 months (p = 0.01). The two patients (2%) diagnosed with chronic HEV infection did not fully respond to the reduction of immunosuppression but responded well to ribavirin treatment. CONCLUSIONS: Seroprevalence of HEV among pediatric LT recipients was not rare in Southeast Asia. Since HEV seropositivity was associated with elevated transaminases of an unknown cause, investigation for the virus should be offered in LT children with hepatitis after excluding other etiologies. Pediatric LT recipients with chronic HEV infection may receive a benefit from a specific antiviral treatment.

Label free electrochemical DNA biosensor for COVID-19 diagnosis.

The COVID-19 pandemic has significantly increased the development of the development of point-of-care (POC) diagnostic tools because they can serve as useful tools for detecting and controlling spread of the disease. Most current methods require sophisticated laboratory instruments and specialists to provide reliable, cost-effective, specific, and sensitive POC testing for COVID-19 diagnosis. Here, a smartphone-assisted Sensit Smart potentiostat (PalmSens) was integrated with a paper-based electrochemical sensor to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). A disposable paper-based device was fabricated, and the working electrode directly modified with a pyrrolidinyl peptide nucleic acid (acpcPNA) as the biological recognition element to capture the target complementary DNA (cDNA). In the presence of the target cDNA, hybridization with acpcPNA probe blocks the redox conversion of a redox reporter, leading to a decrease in electrochemical response correlating to SARS-CoV-2 concentration. Under optimal conditions, a linear range from 0.1 to 200 nM and a detection limit of 1.0 pM were obtained. The PNA-based electrochemical paper-based analytical device (PNA-based ePAD) offers high specificity toward SARS-CoV-2 N gene because of the highly selective PNA-DNA binding. The developed sensor was used for amplification-free SARS-CoV-2 detection in 10 nasopharyngeal swab samples (7 SARS-CoV-2 positive and 3 SARS-CoV-2 negative), giving a 100% agreement result with RT-PCR.

Electrochemical paper-based antigen sensing platform using plant-derived monoclonal antibody for detecting SARS-CoV-2.

The current approaches of diagnostic platforms for detecting SARS-CoV-2 infections mostly relied on adapting the existing technology. In this work, a simple and low-cost electrochemical sensing platform for detecting SAR-CoV-2 antigen was established. The proposed sensor combined the innovative disposable paper-based immunosensor and cost-effective plant-based anti-SARS-CoV-2 monoclonal antibody CR3022, expressed in Nicotiana benthamiana. The cellulose nanocrystal was modified on screen-printed graphene electrode to provide the abundant COOH functional groups on electrode surface, leading to the high ability for antibody immobilization. The quantification of the presence receptor binding domain (RBD) spike protein of SARS-CoV-2 was performed using differential pulse voltammetry by monitoring the changing current of [Fe(CN)6]3-/4- redox solution. The current change of [Fe(CN)6]3-/4- before and after the presence of target RBD could be clearly distinguished, providing a linear relationship with RBD concentration in the range from 0.1 pg/mL to 500 ng/mL with the minimum limit of detection of 2.0 fg/mL. The proposed platform was successfully applied to detect RBD in nasopharyngeal swab samples with satisfactory results. Furthermore, the paper-based immunosensor was extended to quantify the RBD level in spiked saliva samples, demonstrating the broadly applicability of this system. This electrochemical paper-based immunosensor has the potential to be employed as a point-of-care testing for COVID-19 diagnosis.

Risk Factors of Renal Tubular Dysfunction in Thai People Living with HIV Receiving Tenofovir Disoproxil Fumarate.

Tenofovir disoproxil fumarate (TDF) associates with renal tubular dysfunction (RTD) in some people living with HIV (PLWH). We studied clinical and genetic factors associated with RTD in Thai PLWH receiving TDF. RTD was diagnosed in 13 of 65 (20%) patients. The median (interquartile range) age and CD4 cell counts were 43.8 (40.4-50.9) years and 554 (437-716) cells/mm3, respectively. The median duration of TDF use was 46.9 (31.5-54.1) months. Univariate logistic regression demonstrated body mass index (BMI), concomitant use of protease inhibitor (PI), hyperlipidemia, and homozygous C/C SNP rs1059751 of ABCC4 gene as predisposing factors of RTD. In multivariate model, concomitant use of PI [adjusted odds ratio (aOR) 11.39; 95% confidence interval (CI), 1.59- 81.56; P = 0.015], hyperlipidemia (aOR 8.59; 95% CI, 1.46-50.40; P = 0.017), and BMI (aOR 0.76; 95% CI, 0.59-0.98; P = 0.037) remained associated with RTD in patients receiving TDF. PLWH receiving TDF with the presence of these factors should be closely monitored for RTD.

A novel delayed lateral flow immunoassay for enhanced detection of SARS-CoV-2 spike antigen.

A new detection strategy was developed to improve the sensitivity of a lateral flow immunoassay platform utilizing a delayed hydrophobic barrier fabricated with trimethylsilyl cellulose (TMSC). The SARS-CoV-2 spike receptor-binding domain (SARS-CoV-2 SP RBD) antigen was chosen as a model analyte to demonstrate the superior detectability of this scheme. The novel device consists of 2 separate layers, so-called delayed lateral flow immunoassay (d-LFIA). The upper layer is intended for the analyte or sample flow path, where the test solution flows freely straight to the detection zone to bind with the primary antibody. The lower layer, located just underneath, is designed for the SARS-CoV-2 spike receptor-binding domain-conjugated gold nanoparticles (SARS-CoV-2 SP RBD-AuNPs) used for producing a colorimetric signal. This layer is fabricated with a TMSC barrier to time-delay the movement of SARS-CoV-2 SP RBD-AuNPs, thus allowing the antigen to bind with the primary antibody more efficiently. This platform exhibited a 2.6-fold enhancement in the sensitivity and 9.1-fold improvement in the limit of detection (LOD) as compared with the conventional LFIA. In addition, this d-LFIA device was satisfactorily applied to accurate screening of COVID-19 patients.

The Detection of SARS-CoV2 Antigen in Wastewater Using an Automated Chemiluminescence Enzyme Immunoassay.

The SARS-CoV-2 virus, which is driving the current COVID-19 epidemic, has been detected in wastewater and is being utilized as a surveillance tool to establish an early warning system to aid in the management and prevention of future pandemics. qPCR is the method usually used to detect SARS-CoV-2 in wastewater. There has been no study using an immunoassay that is less laboratory-intensive than qPCR with a shorter turnaround time. Therefore, we aimed to evaluate the performance of an automated chemiluminescence enzyme immunoassay (CLEIA) for SARS-CoV-2 antigen in wastewater. The CLEIA assay achieved 100% sensitivity and 66.7% specificity in a field-captured wastewater sample compared to the gold standard RT-qPCR. Our early findings suggest that the SARS-CoV-2 antigen can be identified in wastewater samples using an automated CLEIA, reducing the turnaround time and improving the performance of SARS-CoV-2 wastewater monitoring during the pandemic.

A Prospective Study of Plasma and Bronchoalveolar Lavage Fluid CMV DNA Load Quantification for the Diagnosis and Outcome of CMV Pneumonitis in Immunocompromised Hosts.

BACKGROUND: Molecular testing has been utilized for cytomegalovirus (CMV) pneumonitis (CMVP) diagnosis, although its validity and optimal cut-off values remain limited. METHODS: A prospective study of CMVP diagnosis among immunocompromised patients was conducted by measuring quantitative CMV DNA polymerase chain reaction in plasma and bronchoalveolar lavage fluid (BALF). RESULTS: Forty-five adult immunocompromised patients were investigated. Thirty-two patients (71%) received immunosuppressive therapy. Eleven patients (24%) were confirmed to have CMVP. Of those, three and eight patients were classified as proven and probable CMVP, respectively. Median (IQR) plasma CMV DNA loads in CMVP and non-CMVP were 41,939 (4,424-122,608) and 0 (0-44) IU/mL, respectively (p<0.001). Median (IQR) BALF CMV DNA loads in CMVP and non-CMVP were 379,652 (163,800-1,254,000) and 0 (0-1,348) IU/mL, respectively (p<0.001). A significant correlation was observed between plasma and BALF CMV DNA loads (r=0.887, p<0.001). Plasma CMV DNA load of 831 IU/mL was established as a cut-off value for diagnosing CMVP (AUC 0.9987, sensitivity 100%, specificity 94.1%, positive predictive value 84.5%, negative predictive value 100%). CONCLUSIONS: A strongly positive correlation was observed between CMV DNA loads measured in plasma and BALF. CMV DNA load quantification could potentially assist in diagnosing CMVP in immunocompromised patients, although bronchoscopy remains encouraged for a definitive diagnosis.

Detectable Duration of Viable SARS-CoV-2, Total and Subgenomic SARS-CoV-2 RNA in Noncritically Ill COVID-19 Patients: a Prospective Cohort Study.

Determination of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infectivity is important in guiding the infection control and differentiating between reinfection and persistent viral RNA. Although viral culture is the gold standard to determine viral infectivity, the method is not practical. We studied the kinetics of SARS-CoV-2 total RNAs and subgenomic RNAs (sgRNAs) and their potential role as surrogate markers of viral infectivity. The kinetics of SARS-CoV-2 sgRNAs compared to those of the culture and total RNA shedding in a prospective cohort of patients diagnosed with coronavirus disease 2019 (COVID-19) were investigated. A total of 260 nasopharyngeal swabs from 36 patients were collected every other day after entering the study until the day of viral total RNA clearance, as measured by reverse transcription PCR (RT-PCR). Time to cessation of viral shedding was in order from shortest to longest: by viral culture, sgRNA RT-PCR, and total RNA RT-PCR. The median time (interquartile range) to negativity of viral culture, subgenomic N transcript, and N gene were 7 (5 to 9), 11 (9 to 16), and 18 (13 to 21) days, respectively (P < 0.001). Further analysis identified the receipt of steroid as the factors associated with longer duration of viral infectivity (hazard ratio, 3.28; 95% confidence interval, 1.02 to 10.61; P = 0.047). We propose the potential role of the detection of SARS-CoV-2 subgenomic RNA as the surrogate marker of viral infectivity. Patients with negative subgenomic N RNA RT-PCR could be considered for ending isolation. IMPORTANCE Our study, combined with existing evidence, suggests the feasibility of the use of subgenomic RNA RT-PCR as a surrogate marker for SARS-CoV-2 infectivity. The kinetics of SARS-CoV-2 subgenomic RNA should be further investigated in immunocompromised patients.

Target Enrichment Metagenomics Reveals Human Pegivirus-1 in Pediatric Hematopoietic Stem Cell Transplantation Recipients.

Human pegivirus-1 (HPgV-1) is a lymphotropic human virus, typically considered nonpathogenic, but its infection can sometimes cause persistent viremia both in immunocompetent and immunosuppressed individuals. In a viral discovery research program in hematopoietic stem cell transplant (HSCT) pediatric patients, HPgV-1 was detected in 3 out of 14 patients (21.4%) using a target enrichment next-generation sequencing method, and the presence of the viruses was confirmed by agent-specific qRT-PCR assays. For the first time in this patient cohort, complete genomes of HPgV-1 were acquired and characterized. Phylogenetic analyses indicated that two patients had HPgV-1 genotype 2 and one had HPgV-1 genotype 3. Intra-host genomic variations were described and discussed. Our results highlight the necessity to screen HSCT patients and blood and stem cell donors to reduce the potential risk of HPgV-1 transmission.

SARS-CoV-2 RT-PCR positivity of individuals subsequent to completing quarantine upon entry into a country during a transmission-free period.

BACKGROUND: During the current coronavirus disease 2019 (COVID-19) pandemic, many countries require travellers to undergo a reverse transcription-polymerase chain reaction (RT-PCR) testing for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) before travelling across borders. However, in persons having recovered from COVID-19, RT-PCR positivity can persist for an extended period. MATERIALS AND METHODS: We describe three cases who sought fit-to-fly certificates in Thailand during the period free of local transmission but were tested positive for RT-PCR for SARS-CoV-2. All had returned from a country with an active outbreak of COVID-19. Their clinical courses are described; positive nasopharyngeal swab samples were processed for viral isolation and whole-genome sequencing (WGS); and serology as well as neutralizing antibody were assessed. The contact tracing was carried out for determining evidence of indigenous transmission among close contacts of those three cases. RESULTS: All three cases were completely asymptomatic. Chest computerized tomography was not compatible with COVID-19 pneumonia; cell cultures failed to rescue replication-competent virus; WGS revealed fragmented viral genetic material from nasopharyngeal swab samples; and serological tests demonstrated stable levels of antibodies, together with the presence of neutralizing antibody, suggesting past infection with negligible transmission risk. Contact tracing identified no transmission in high-risk close contact individuals. CONCLUSION: RT-PCR positivity for SARS-CoV-2 might detect fragmented viral genome. Issuance of a travel certificate in these circumstances is problematic. Serology tests can help to define past infection. A practical acceptable set of guidelines for issuance of a COVID-19 safety travel certification is a necessity.