CRISPR/Cas9-mediated genome editing of the thymidine kinase gene in a clinical HSV-1 isolate identifies F289S as novel acyclovir-resistant mutation.

Herpes simplex virus type 1 (HSV-1) is a neurotropic alphaherpesvirus that establishes a lifelong infection in sensory neurons of infected individuals, accompanied with intermittent reactivation of latent virus causing (a)symptomatic virus shedding. Whereas acyclovir (ACV) is a safe and highly effective antiviral to treat HSV-1 infections, long-term usage can lead to emergence of ACV resistant (ACVR) HSV-1 and subsequently ACV refractory disease. Here, we isolated an HSV-1 strain from a patient with reactivated herpetic eye disease that did not respond to ACV treatment. The isolate carried a novel non-synonymous F289S mutation in the viral UL23 gene encoding the thymidine kinase (TK) protein. Because ACV needs conversion by viral TK and subsequently cellular kinases to inhibit HSV-1 replication, the UL23 gene is commonly mutated in ACVR HSV-1 strains. The potential role of the F289S mutation causing ACVR was investigated using CRISPR/Cas9-mediated HSV-1 genome editing. Reverting the F289S mutation in the original clinical isolate to the wild-type sequence S289F resulted in an ACV-sensitive (ACVS) phenotype, and introduction of the F289S substitution in an ACVS HSV-1 reference strain led to an ACVR phenotype. In summary, we identified a new HSV-1 TK mutation in the eye of a patient with ACV refractory herpetic eye disease, which was identified as the causative ACVR mutation with the aid of CRISPR/Cas9-mediated genome engineering technology. Direct editing of clinical HSV-1 isolates by CRISPR/Cas9 is a powerful strategy to assess whether single residue substitutions are causative to a clinical ACVR phenotype.

COVID-19 epidemiology and performance of the WHO clinical algorithm to diagnose COVID-19 in people with HIV from Ukraine.

BACKGROUND: The two main objectives were to evaluate the COVID-19 point prevalence and the test performance of the WHO case definition to diagnose COVID-19 clinically in people with HIV in West Ukraine. METHODS: Multicenter cross-sectional study in Lviv, Ukraine, from October 2020-November 2021. COVID-19 unvaccinated people with HIV were included regardless of COVID-19 symptoms at routine clinical visits and had standardized medical, quality of life (EQ(5D)) and SARS-CoV-2 serology assessments. Reported symptoms indicating potential COVID-19 events at inclusion or between March 2020 and inclusion were classified by the WHO case definition as suspected, probable or confirmed. A clinical COVID-19 case was defined as being SARS-CoV-2 seropositive with at least a suspected COVID-19 according to the WHO case definition. The primary endpoints were the clinical COVID-19 prevalence and the test characteristics of the WHO case definition with SARS-CoV-2 serology as reference. (Clinicaltrials.gov:NCT04711954). RESULTS: The 971 included people with HIV were median 40 years, 38.8% women, 44.8% had prior AIDS, and 55.6% had comorbidities. SARS-CoV-2 seroprevalence was 40.1% (95%CI:37.0-43.1) and 20.5% (95%CI:18.0-23.1) had clinical COVID-19 median 4 months (IQR:2-7) before inclusion. Clinical COVID-19 occurred less frequently in people with HIV with tuberculosis history, injecting drug use, CD4+ T-cells <200/mL and unemployment. The quality of life was not impacted after COVID-19. An at least probable COVID-19 classification by the WHO case definition had 44.1% sensitivity (95%CI:38.7-49.7), 85.2% specificity (95%CI:81.5-88.4), 66.6% positive predictive value (95%CI:59.8-73.0) and 69.5% negative predictive value (95%CI:65.5-73.3) to diagnose COVID-19. CONCLUSIONS: COVID-19 unvaccinated people with HIV from Ukraine had a significant COVID-19 rate and using the WHO case definition had insufficient diagnostic accuracy to diagnose these cases. The lower burden in vulnerable people with HIV was unexpected but might reflect a shielding effect.

Case Report: Late Reactivation of Herpes B Virus After a Monkey Bite: A Case of Severe Meningoencephalitis.

Macacine alphaherpesvirus 1, also known as herpes B virus (BV), is an alphaherpesvirus endemic to several macaque species, capable of causing zoonotic infections in humans, with high mortality rates. Evidence of reactivation in humans has rarely been reported. Here we depict a case of BV reactivation after 54 years, leading to severe meningoencephalitis. This case supports the use of antiviral prophylaxis in patients surviving a confirmed BV central nervous system infection. We sequenced DNA from BV obtained from the patient's cerebrospinal fluid. Phylogenetic analysis showed significant divergence in the clustering of this particular BV strain compared with other known BVs. Therefore, additional efforts are needed to obtain a broader sequence landscape from BVs circulating in monkeys.

Current Perspectives on the Management of Herpesvirus Infections in Solid Organ Transplant Recipients.

Solid organ transplant recipients (SOTRs) are at high risk of human herpesvirus (HHV)-related morbidity and mortality due to the use of immunosuppressive therapy. We aim to increase awareness and understanding of HHV disease burden in SOTRs by providing an overview of current prevention and management strategies as described in the literature and guidelines. We discuss challenges in both prevention and treatment as well as future perspectives.

The BAF complex inhibitor pyrimethamine reverses HIV-1 latency in people with HIV-1 on antiretroviral therapy.

Reactivation of the latent HIV-1 reservoir is a first step toward triggering reservoir decay. Here, we investigated the impact of the BAF complex inhibitor pyrimethamine on the reservoir of people living with HIV-1 (PLWH). Twenty-eight PLWH on suppressive antiretroviral therapy were randomized (1:1:1:1 ratio) to receive pyrimethamine, valproic acid, both, or no intervention for 14 days. The primary end point was change in cell-associated unspliced (CA US) HIV-1 RNA at days 0 and 14. We observed a rapid, modest, and significant increase in (CA US) HIV-1 RNA in response to pyrimethamine exposure, which persisted throughout treatment and follow-up. Valproic acid treatment alone did not increase (CA US) HIV-1 RNA or augment the effect of pyrimethamine. Pyrimethamine treatment did not result in a reduction in the size of the inducible reservoir. These data demonstrate that the licensed drug pyrimethamine can be repurposed as a BAF complex inhibitor to reverse HIV-1 latency in vivo in PLWH, substantiating its potential advancement in clinical studies.

Emergence and Potential Extinction of Genetic Lineages of Human Metapneumovirus between 2005 and 2021.

Human metapneumovirus (HMPV) is one of the leading causes of respiratory illness (RI), primarily in infants. Worldwide, two genetic lineages (A and B) of HMPV are circulating that are antigenically distinct and can each be further divided into genetic sublineages. Surveillance combined with large-scale whole-genome sequencing studies of HMPV are scarce but would help to identify viral evolutionary dynamics. Here, we analyzed 130 whole HMPV genome sequences obtained from samples collected from individuals hospitalized with RI and partial fusion (n = 144) and attachment (n = 123) protein gene sequences obtained from samples collected from patients with RI visiting general practitioners between 2005 and 2021 in the Netherlands. Phylogenetic analyses demonstrated that HMPV continued to group in the four sublineages described in 2004 (A1, A2, B1, and B2). However, one sublineage (A1) was no longer detected in the Netherlands after 2006, while the others continued to evolve. No differences were observed in dominant (sub)lineages between samples obtained from patients with RI being hospitalized and those consulting general practitioners. In both populations, viruses of lineage A2 carrying a 180-nucleotide or 111-nucleotide duplication in the attachment protein gene became the most frequently detected genotypes. In the past, different names for the newly energing lineages have been proposed, demonstrating the need for a consistent naming convention. Here, criteria are proposed for the designation of new genetic lineages to aid in moving toward a systematic HMPV classification. IMPORTANCE Human metapneumovirus (HMPV) is one of the major causative agents of human respiratory tract infections. Monitoring of virus evolution could aid toward the development of new antiviral treatments or vaccine designs. Here, we studied HMPV evolution between 2005 and 2021, with viruses obtained from samples collected from hospitalized individuals and patients with respiratory infections consulting general practitioners. Phylogenetic analyses demonstrated that HMPV continued to group in the four previously described sublineages (A1, A2, B1, and B2). However, one sublineage (A1) was no longer detected after 2006, while the others continued to evolve. No differences were observed in dominant (sub)lineages between patients being hospitalized and those consulting general practitioners. In both populations, viruses of lineage A2 carrying a 180-nucleotide or 111-nucleotide duplication in the attachment protein gene became the most frequently detected genotypes. These data were used to propose criteria for the designation of new genetic lineages to aid toward a systematic HMPV classification.

HIV transmission among acutely infected participants of a Dutch cohort study 2015-2021 is not associated with large, clustered outbreaks.

OBJECTIVE: Timely identification of acute or early HIV infection (AEHI) is important to help prevent onward transmission, and understanding the number of secondary infections resulting from individuals with AEHI is key to planning HIV prevention services and case finding. DESIGN: We performed a phylogenetic investigation of a dense sample of individuals with AEHI who took part in the Netherlands Cohort Study on Acute HIV infection (NOVA) in the Netherlands during 2015-2021. METHODS: Transmission clusters were identified using phylogenetic analyses based on HIV pol sequences. The Tamura-Nei model was used to estimate genetic distance. A number of 1000 bootstraps was used to check the reliability of clustering using maximum likelihood. A cluster was defined as having a bootstrap value of at least 95% and a genetic distance of at most 1.5%. Sensitivity analyses using different values for the bootstrap and genetic distance were performed to study the reproducibility of the clustering. RESULTS: Of the 156 participants included in NOVA between July 2015 and April 2021, 134 individuals for whom baseline characteristics and genotypic resistance data at baseline were available could be included. We identified 10 clusters, but the majority of persons (111/134) were not part of a cluster, suggesting mainly independent transmission events. CONCLUSION: Mainly independent transmission events among a study population consisting predominantly of MSM in a low-incidence high-resource setting is likely the result of active AEHI case finding and direct start of treatment, and the roll-out over recent years of preventive measures such as preexposure prophylaxis.

Clinical Characteristics and Outcomes of Immunocompromised Patients With Coronavirus Disease 2019 Caused by the Omicron Variant: A Prospective, Observational Study.

BACKGROUND: Illness after infection with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron variant is less severe compared with previous variants. Data on the disease burden in immunocompromised patients are lacking. We investigated the clinical characteristics and outcomes of immunocompromised patients with coronavirus disease 2019 (COVID-19) caused by Omicron. METHODS: Organ transplant recipients, patients on anti-CD20 therapy, and allogenic hematopoietic stem cell transplantation recipients infected with the Omicron variant were included. Characteristics of consenting patients were collected and patients were contacted regularly until symptom resolution. To identify possible risk factors for hospitalization, a univariate logistic analysis was performed. RESULTS: 114 consecutive immunocompromised patients were enrolled. Eighty-nine percent had previously received 3 mRNA vaccinations. While only 1 patient died, 23 (20%) were hospitalized for a median of 11 days. A low SARS-CoV-2 immunoglobulin G (IgG) antibody response (<300 BAU [binding antibody units]/mL) at diagnosis, being older, being a lung transplant recipient, having more comorbidities, and having a higher frailty score were associated with hospital admission (all P < .01). At the end of follow-up, 25% had still not fully recovered. Of the 23 hospitalized patients, 70% had a negative and 92% had a low IgG (<300 BAU/mL) antibody response at admission. Sotrovimab was administered to 17 of these patients, and 1 died. CONCLUSIONS: While the mortality in immunocompromised patients infected with Omicron was low, hospital admission was frequent and the duration of symptoms often prolonged. In addition to vaccination, other interventions are needed to limit the morbidity from COVID-19 in immunocompromised patients.

HIV-1 resistance against dolutegravir fluctuates rapidly alongside erratic treatment adherence: a case report.

OBJECTIVES: We report a case of incomplete HIV-1 suppression on a dolutegravir, lamivudine, and abacavir single-tablet regimen with the emergence of the H51Y and G118R integrase resistance mutations. METHODS: Integrase sequencing was performed retrospectively by Sanger and next-generation sequencing. Rates of emergence and decline of resistance mutations were calculated using next-generation sequencing data. Dolutegravir plasma concentrations were measured by ultra-performance liquid chromatography-tandem mass spectrometry. The effects of H51Y and G118R on infectivity, fitness, and susceptibility to dolutegravir were quantified using cell-based assays. RESULTS: During periods of non-adherence to treatment, mutations were retrospectively documented only by next-generation sequencing. Misdiagnosis by Sanger sequencing was caused by the rapid decline of mutant strains within the retroviral population. This observation was also true for a M184V lamivudine-resistant reverse transcriptase mutation found in association with integrase mutations on single HIV genomes. Resistance rebound upon treatment re-initiation was swift (>8000 copies per day). Next-generation sequencing indicated cumulative adherence to treatment. Compared to WT HIV-1, relative infectivity was 73%, 38%, and 43%; relative fitness was 100%, 35%, and 10% for H51Y, G118R, and H51Y+G118R viruses, respectively. H51Y did not change the susceptibility to dolutegravir, but G188R and H51Y+G118R conferred 7- and 28-fold resistance, respectively. CONCLUSION: This case illustrates how poorly-fit drug-resistant viruses wax and wane alongside erratic treatment adherence and are easily misdiagnosed by Sanger sequencing. We recommend next-generation sequencing to improve the clinical management of incomplete virological suppression with dolutegravir.

Clinical and In Vitro Evidence Favoring Immunoglobulin Treatment of a Chronic Norovirus Infection in a Patient With Common Variable Immunodeficiency.

BACKGROUND: Immunocompromised individuals can become chronically infected with norovirus, but effective antiviral therapies are not yet available. METHODS: Treatments with nitazoxanide, ribavirin, interferon alpha-2a, and nasoduodenally administered immunoglobulins were evaluated sequentially in an immunocompromised patient chronically infected with norovirus. In support, these components were also applied to measure norovirus inhibition in intestinal enteroid cultures in vitro. Viral RNA levels were determined in fecal and plasma samples during each treatment and viral genomes were sequenced. RESULTS: None of the antivirals resulted in a reduction of viral RNA levels in feces or plasma. However, during ribavirin treatment, there was an increased accumulation of virus genome mutations. In vitro, an effect of interferon alpha-2a on virus replication was observed and a genetically related strain was neutralized effectively in vitro using immunoglobulins and post-norovirus-infection antiserum. In agreement, after administration of immunoglobulins, the patient cleared the infection. CONCLUSIONS: Intestinal enteroid cultures provide a relevant system to evaluate antivirals and the neutralizing potential of immunoglobulins. We successfully treated a chronically infected patient with immunoglobulins, despite varying results reported by others. This case study provides in-depth, multifaceted exploration of norovirus treatment that can be used as a guidance for further research towards norovirus treatments.

From more testing to smart testing: data-guided SARS-CoV-2 testing choices, the Netherlands, May to September 2020.

BackgroundSARS-CoV-2 RT-PCR assays are more sensitive than rapid antigen detection assays (RDT) and can detect viral RNA even after an individual is no longer infectious. RDT can reduce the time to test and the results might better correlate with infectiousness.AimWe assessed the ability of five RDT to identify infectious COVID-19 cases and systematically recorded the turnaround time of RT-PCR testing.MethodsSensitivity of RDT was determined using a serially diluted SARS-CoV-2 stock with known viral RNA concentration. The probability of detecting infectious virus at a given viral load was calculated using logistic regression of viral RNA concentration and matched culture results of 78 specimens from randomly selected non-hospitalised cases. The probability of each RDT to detect infectious cases was calculated as the sum of the projected probabilities for viral isolation success for every viral RNA load found at the time of diagnosis in 1,739 confirmed non-hospitalised COVID-19 cases.ResultsThe distribution of quantification cycle values and estimated RNA loads for patients reporting to drive-through testing was skewed to high RNA loads. With the most sensitive RDT (Abbott and SD Biosensor), 97.30% (range: 88.65-99.77) of infectious individuals would be detected. This decreased to 92.73% (range: 60.30-99.77) for Coris BioConcept and GenBody, and 75.53% (range: 17.55-99.77) for RapiGEN. Only 32.9% of RT-PCR results were available on the same day as specimen collection.ConclusionThe most sensitive RDT detected infectious COVID-19 cases with high sensitivity and may considerably improve containment through more rapid isolation and contact tracing.

Diminished amplification of SARS-CoV-2 ORF1ab in a commercial dual-target qRT-PCR diagnostic assay.

Here we describe a SARS-CoV-2 variant with diminished amplification of the ORF ORF1ab target in the Cobas® dual-target SARS-CoV-2 assay resulting in a discrepancy of Ct-values (Ct-value 20.7 for the E-gene and Ct-value 30.2 for ORF1ab). Five unique nucleotide mutations were identified in ORF1ab: C11450A (nsp10) C14178T (RdRp), G15006T (RdRp), G18394T (Hel), and G20995T (Hel). This case highlights the importance of surveillance of genomic regions used in molecular diagnostics and the importance of the public release of target regions used to update commercial and in-house developed SARS-CoV-2 PCR tests. This work underpins the importance of using dual-targets in molecular diagnostic assays to limit the change of false-negative results due to primer and/or probe mismatches.

Significant Impact of Coronavirus Disease 2019 (COVID-19) on Human Immunodeficiency Virus (HIV) Care in Hospitals Affecting the First Pillar of the HIV Care Continuum.

During COVID-19 lockdown, the in-hospital number of HIV indicator conditions decreased disproportionally compared with other non-COVID-19 diseases, which was accompanied by reduced HIV testing rates, number and proportion of positive HIV tests, and new HIV referrals, with more late presentation after lockdown cessation, indicating a significantly impacted HIV care continuum.

Many but small HIV-1 non-B transmission chains in the Netherlands.

OBJECTIVE: The aim of this study was to investigate introductions and spread of different HIV-1 subtypes in the Netherlands. DESIGN: We identified distinct HIV-1 transmission chains in the Netherlands within the global epidemic context through viral phylogenetic analysis of partial HIV-1 polymerase sequences from individuals enrolled in the ATHENA national HIV cohort of all persons in care since 1996, and publicly available international background sequences. METHODS: Viral lineages circulating in the Netherlands were identified through maximum parsimony phylogeographic analysis. The proportion of HIV-1 infections acquired in-country among heterosexuals and MSM was estimated from phylogenetically observed, national transmission chains using a branching process model that accounts for incomplete sampling. RESULTS: As of 1 January 2019, 2589 (24%) of 10 971 (41%) HIV-1 sequenced individuals in ATHENA had non-B subtypes (A1, C, D, F, G) or circulating recombinant forms (CRF01AE, CRF02AG, CRF06-cpx). The 1588 heterosexuals were in 1224, and 536 MSM in 270 phylogenetically observed transmission chains. After adjustments for incomplete sampling, most heterosexual (75%) and MSM (76%) transmission chains were estimated to include only the individual introducing the virus (size = 1). Onward transmission occurred mostly in chains size 2-5 amongst heterosexuals (62%) and in chains size at least 10 amongst MSM (64%). Considering some chains originated in-country from other risk-groups, 40% (95% confidence interval: 36-44) of non-B-infected heterosexuals and 62% (95% confidence interval: 49-73) of MSM-acquired infection in-country. CONCLUSION: Although most HIV-1 non-B introductions showed no or very little onward transmission, a considerable proportion of non-B infections amongst both heterosexuals and MSM in the Netherlands have been acquired in-country.

Targeted proteomics as a tool to detect SARS-CoV-2 proteins in clinical specimens.

The rapid, sensitive and specific detection of SARS-CoV-2 is critical in responding to the current COVID-19 outbreak. In this proof-of-concept study, we explored the potential of targeted mass spectrometry (MS) based proteomics for the detection of SARS-CoV-2 proteins in both research samples and clinical specimens. First, we assessed the limit of detection for several SARS-CoV-2 proteins by parallel reaction monitoring (PRM) MS in infected Vero E6 cells. For tryptic peptides of Nucleocapsid protein, the limit of detection was estimated to be in the mid-attomole range (9E-13 g). Next, this PRM methodology was applied to the detection of viral proteins in various COVID-19 patient clinical specimens, such as sputum and nasopharyngeal swabs. SARS-CoV-2 proteins were detected in these samples with high sensitivity in all specimens with PCR Ct values <24 and in several samples with higher CT values. A clear relationship was observed between summed MS peak intensities for SARS-CoV-2 proteins and Ct values reflecting the abundance of viral RNA. Taken together, these results suggest that targeted MS based proteomics may have the potential to be used as an additional tool in COVID-19 diagnostics.

The Role of the Respiratory Microbiome and Viral Presence in Lower Respiratory Tract Infection Severity in the First Five Years of Life.

Lower respiratory tract infections (LRTIs) in children are common and, although often mild, a major cause of mortality and hospitalization. Recently, the respiratory microbiome has been associated with both susceptibility and severity of LRTI. In this current study, we combined respiratory microbiome, viral, and clinical data to find associations with the severity of LRTI. Nasopharyngeal aspirates of children aged one month to five years included in the STRAP study (Study to Reduce Antibiotic prescription in childhood Pneumonia), who presented at the emergency department (ED) with fever and cough or dyspnea, were sequenced with nanopore 16S-rRNA gene sequencing and subsequently analyzed with hierarchical clustering to identify respiratory microbiome profiles. Samples were also tested using a panel of 15 respiratory viruses and Mycoplasma pneumoniae, which were analyzed in two groups, according to their reported virulence. The primary outcome was hospitalization, as measure of disease severity. Nasopharyngeal samples were isolated from a total of 167 children. After quality filtering, microbiome results were available for 54 children and virology panels for 158 children. Six distinct genus-dominant microbiome profiles were identified, with Haemophilus-, Moraxella-, and Streptococcus-dominant profiles being the most prevalent. However, these profiles were not found to be significantly associated with hospitalization. At least one virus was detected in 139 (88%) children, of whom 32.4% had co-infections with multiple viruses. Viral co-infections were common for adenovirus, bocavirus, and enterovirus, and uncommon for human metapneumovirus (hMPV) and influenza A virus. The detection of enteroviruses was negatively associated with hospitalization. Virulence groups were not significantly associated with hospitalization. Our data underlines high detection rates and co-infection of viruses in children with respiratory symptoms and confirms the predominant presence of Haemophilus-, Streptococcus-, and Moraxella-dominant profiles in a symptomatic pediatric population at the ED. However, we could not assess significant associations between microbiome profiles and disease severity measures.

Small quantities of respiratory syncytial virus RNA only in large droplets around infants hospitalized with acute respiratory infections.

BACKGROUND: Respiratory syncytial virus (RSV) is a major cause of respiratory tract infections in young children. The predominant transmission routes for RSV are still a matter of debate. Specifically, it remains unclear if RSV can be transmitted through the air and what the correlation is between the amount of RSV in nasopharynx samples and in the air. METHODS: The amount of RSV in the air around hospitalized RSV infected infants in single-patient rooms was quantified using a six-stage Andersen cascade impactor that collects and fractionates aerosols and droplets according to size. RSV shedding in the nasopharynx of patients was followed longitudinally by quantifying RSV RNA levels and infectious virus in nasopharyngeal aspirates. Nose and throat swabs of parents and swabs of the patient's bedrail and a datalogger were also collected. RESULTS: Patients remained RSV positive during the air sampling period and infectious virus was isolated up to 9 days post onset of symptoms. In three out of six patients, low levels of RSV RNA, but no infectious virus, were recovered from impactor collection plates that capture large droplets > 7 µm. For four of these patients, one or both parents were also positive for RSV. All surface swabs were RSV-negative. CONCLUSIONS: Despite the prolonged detection of infectious RSV in the nasopharynx of patients, only small amounts of RSV RNA were collected from the air around three out of six patients, which were primarily contained in large droplets which do not remain suspended in the air for long periods of time.

Effect of Methylprednisolone on Inflammation and Coagulation in Patients with Severe COVID-19: A Retrospective Cohort Study.

Corticosteroids reduced mortality rate in patients with coronavirus disease 2019 (COVID-19). Previously, we hypothesized that corticosteroids mitigate the inflammation response resulting in reduced coagulation and thrombosis. In this retrospective study, we included 27 patients with COVID-19 that received high-dose corticosteroids (methylprednisolone 1000 mg i.v. daily for 3 days) for persistent respiratory failure or an excessive inflammation response. We found that inflammation, coagulation, and ventilation parameters improved significantly after methylprednisolone. The viral loads of SARS-CoV-2 remained stable or decreased. These results provides insight into the reduced mortality rate observed in patients with COVID-19 treated with corticosteroids.

Effects of potent neutralizing antibodies from convalescent plasma in patients hospitalized for severe SARS-CoV-2 infection.

In a randomized clinical trial of 86 hospitalized COVID-19 patients comparing standard care to treatment with 300mL convalescent plasma containing high titers of neutralizing SARS-CoV-2 antibodies, no overall clinical benefit was observed. Using a comprehensive translational approach, we unravel the virological and immunological responses following treatment to disentangle which COVID-19 patients may benefit and should be the focus of future studies. Convalescent plasma is safe, does not improve survival, has no effect on the disease course, nor does plasma enhance viral clearance in the respiratory tract, influence SARS-CoV-2 antibody development or serum proinflammatory cytokines levels. Here, we show that the vast majority of patients already had potent neutralizing SARS-CoV-2 antibodies at hospital admission and with comparable titers to carefully selected plasma donors. This resulted in the decision to terminate the trial prematurely. Treatment with convalescent plasma should be studied early in the disease course or at least preceding autologous humoral response development.

Temporal Kinetics of RNAemia and Associated Systemic Cytokines in Hospitalized COVID-19 Patients.

COVID-19 is associated with a wide range of extrarespiratory complications, of which the pathogenesis is currently not fully understood. However, both systemic spread and systemic inflammatory responses are thought to contribute to the systemic pathogenesis. In this study, we determined the temporal kinetics of viral RNA in serum (RNAemia) and the associated inflammatory cytokines and chemokines during the course of COVID-19 in hospitalized patients. We show that RNAemia can be detected in 90% of the patients who develop critical disease, compared to 50% of the patients who develop moderate or severe disease. Furthermore, RNAemia lasts longer in patients who develop critical disease. Elevated levels of interleukin-10 (IL-10) and MCP-1-but not IL-6-are associated with viral load in serum, whereas higher levels of IL-6 in serum were associated with the development of critical disease. In conclusion, RNAemia is common in hospitalized patients, with the highest frequency and duration in patients who develop critical disease. The fact that several cytokines or chemokines are directly associated with the presence of viral RNA in the circulation suggests that the development of RNAemia is an important factor in the systemic pathogenesis of COVID-19. IMPORTANCE Severe COVID-19 can be considered a systemic disease as many extrarespiratory complications occur. However, the systemic pathogenesis is poorly understood. Here, we show that the presence of viral RNA in the blood (RNAemia) occurs more frequently in patients who develop critical disease, compared to patients with moderate or severe disease. In addition, RNAemia is associated with increased levels of inflammatory cytokines and chemokines, like MCP-1 and IL-10, in serum during the course of disease. This suggests that extrarespiratory spread of SARS-CoV-2 contributes to systemic inflammatory responses, which are an important factor in the systemic pathogenesis of COVID-19.