Viral persistence, reactivation, and mechanisms of long COVID.

The COVID-19 global pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has infected hundreds of millions of individuals. Following COVID-19 infection, a subset can develop a wide range of chronic symptoms affecting diverse organ systems referred to as post-acute sequelae of SARS-CoV-2 infection (PASC), also known as long COVID. A National Institutes of Health-sponsored initiative, RECOVER: Researching COVID to Enhance Recovery, has sought to understand the basis of long COVID in a large cohort. Given the range of symptoms that occur in long COVID, the mechanisms that may underlie these diverse symptoms may also be diverse. In this review, we focus on the emerging literature supporting the role(s) that viral persistence or reactivation of viruses may play in PASC. Persistence of SARS-CoV-2 RNA or antigens is reported in some organs, yet the mechanism by which they do so and how they may be associated with pathogenic immune responses is unclear. Understanding the mechanisms of persistence of RNA, antigen or other reactivated viruses and how they may relate to specific inflammatory responses that drive symptoms of PASC may provide a rationale for treatment.

Comparison of Reverse Transcription (RT)-Quantitative PCR and RT-Droplet Digital PCR for Detection of Genomic and Subgenomic SARS-CoV-2 RNA.

Most individuals acutely infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) exhibit mild symptoms. However, 10 to 20% of those infected develop long-term symptoms, referred to as post-coronavirus disease 2019 (COVID-19) condition (PCC). One hypothesis is that PCC might be exacerbated by viral persistence in tissue sanctuaries. Therefore, the accurate detection and quantification of SARS-CoV-2 are not only necessary for viral load monitoring but also crucial for detecting long-term viral persistence and determining whether viral replication is occurring in tissue reservoirs. In this study, the sensitivity and robustness of reverse transcription (RT)-droplet digital PCR (ddPCR) and RT-quantitative PCR (qPCR) techniques have been compared for the detection and quantification of SARS-CoV-2 genomic and subgenomic RNAs from oropharyngeal swabs taken from confirmed SARS-CoV-2-positive, SARS-CoV-2-exposed, and nonexposed individuals as well as from samples from mice infected with SARS-CoV-2. Our data demonstrated that both techniques presented equivalent results in the mid- and high-viral-load ranges. Additionally, RT-ddPCR was more sensitive than RT-qPCR in the low-viral-load range, allowing the accurate detection of positive results in individuals exposed to the virus. Overall, these data suggest that RT-ddPCR might be an alternative to RT-qPCR for detecting low viral loads in samples and for assessing viral persistence in samples from individuals with PCC. IMPORTANCE We developed one-step reverse transcription (RT)-droplet digital PCR (ddPCR) protocols to detect SARS-CoV-2 RNA and compared them to the gold-standard RT-quantitative PCR (RT-qPCR) method. RT-ddPCR was more sensitive than RT-qPCR in the low-viral-load range, while both techniques were equivalent in the mid- and high-viral-load ranges. Overall, these results suggest that RT-ddPCR might be a viable alternative to RT-qPCR when it comes to detecting low viral loads in samples, which is a highly relevant issue for determining viral persistence in as-yet-unknown tissue reservoirs in individuals suffering from post-COVID conditions or long COVID.

Rate of shed of SARS COV-2 viral RNA from COVID-19 cadavers.

BACKGROUND: At what rate does the RNA of SARS CoV-2 shed from cadavers? Although, there have been numerous studies which have demonstrated the persistence of the virus on dead bodies, there is a lack of conclusive evidence regarding the variation of viral RNA content in cadavers. This has led to a knowledge gap regarding the safe handling/management of COVID-19 decedents, posing a barrier in forensic investigations. METHODS: In this study, we report the presence of RNA of SARS CoV-2 by real time RT-PCR, in nasopharyngeal swabs collected after death from two groups of bodies - one who died due to COVID-19 and the other who died due to other diagnoses. A prospective study on 199 corpses, who had tested positive for COVID-19 ante-mortem, was conducted at a tertiary care center. RNA testing was conducted at different time intervals (T1-T5). RESULTS: 112(56.3%) died primarily due to COVID-19 and 87(43.7%) died due to other diagnoses. 144(72.4%) were male and 55(27.6%) were female. A total of 115 (57.8%) tested positive for COVID-19 after death at different time points. The mean age was 50.7 ± 18.9 years and the length of hospitalization ranged from 1 to 50 days with a mean of 9.2 ± 7.6 days. Realtime RT-PCR positivity of SARS CoV-2 RNA decreases with time. CONCLUSION: We observed that real time RT-PCR positivity, indicating viral RNA detection, decreases with time. Therefore, it is advisable to follow appropriate COVID-19 precautions to carry out scientific studies, medico-legal investigations and mortuary services on suspected/confirmed COVID-19 corpses.

Corrigendum: Case report: Persistence of residual antigen and RNA of the SARS-CoV-2 virus in tissues of two patients with long COVID.

[This corrects the article DOI: 10.3389/fimmu.2022.939989.].

Case report: Persistence of residual antigen and RNA of the SARS-CoV-2 virus in tissues of two patients with long COVID.

The World Health Organization has defined long COVID-19 (LC) as a condition that occurs in individuals with a history of SARS-CoV-2 infection who exhibit persistent symptoms after its acute phase that last for at least two months and cannot be explained by an alternative diagnosis. Since we had previously reported residual viral antigens in tissues of convalescent patients, we aimed to assess the presence of such antigens in long COVID tissues. Here, we established the presence of the residual virus in the appendix, skin, and breast tissues of 2 patients who exhibited LC symptoms 163 and 426 days after symptom onset. With multiplex immunohistochemistry, we detected viral nucleocapsid protein in all three tissues. The nucleocapsid protein was further observed to colocalize with macrophage marker CD68, suggesting that immune cells were direct targets of SARS-CoV-2. Additionally, using RNAscope, the presence of viral RNA was also detected. Our positive finding in the breast tissue is corroborated by the recent reports of immunocompromised patients experiencing LC symptoms and persistent viral replication. Overall, our findings and emerging LC studies raise the possibility that the gastrointestinal tract may function as a reservoir for SARS-CoV-2.

Chronic biopsy proven post-COVID myoendocarditis with SARS-Cov-2 persistence and high level of antiheart antibodies.

PURPOSE: To study the clinical signs and mechanisms (viral and autoimmune) of myoendocarditis in the long-term period after COronaVIrus Disease 2019 (COVID-19). METHODS: Fourteen patients (nine male, 50.1 ± 10.2 y.o.) with biopsy proven post-COVID myocarditis were observed. The diagnosis of COVID-19 was confirmed by IgG seroconversion. The average time of admission after COVID-19 was 5.5 [2; 10] months. An endomyocardial biopsy (EMB) of the right ventricle was obtained. The biopsy analysis included polymerase chain reaction diagnosis of viral infection, morphological, immunohistochemical (IHC) examination with antibodies to CD3, CD45, CD68, CD20, SARS-Cov-2 spike, and nucleocapsid antigens. Coronary atherosclerosis was ruled out in all patients over 40 years. RESULTS: The new cardiac symptoms (congestive heart failure 3-4 New York Heart Association class with severe right ventricular involvement, various rhythm, and conduction disturbances) appeared 1-5 months following COVID-19. Magnetic resonance imaging showed disseminated or focal subepicardial and intramyocardial late gadolinium enhancement, hyperemia, edema, and increased myocardial native T1 relaxation time. Antiheart antibodies levels were increased 3-4 times in 92.9% of patients. The mean left ventricular (LV) ejection fraction (EF) was 28% (24.5; 37.8). Active lymphocytic myocarditis was diagnosed in 12 patients, eosinophilic myocarditis in two patients. SARS-Cov-2 RNA was detected in 12 cases (85.7%), in association with parvovirus B19 DNA-in one. Three patients had also endocarditis (infective and nonbacterial, with parietal thrombosis). As a result of steroid and chronic heart failure therapy, the EF increased to 47% (37.5; 52.5). CONCLUSIONS: COVID-19 can lead to long-term severe post-COVID myoendocarditis, that is characterized by prolonged persistence of coronavirus in cardiomyocytes, endothelium, and macrophages (up to 18 months) in combination with high immune activity. Corticosteroids and anticoagulants should be considered as a treatment option of post-COVID myoendocarditis.

Identification of Distinct Immune Cell Subsets Associated With Asymptomatic Infection, Disease Severity, and Viral Persistence in COVID-19 Patients.

The cell-mediated protective and pathogenic immune responses to SARS-CoV-2 infection remain largely elusive. Here we identified 76 distinct cell subsets in the PBMC samples that were associated with various clinical presentations of COVID-19 using scRNA-seq technology coupled with a deep and comprehensive analysis of unique cell surface markers and differentially expressed genes. We revealed that (TRAV1-2+CD8+)MAIT cells and (NCAM1hiCD160+)NK cells significantly enriched in the asymptomatic subjects whereas (LAG3+CD160+CD8+)NKT cells increased in the symptomatic patients. We also observed that (CD68-CSF1R-IL1BhiCD14+)classical monocytes were positively correlated with the disease severity. Moreover, (CD33-HLA-DMA-CD14+)classical monocytes and (CLEC10A-S100A9lo)pDC were associated with the viral persistence. The GO and KEGG analyses identified enriched pathways related to immune responses, inflammation, and apoptosis. These findings may enhance our understanding of the immunopathogenesis of COVID-19 and help develop novel strategies against SARS-CoV-2 infection.

Sentinel Cards Provide Practical SARS-CoV-2 Monitoring in School Settings.

A promising approach to help students safely return to in person learning is through the application of sentinel cards for accurate high resolution environmental monitoring of SARS-CoV-2 traces indoors. Because SARS-CoV-2 RNA can persist for up to a week on several indoor surface materials, there is a need for increased temporal resolution to determine whether consecutive surface positives arise from new infection events or continue to report past events. Cleaning sentinel cards after sampling would provide the needed resolution but might interfere with assay performance. We tested the effect of three cleaning solutions (BZK wipes, Wet Wipes, RNase Away) at three different viral loads: "high" (4 × 104 GE/mL), "medium" (1 × 104 GE/mL), and "low" (2.5 × 103 GE/mL). RNase Away, chosen as a positive control, was the most effective cleaning solution on all three viral loads. Wet Wipes were found to be more effective than BZK wipes in the medium viral load condition. The low viral load condition was easily reset with all three cleaning solutions. These findings will enable temporal SARS-CoV-2 monitoring in indoor environments where transmission risk of the virus is high and the need to avoid individual-level sampling for privacy or compliance reasons exists. IMPORTANCE Because SARS-CoV-2, the virus that causes COVID-19, persists on surfaces, testing swabs taken from surfaces is useful as a monitoring tool. This approach is especially valuable in school settings, where there are cost and privacy concerns that are eliminated by taking a single sample from a classroom. However, the virus persists for days to weeks on surface samples, so it is impossible to tell whether positive detection events on consecutive days are a persistent signal or new infectious cases and therefore whether the positive individuals have been successfully removed from the classroom. We compare several methods for cleaning "sentinel cards" to show that this approach can be used to identify new SARS-CoV-2 signals day to day. The results are important for determining how to monitor classrooms and other indoor environments for SARS-CoV-2 virus.

Human Nasal Epithelial Cells Sustain Persistent SARS-CoV-2 Infection In Vitro, despite Eliciting a Prolonged Antiviral Response

The dynamics of SARS-CoV-2 infection in COVID-19 patients are highly variable, with a subset of patients demonstrating prolonged virus shedding, which poses a significant challenge for disease management and transmission control. In this study, the long-term dynamics of SARS-CoV-2 infection were investigated using a human well-differentiated nasal epithelial cell (NEC) model of infection. NECs were observed to release SARS-CoV-2 virus onto the apical surface for up to 28 days post-infection (dpi), further corroborated by viral antigen staining. Single-cell transcriptome sequencing (sc-seq) was utilized to explore the host response from infected NECs after short-term (3-dpi) and long-term (28-dpi) infection. We identified a unique population of cells harboring high viral loads present at both 3 and 28 dpi, characterized by expression of cell stress-related genes DDIT3 and ATF3 and enriched for genes involved in tumor necrosis factor alpha (TNF-alpha) signaling and apoptosis. Remarkably, this sc-seq analysis revealed an antiviral gene signature within all NEC cell types even at 28 dpi. We demonstrate increased replication of basal cells, absence of widespread cell death within the epithelial monolayer, and the ability of SARS-CoV-2 to replicate despite a continuous interferon response as factors likely contributing to SARS-CoV-2 persistence. This study provides a model system for development of therapeutics aimed at improving viral clearance in immunocompromised patients and implies a crucial role for immune cells in mediating viral clearance from infected epithelia. IMPORTANCE Increasing medical attention has been drawn to the persistence of symptoms (long-COVID syndrome) or live virus shedding from subsets of COVID-19 patients weeks to months after the initial onset of symptoms. In vitro approaches to model viral or symptom persistence are needed to fully dissect the complex and likely varied mechanisms underlying these clinical observations. We show that in vitro differentiated human NECs are persistently infected with SARS-CoV-2 for up to 28 dpi. This viral replication occurred despite the presence of an antiviral gene signature across all NEC cell types even at 28 dpi. This indicates that epithelial cell intrinsic antiviral responses are insufficient for the clearance of SARS-CoV-Z implying an essential role for tissue-resident and infiltrating immune cells for eventual viral clearance from infected airway tissue in COVID-19 patients.

Human Nasal Epithelial Cells Sustain Persistent SARS-CoV-2 Infection In Vitro, despite Eliciting a Prolonged Antiviral Response.

The dynamics of SARS-CoV-2 infection in COVID-19 patients are highly variable, with a subset of patients demonstrating prolonged virus shedding, which poses a significant challenge for disease management and transmission control. In this study, the long-term dynamics of SARS-CoV-2 infection were investigated using a human well-differentiated nasal epithelial cell (NEC) model of infection. NECs were observed to release SARS-CoV-2 virus onto the apical surface for up to 28 days postinfection (dpi), further corroborated by viral antigen staining. Single-cell transcriptome sequencing (sc-seq) was utilized to explore the host response from infected NECs after short-term (3-dpi) and long-term (28-dpi) infection. We identified a unique population of cells harboring high viral loads present at both 3 and 28 dpi, characterized by expression of cell stress-related genes DDIT3 and ATF3 and enriched for genes involved in tumor necrosis factor alpha (TNF-α) signaling and apoptosis. Remarkably, this sc-seq analysis revealed an antiviral gene signature within all NEC cell types even at 28 dpi. We demonstrate increased replication of basal cells, absence of widespread cell death within the epithelial monolayer, and the ability of SARS-CoV-2 to replicate despite a continuous interferon response as factors likely contributing to SARS-CoV-2 persistence. This study provides a model system for development of therapeutics aimed at improving viral clearance in immunocompromised patients and implies a crucial role for immune cells in mediating viral clearance from infected epithelia. IMPORTANCE Increasing medical attention has been drawn to the persistence of symptoms (long-COVID syndrome) or live virus shedding from subsets of COVID-19 patients weeks to months after the initial onset of symptoms. In vitro approaches to model viral or symptom persistence are needed to fully dissect the complex and likely varied mechanisms underlying these clinical observations. We show that in vitro differentiated human NECs are persistently infected with SARS-CoV-2 for up to 28 dpi. This viral replication occurred despite the presence of an antiviral gene signature across all NEC cell types even at 28 dpi. This indicates that epithelial cell intrinsic antiviral responses are insufficient for the clearance of SARS-CoV-2, implying an essential role for tissue-resident and infiltrating immune cells for eventual viral clearance from infected airway tissue in COVID-19 patients.

Low-molecular-weight heparin use in coronavirus disease 2019 is associated with curtailed viral persistence: a retrospective multicentre observational study.

AIMS: Anticoagulation was associated with improved survival of hospitalized coronavirus disease 2019 (COVID-19) patients in large-scale studies. Yet, the development of COVID-19-associated coagulopathy (CAC) and the mechanism responsible for improved survival of anticoagulated patients with COVID-19 remain largely elusive. This investigation aimed to explore the effects of anticoagulation and low-molecular-weight heparin (LMWH) in particular on patient outcome, CAC development, thromboinflammation, cell death, and viral persistence. METHODS AND RESULTS: Data of 586 hospitalized COVID-19 patients from three different regions of Austria were evaluated retrospectively. Of these, 419 (71.5%) patients received LMWH and 62 (10.5%) received non-vitamin-K oral anticoagulants (NOACs) during hospitalization. Plasma was collected at different time points in a subset of 106 patients in order to evaluate markers of thromboinflammation (H3Cit-DNA) and the cell death marker cell-free DNA (cfDNA). Use of LMWH was associated with improved survival upon multivariable Cox regression (hazard ratio = 0.561, 95% confidence interval: 0.348-0.906). Interestingly, neither LMWH nor NOAC was associated with attenuation of D-dimer increase over time, or thromboinflammation. In contrast, anticoagulation was associated with a decrease in cfDNA during hospitalization, and curtailed viral persistence was observed in patients using LMWH leading to a 4-day reduction of virus positivity upon quantitative polymerase chain reaction [13 (interquartile range: 6-24) vs. 9 (interquartile range: 5-16) days, P = 0.009]. CONCLUSION: Time courses of haemostatic and thromboinflammatory biomarkers were similar in patients with and without LMWH, indicating either no effects of LMWH on haemostasis or that LMWH reduced hypercoagulability to levels of patients without LMWH. Nonetheless, anticoagulation with LMWH was associated with reduced mortality, improved markers of cell death, and curtailed viral persistence, indicating potential beneficial effects of LMWH beyond haemostasis, which encourages use of LMWH in COVID-19 patients without contraindications.

Findings from Studies Are Congruent with Obesity Having a Viral Origin, but What about Obesity-Related NAFLD?

Infection has recently started receiving greater attention as an unusual causative/inducing factor of obesity. Indeed, the biological plausibility of infectobesity includes direct roles of some viruses to reprogram host metabolism toward a more lipogenic and adipogenic status. Furthermore, the probability that humans may exchange microbiota components (virome/virobiota) points out that the altered response of IFN and other cytokines, which surfaces as a central mechanism for adipogenesis and obesity-associated immune suppression, is due to the fact that gut microbiota uphold intrinsic IFN signaling. Last but not least, the adaptation of both host immune and metabolic system under persistent viral infections play a central role in these phenomena. We hereby discuss the possible link between adenovirus and obesity-related nonalcoholic fatty liver disease (NAFLD). The mechanisms of adenovirus-36 (Ad-36) involvement in hepatic steatosis/NAFLD consist in reducing leptin gene expression and insulin sensitivity, augmenting glucose uptake, activating the lipogenic and pro-inflammatory pathways in adipose tissue, and increasing the level of macrophage chemoattractant protein-1, all of these ultimately leading to chronic inflammation and altered lipid metabolism. Moreover, by reducing leptin expression and secretion Ad-36 may have in turn an obesogenic effect through increased food intake or decreased energy expenditure via altered fat metabolism. Finally, Ad-36 is involved in upregulation of cAMP, phosphatidylinositol 3-kinase, and p38 signaling pathways, downregulation of Wnt10b expression, increased expression of CCAAT/enhancer binding protein-beta, and peroxisome proliferator-activated receptor gamma 2 with consequential lipid accumulation.

SARS-CoV-2 persistence in immunocompromised children.

OBJECTIVES: We evaluated the length of time immunocompromised children (ICC) remain positive for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), identified factors associated with viral persistence, and determined cycle threshold (CT ) values of children with viral persistence as a surrogate of viral load. METHODS: We conducted a retrospective cohort study of ICC at a pediatric hospital from March 2020 to March 2021. Immunocompromised status was defined as primary, secondary, or acquired due to medical comorbidities/immunosuppressive treatment. The primary outcome was time to first of two consecutive negative SARS-CoV-2 polymerase chain reaction (PCR) tests at least 24 hours apart. Testing of sequential clinical specimens from the same subject was conducted using the Centers for Disease Control (CDC) 2019-nCoV real-time reverse transcriptase (RT)-PCR Diagnostic Panel assay. Descriptive statistics, Kaplan-Meier curve median event times and log-rank tests were used to compare outcomes between groups. RESULTS: Ninety-one children met inclusion criteria. Median age was 15.5 years (interquartile range [IQR] 8-18), 64% were male, 58% were White, and 43% were Hispanic/Latinx. Most (67%) were tested in outpatient settings and 58% were asymptomatic. The median time to two negative tests was 42 days (IQR 25.0-55.0), with no differences in median time by illness presentation or level of immunosuppression. Seven children had more than one sample available for repeat testing, and five of seven (71%) children had initial CT values of <30 (moderate to high viral load); four children had CT values of <30, 3-4 weeks later, suggesting persistent moderate to high viral loads. CONCLUSIONS: Most ICC with SARS-CoV-2 infection had mild disease, with prolonged viral persistence >6 weeks and moderate to high viral load.

Relative COVID-19 Viral Persistence and Antibody Kinetics.

A total of 15 RT-PCR confirmed COVID-19 patients were admitted to our hospital during the in-itial outbreak in Taiwan. The average time of virus clearance was delayed in seven patients, 24.14 ± 4.33 days compared to 10.25 ± 0.56 days post-symptom onset (PSO) in the other eight pa-tients. There was strong antibody response in patients with viral persistence at the pharynx, with peak values of serum antibody 677.2 ± 217.8 vs. 76.70 ± 32.11 in patients with delayed versus rapid virus clearance. The patients with delayed viral clearance had excessive antibodies of compromised quality in an early stage with the delay in peak virus neutralization efficacy, 34.14 ± 7.15 versus 12.50 ± 2.35 days PSO in patients with rapid virus clearance. Weak antibody re-sponse of patients with rapid viral clearance was also effective, with substantial and comparable neutralization efficacy, 35.70 ± 8.78 versus 41.37 ± 11.49 of patients with delayed virus clearance. Human Cytokine 48-Plex Screening of the serial sera samples revealed elevated concentrations of proinflammatory cytokines and chemokines in a deceased patient with delayed virus clear-ance and severe disease. The levels were comparatively less in the other two patients who suf-fered from severe disease but eventually survived.

SARS-CoV-2 Viral Persistence Based on Cycle Threshold Value and Liver Injury in Patients With COVID-19.

BACKGROUND: Liver injury in patients with coronavirus disease 2019 (COVID-19) is common and prognostic. Direct viral tropism of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) for angiotensin-converting enzyme 2 receptors in hepatocytes may be one of the mechanisms of liver injury. We aimed to determine the role of viral persistence of SARS-CoV-2, based on cycle threshold (Ct) value, in liver injury in COVID-19. METHODS: This was a territory-wide retrospective cohort study of all public hospitals in Hong Kong. Laboratory-confirmed COVID-19 was identified. Serial liver biochemistries and Ct values of SARS-CoV-2 RNA were analyzed. RESULTS: We identified 7622 COVID-19 patients (mean age, 47 years; 48.2% male) diagnosed from March 24 to January 1, 2021, who had serial liver biochemistries and Ct values. A total of 1363 (17.9%) COVID-19 patients had alanine transferase (ALT)/aspartate aminotransferase (AST) elevations with 2 temporal patterns-early (within first 14 days of symptom onset) and late (>14 days from symptom onset). COVID-19 patients with ALT/AST elevations had a lower Ct value at admission (23 vs 25; P < .001), day 5 (24 vs 26; P < .001), and day 20 (31 vs 32; P < .001) after admission, compared with those without ALT/AST elevations. COVID-19 patients with ALT/AST elevations had a longer duration from first positive to first negative reverse transcription polymerase chain reaction test for SARS-CoV-2 (13 vs 9 days; P < .001). ALT/AST elevation and presence of diabetes were independent risk factors of viral persistence. CONCLUSIONS: Liver injury in COVID-19 is linked to a higher SARS-CoV-2 viral load during the early phase of infection, signifying a possible direct viral injury to the liver. Prolonged viral persistence of SARS-CoV-2 is associated with liver injury.

Insights into SARS-CoV-2 Persistence and Its Relevance.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), continues to wreak havoc, threatening the public health services and imposing economic collapse worldwide. Tailoring public health responses to the SARS-CoV-2 pandemic depends on understanding the mechanism of viral replication, disease pathogenesis, accurately identifying acute infections, and mapping the spreading risk of hotspots across the globe. However, effective identification and isolation of persons with asymptomatic and mild SARS-CoV-2 infections remain the major obstacles to efforts in controlling the SARS-CoV-2 spread and hence the pandemic. Understanding the mechanism of persistent viral shedding, reinfection, and the post-acute sequalae of SARS-CoV-2 infection (PASC) is crucial in our efforts to combat the pandemic and provide better care and rehabilitation to survivors. Here, we present a living literature review (January 2020 through 15 March 2021) on SARS-CoV-2 viral persistence, reinfection, and PASC. We also highlight potential areas of research to uncover putative links between viral persistence, intra-host evolution, host immune status, and protective immunity to guide and direct future basic science and clinical research priorities.

SARS-CoV-2 Persistent Viral Shedding in the Context of Hydroxychloroquine-Azithromycin Treatment.

SARS-CoV-2 nasopharyngeal shedding contributes to the spread of the COVID-19 epidemic. Among 3271 COVID-19 patients treated at the Hospital University Institute Méditerranée Infection, Marseille, France from 3 March to 27 April 2020, tested at least twice by qRT-PCR, the median SARS-CoV-2 nasopharyngeal shedding duration was 6 days (range 2-54 days). Compared with short shedders (qRT-PCR positivity < 10 days), 34 (1.04%) persistent shedders (qRT-PCR positivity ≥ 17 days; mean ± SD: 23.3 ± 3.8 days) were significantly older, with associated comorbidities, exhibiting lymphopenia, eosinopenia, increased D-dimer and increased troponin (p < 0.05), and were hospitalized in intensive care unit in 17.7% vs. 1.1% of cases (p < 0.0001). Viral culture was positive in six persistent shedders after day 10, including in one patient after day 17, and no viral co-pathogen was detected in 33 tested patients. Persistent shedders received azithromycin plus hydroxychloroquine ≥ 3 days in 26/34 (76.5%) patients, a figure significantly lower than in short shedders (86.6%) (p = 0.042). Accordingly, mortality was 14.7% vs. 0.5% (p < 0.0001). Persistent shedding was significantly associated with persistent dyspnea and anosmia/ageusia (p < 0.05). In the context of COVID-19 treatment, including treatment with azithromycin plus hydroxychloroquine, the persistence of SARS-CoV-2 nasopharyngeal shedding was a rare event, most frequently encountered in elderly patients with comorbidities and lacking azithromycin plus hydroxychloroquine treatment.

Viral infections and implications for male reproductive health.

Viral infections have haunted humankind since times immemorial. Overpopulation, globalization, and extensive deforestation have created an ideal environment for a viral spread with unknown and multiple shedding routes. Many viruses can infect the male reproductive tract, with potential adverse consequences to male reproductive health, including infertility and cancer. Moreover, some genital tract viral infections can be sexually transmitted, potentially impacting the resulting offspring's health. We have summarized the evidence concerning the presence and adverse effects of the relevant viruses on the reproductive tract (mumps virus, human immunodeficiency virus, herpes virus, human papillomavirus, hepatitis B and C viruses, Ebola virus, Zika virus, influenza virus, and coronaviruses), their routes of infection, target organs and cells, prevalence and pattern of virus shedding in semen, as well as diagnosis/testing and treatment strategies. The pathophysiological understanding in the male genital tract is essential to assess its clinical impact on male reproductive health and guide future research.

Persistent Viral Presence Determines the Clinical Course of the Disease in COVID-19.

BACKGROUND: The clinical management of coronavirus disease 2019 (COVID-19) is dependent on understanding the underlying factors that contribute to the disease severity. In the absence of effective antiviral therapies, other host immunomodulatory therapies such as targeting inflammatory response are currently being used without clear evidence of their effectiveness. Because inflammation is an essential component of host antiviral mechanisms, therapies targeting inflammation may adversely affect viral clearance and disease outcome. OBJECTIVE: To understand whether the persistent presence of the virus is a key determinant in the disease severity during COVID-19 and to determine whether the viral reactivation in some patients is associated with infectious viral particles. METHODS: The data for patients were available including the onset of the disease, duration of viral persistence, measurements of inflammatory markers such as IL-6 and C-reactive protein, chest imaging, disease symptoms, and their durations among others. Follow-up tests were performed to determine whether the viral negative status persists after their recovery. RESULTS: Our data show that patients with persistent viral presence (>16 days) have more severe disease outcomes including extensive lung involvement and requirement of respiratory support. Two patients who died of COVID-19 were virus-positive at the time of their death. Four patients demonstrated virus-positive status on the follow-up tests, and these patient samples were sent to viral culture facility where virus culture could not be established. CONCLUSIONS: These data suggest that viral persistence is the key determining factor of the disease severity. Therapies that may impair the viral clearance may impair the host recovery from COVID-19.

Gastrointestinal Manifestations of SARS-CoV-2 Infection and Virus Load in Fecal Samples From a Hong Kong Cohort: Systematic Review and Meta-analysis.

BACKGROUND & AIMS: Infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes coronavirus disease 2019 (COVID-19), which has been characterized by fever, respiratory, and gastrointestinal symptoms as well as shedding of virus RNA into feces. We performed a systematic review and meta-analysis of published gastrointestinal symptoms and detection of virus in stool and also summarized data from a cohort of patients with COVID-19 in Hong Kong. METHODS: We collected data from the cohort of patients with COVID-19 in Hong Kong (N = 59; diagnosis from February 2 through February 29, 2020),and searched PubMed, Embase, Cochrane, and 3 Chinese databases through March 11, 2020, according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. We analyzed pooled data on the prevalence of overall and individual gastrointestinal symptoms (loss of appetite, nausea, vomiting, diarrhea, and abdominal pain or discomfort) using a random effects model. RESULTS: Among the 59 patients with COVID-19 in Hong Kong, 15 patients (25.4%) had gastrointestinal symptoms, and 9 patients (15.3%) had stool that tested positive for virus RNA. Stool viral RNA was detected in 38.5% and 8.7% among those with and without diarrhea, respectively (P = .02). The median fecal viral load was 5.1 log10 copies per milliliter in patients with diarrhea vs 3.9 log10 copies per milliliter in patients without diarrhea (P = .06). In a meta-analysis of 60 studies comprising 4243 patients, the pooled prevalence of all gastrointestinal symptoms was 17.6% (95% confidence interval [CI], 12.3-24.5); 11.8% of patients with nonsevere COVID-19 had gastrointestinal symptoms (95% CI, 4.1-29.1), and 17.1% of patients with severe COVID-19 had gastrointestinal symptoms (95% CI, 6.9-36.7). In the meta-analysis, the pooled prevalence of stool samples that were positive for virus RNA was 48.1% (95% CI, 38.3-57.9); of these samples, 70.3% of those collected after loss of virus from respiratory specimens tested positive for the virus (95% CI, 49.6-85.1). CONCLUSIONS: In an analysis of data from the Hong Kong cohort of patients with COVID-19 and a meta-analysis of findings from publications, we found that 17.6% of patients with COVID-19 had gastrointestinal symptoms. Virus RNA was detected in stool samples from 48.1% patients, even in stool collected after respiratory samples had negative test results. Health care workers should therefore exercise caution in collecting fecal samples or performing endoscopic procedures in patients with COVID-19, even during patient recovery.