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.

Impact of chemotherapy and/or immunotherapy on neutralizing antibody response to SARS-CoV-2 mRNA-1237 vaccine in patients with solid tumors.

Patients with solid tumors have been a risk group since the beginning of the SARS-CoV-2 pandemic due to more significant complications, hospitalizations or deaths. The immunosuppressive state of cancer treatments or the tumor itself could influence the development of post-vaccination antibodies. This study prospectively analyzed 89 patients under chemotherapy and/or immunotherapy, who received two doses of the mRNA-1237 vaccine, and were compared with a group of 26 non-cancer individuals. Information on adverse events and neutralizing antibodies against the ancestral strain of SARS-CoV-2 (WH1) have been analyzed. Local reactions accounted for 65%, while systemic reactions accounted for 46% of oncologic individuals/cancer patients. Regarding the response to vaccination, 6.7% of cancer patients developed low neutralizing antibody levels. Lower levels of neutralizing antibodies between cancer and non-cancer groups were significant in individuals without previous SARS-CoV-2 infection, but not in previously infected individuals. We also observed that patients receiving chemotherapy or chemoimmunotherapy have significantly lower levels of neutralizing antibodies than non-cancer individuals. In conclusion, our study confirms the importance of prioritizing cancer patients receiving anticancer treatment in SARS-CoV-2 vaccination programs.

Performance of SARS-CoV-2 Antigen-Detecting Rapid Diagnostic Tests for Omicron and Other Variants of Concern.

The SARS-CoV-2 antigen-detecting rapid diagnostic test (Ag-RDTs) is an easy-to-use diagnostic tool to identify the contagious individuals and reduce the new infections. However, to be effective, Ag-RDTs require the detection of distinct variants of concern (VOC) with high analytical sensitivity. Here, we found that the VOC diverge at the nucleocapsid protein used by four commercial Ag-RDTs for the viral detection. Relative to the original D614G variant, there was a 10-fold loss of detection for the Delta and Alpha variants in certain Ag-RDTs, a reduction above the threshold required to isolate the viable virus. However, Beta and Omicron variants did not lose the detection capacity. As the new VOC arise, successful contact tracing requires continuous monitoring of Ag-RDTs performance.

Heterogeneous Infectivity and Pathogenesis of SARS-CoV-2 Variants Beta, Delta and Omicron in Transgenic K18-hACE2 and Wildtype Mice.

The emerging SARS-CoV-2 variants of concern (VOCs) may display enhanced transmissibility, more severity and/or immune evasion; however, the pathogenesis of these new VOCs in experimental SARS-CoV-2 models or the potential infection of other animal species is not completely understood. Here we infected K18-hACE2 transgenic mice with B.1, B.1.351/Beta, B.1.617.2/Delta and BA.1.1/Omicron isolates and demonstrated heterogeneous infectivity and pathogenesis. B.1.351/Beta variant was the most pathogenic, while BA.1.1/Omicron led to lower viral RNA in the absence of major visible clinical signs. In parallel, we infected wildtype (WT) mice and confirmed that, contrary to B.1 and B.1.617.2/Delta, B.1.351/Beta and BA.1.1/Omicron can infect them. Infection in WT mice coursed without major clinical signs and viral RNA was transient and undetectable in the lungs by day 7 post-infection. In silico modeling supported these findings by predicting B.1.351/Beta receptor binding domain (RBD) mutations result in an increased affinity for both human and murine ACE2 receptors, while BA.1/Omicron RBD mutations only show increased affinity for murine ACE2.

Virological and Clinical Determinants of the Magnitude of Humoral Responses to SARS-CoV-2 in Mild-Symptomatic Individuals.

Background: Evidence on the determinants of the magnitude of humoral responses and neutralizing titers in individuals with mild COVID-19 is scarce. Methods: In this cohort study of mild COVID-19 patients, we assessed viral load (VL) by RT-qPCR at two/three time points during acute infection, and anti-SARS-CoV-2 antibodies by ELISA and plasma neutralizing responses using a pseudovirus assay at day 60. Results: Seventy-one individuals (65% female, median 42 years old) were recruited and grouped into high viral load (VL) >7.5 Log10 copies/mL (n=20), low, VL ≤7.5 Log10 copies/mL (n=22), or as Non-early seroconverters with a positive PCR (n=20), and healthy individuals with a negative PCR (n=9). Individuals with high or low VL showed similar titers of total neutralizing antibodies at day 60, irrespective of maximal VL or viral dynamics. Non-early seroconverters had lower antibody titers on day 60, albeit similar neutralizing activity as the groups with high or low VL. Longer symptom duration and older age were independently associated with increased humoral responses. Conclusions: In mild SARS-CoV-2-infected individuals, the duration of symptoms and age (but not VL) contribute to higher humoral responses.

Small form factor flow virometer for SARS-CoV-2.

Current diagnostics of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection heavily rely on reverse transcription-polymerase chain reaction (RT-PCR) or on rapid antigen detection tests. The former suffers from long time-to-result and high cost while the latter from poor sensitivity. Therefore, it is crucial to develop rapid, sensitive, robust, and inexpensive methods for SARS-CoV-2 testing. Herein, we report a novel optofluidic technology, a flow-virometry reader (FVR), for fast and reliable SARS-CoV-2 detection in saliva samples. A small microfluidic chip together with a laser-pumped optical head detects the presence of viruses tagged with fluorescent antibodies directly from saliva samples. The technology has been validated using clinical samples with high sensitivity (91.2%) and specificity (90%). Thanks also to its short time-to-result (<30 min) and small size (25 × 30 × 13 cm), which can be further reduced in the future, it is a strong alternative to existing tests, especially for point-of-care (POC) and low resource settings.

JAK-STAT Pathway: A Novel Target to Tackle Viral Infections.

Modulation of the antiviral innate immune response has been proposed as a putative cellular target for the development of novel pan-viral therapeutic strategies. The Janus kinase-signal transducer and activator of transcription (JAK-STAT) pathway is especially relevant due to its essential role in the regulation of local and systemic inflammation in response to viral infections, being, therefore, a putative therapeutic target. Here, we review the extraordinary diversity of strategies that viruses have evolved to interfere with JAK-STAT signaling, stressing the relevance of this pathway as a putative antiviral target. Moreover, due to the recent remarkable progress on the development of novel JAK inhibitors (JAKi), the current knowledge on its efficacy against distinct viral infections is also discussed. JAKi have a proven efficacy against a broad spectrum of disorders and exhibit safety profiles comparable to biologics, therefore representing good candidates for drug repurposing strategies, including viral infections.

Hydroxychloroquine for Early Treatment of Adults With Mild Coronavirus Disease 2019: A Randomized, Controlled Trial.

BACKGROUND: No effective treatments for coronavirus disease 2019 (COVID-19) exist. We aimed to determine whether early treatment with hydroxychloroquine (HCQ) would be efficacious for outpatients with COVID-19. METHODS: Multicenter open-label, randomized, controlled trial conducted in Catalonia, Spain, between 17 March and 26 May 2020. Patients recently diagnosed with <5-day of symptom onset were assigned to receive HCQ (800 mg on day 1 followed by 400 mg once daily for 6 days) or usual care. Outcomes were reduction of viral load in nasopharyngeal swabs up to 7 days after treatment start, disease progression up to 28 days, and time to complete resolution of symptoms. Adverse events were assessed up to 28 days. RESULTS: A total of 293 patients were eligible for intention-to-treat analysis: 157 in the control arm and 136 in the intervention arm. The mean age was 41.6 years (SD, 12.6), mean viral load at baseline was 7.90 log10 copies/mL (SD, 1.82), and median time from symptom onset to randomization was 3 days. No differences were found in the mean reduction of viral load at day 3 (-1.41 vs -1.41 log10 copies/mL in the control and intervention arm, respectively) or at day 7 (-3.37 vs -3.44). Treatment did not reduce risk of hospitalization (7.1% control vs 5.9% intervention) nor shorten the time to complete resolution of symptoms (12 days, control vs 10 days, intervention). No relevant adverse events were reported. CONCLUSIONS: In patients with mild COVID-19, no benefit was observed with HCQ beyond the usual care.

Transmission of COVID-19 in 282 clusters in Catalonia, Spain: a cohort study.

BACKGROUND: Scarce data are available on what variables affect the risk of transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the development of symptomatic COVID-19, and, particularly, the relationship with viral load. We aimed to analyse data from linked index cases of COVID-19 and their contacts to explore factors associated with transmission of SARS-CoV-2. METHODS: In this cohort study, patients were recruited as part of a randomised controlled trial done between March 17 and April 28, 2020, that aimed to assess if hydroxychloroquine reduced transmission of SARS-CoV-2. Patients with COVID-19 and their contacts were identified by use of the electronic registry of the Epidemiological Surveillance Emergency Service of Catalonia (Spain). Patients with COVID-19 included in our analysis were aged 18 years or older, not hospitalised, had quantitative PCR results available at baseline, had mild symptom onset within 5 days before enrolment, and had no reported symptoms of SARS-CoV-2 infections in their accommodation or workplace within the 14 days before enrolment. Contacts included were adults with a recent history of exposure and absence of COVID-19-like symptoms within the 7 days preceding enrolment. Viral load of contacts, measured by quantitative PCR from a nasopharyngeal swab, was assessed at enrolment, at day 14, and whenever the participant reported COVID-19-like symptoms. We assessed risk of transmission and developing symptomatic disease and incubation dynamics using regression analysis. We assessed the relationship of viral load and characteristics of cases (age, sex, number of days from reported symptom onset, and presence or absence of fever, cough, dyspnoea, rhinitis, and anosmia) and associations between risk of transmission and characteristics of the index case and contacts. FINDINGS: We identified 314 patients with COVID-19, with 282 (90%) having at least one contact (753 contacts in total), resulting in 282 clusters. 90 (32%) of 282 clusters had at least one transmission event. The secondary attack rate was 17% (125 of 753 contacts), with a variation from 12% when the index case had a viral load lower than 1 × 106 copies per mL to 24% when the index case had a viral load of 1 × 1010 copies per mL or higher (adjusted odds ratio per log10 increase in viral load 1·3, 95% CI 1·1-1·5). Increased risk of transmission was also associated with household contact (3·0, 1·59-5·65) and age of the contact (per year: 1·02, 1·01-1·04). 449 contacts had a positive PCR result at baseline. 28 (6%) of 449 contacts had symptoms at the first visit. Of 421 contacts who were asymptomatic at the first visit, 181 (43%) developed symptomatic COVID-19, with a variation from approximately 38% in contacts with an initial viral load lower than 1 × 107 copies per mL to greater than 66% for those with an initial viral load of 1 × 1010 copies per mL or higher (hazard ratio per log10 increase in viral load 1·12, 95% CI 1·05-1·20; p=0·0006). Time to onset of symptomatic disease decreased from a median of 7 days (IQR 5-10) for individuals with an initial viral load lower than 1 × 107 copies per mL to 6 days (4-8) for those with an initial viral load between 1 × 107 and 1 × 109 copies per mL, and 5 days (3-8) for those with an initial viral load higher than 1 × 109 copies per mL. INTERPRETATION: In our study, the viral load of index cases was a leading driver of SARS-CoV-2 transmission. The risk of symptomatic COVID-19 was strongly associated with the viral load of contacts at baseline and shortened the incubation time of COVID-19 in a dose-dependent manner. FUNDING: YoMeCorono, Generalitat de Catalunya. TRANSLATIONS: For the Catalan translation of the abstract see Supplementary Materials section.

SARS-CoV-2 Infection Modulates ACE2 Function and Subsequent Inflammatory Responses in Swabs and Plasma of COVID-19 Patients.

Angiotensin converting enzyme 2 (ACE2) is a host ectopeptidase and the receptor for the SARS-CoV-2 virus, albeit virus-ACE2 interaction goes far beyond viral entry into target cells. Controversial data exists linking viral infection to changes in ACE2 expression and function, which might influence the subsequent induction of an inflammatory response. Here, we tested the significance of soluble ACE2 enzymatic activity longitudinally in nasopharyngeal swabs and plasma samples of SARS-CoV-2 infected patients, along with the induction of inflammatory cytokines. Release of soluble functional ACE2 increases upon SARS-CoV-2 infection in swabs and plasma of infected patients, albeit rapidly decreasing during infection course in parallel with ACE2 gene expression. Similarly, SARS-CoV-2 infection also induced the expression of inflammatory cytokines. These changes positively correlated with the viral load. Overall, our results demonstrate the existence of mechanisms by which SARS-CoV-2 modulates ACE2 expression and function, intracellular viral sensing and subsequent inflammatory response, offering new insights into ACE2 dynamics in the human upper respiratory tract and pointing towards soluble ACE2 levels as a putative early biomarker of infection severity.

Critical Presentation of a Severe Acute Respiratory Syndrome Coronavirus 2 Reinfection: A Case Report.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) reinfections have been reported; however, most cases are milder than the primary infection. We report the first case of a life-threatening critical presentation of a SARS-CoV-2 reinfection. METHODS: A 62-year-old man from Palamós (Spain) suffered a first mild coronavirus disease 2019 (COVID-19) episode in March 2020, confirmed by 2 independent SARS-CoV-2 nasopharyngeal polymerase chain reaction (PCR) assays and a normal radiograph. He recovered completely and tested negative on 2 consecutive PCRs. In August 2020, the patient developed a second SARS-CoV-2 infection with life-threatening bilateral pneumonia and Acute respiratory distress syndrome criteria, requiring COVID-19-specific treatment (remdesivir + dexamethasone) plus high-flow oxygen therapy. Nasopharyngeal swabs from the second episode were obtained for virus quantification by real-time PCR, for virus outgrowth and sequencing. In addition, plasma and peripheral blood mononuclear cells during the hospitalization period were used to determine SARS-CoV-2-specific humoral and T-cell responses. RESULTS: Genomic analysis of SARS-CoV-2 showed that the virus had probably originated shortly before symptom onset. When the reinfection occurred, the subject showed a weak immune response, with marginal humoral and specific T-cell responses against SARS-CoV-2. All antibody isotypes tested as well as SARS-CoV-2 neutralizing antibodies increased sharply after day 8 postsymptoms. A slight increase of T-cell responses was observed at day 19 after symptom onset. CONCLUSIONS: The reinfection was firmly documented and occurred in the absence of robust preexisting humoral and cellular immunity. SARS-CoV-2 immunity in some subjects is unprotective and/or short-lived; therefore, SARS-CoV-2 vaccine schedules inducing long-term immunity will be required to bring the pandemic under control.

Cyclin-dependent Kinases as Emerging Targets for Developing Novel Antiviral Therapeutics.

Besides its prominent role in cell proliferation, cyclin-dependent kinases (CDKs) are key players in viral infections as both DNA and RNA viruses modify CDK function to favor viral replication. Recently, a number of specific pharmacological CDK inhibitors have been developed and approved for cancer treatment. The repurposing of these specific CDK inhibitors for the treatment of viral infections may represent a novel effective therapeutic strategy to combat old and emergent viruses. In this review, we describe the role, mechanisms of action, and potential of CDKs as antiviral drug targets. We also discuss the current clinical state of novel specific CDK inhibitors, focusing on their putative use as antivirals, especially against new emerging viruses.

A Cluster-Randomized Trial of Hydroxychloroquine for Prevention of Covid-19.

BACKGROUND: Current strategies for preventing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection are limited to nonpharmacologic interventions. Hydroxychloroquine has been proposed as a postexposure therapy to prevent coronavirus disease 2019 (Covid-19), but definitive evidence is lacking. METHODS: We conducted an open-label, cluster-randomized trial involving asymptomatic contacts of patients with polymerase-chain-reaction (PCR)-confirmed Covid-19 in Catalonia, Spain. We randomly assigned clusters of contacts to the hydroxychloroquine group (which received the drug at a dose of 800 mg once, followed by 400 mg daily for 6 days) or to the usual-care group (which received no specific therapy). The primary outcome was PCR-confirmed, symptomatic Covid-19 within 14 days. The secondary outcome was SARS-CoV-2 infection, defined by symptoms compatible with Covid-19 or a positive PCR test regardless of symptoms. Adverse events were assessed for up to 28 days. RESULTS: The analysis included 2314 healthy contacts of 672 index case patients with Covid-19 who were identified between March 17 and April 28, 2020. A total of 1116 contacts were randomly assigned to receive hydroxychloroquine and 1198 to receive usual care. Results were similar in the hydroxychloroquine and usual-care groups with respect to the incidence of PCR-confirmed, symptomatic Covid-19 (5.7% and 6.2%, respectively; risk ratio, 0.86 [95% confidence interval, 0.52 to 1.42]). In addition, hydroxychloroquine was not associated with a lower incidence of SARS-CoV-2 transmission than usual care (18.7% and 17.8%, respectively). The incidence of adverse events was higher in the hydroxychloroquine group than in the usual-care group (56.1% vs. 5.9%), but no treatment-related serious adverse events were reported. CONCLUSIONS: Postexposure therapy with hydroxychloroquine did not prevent SARS-CoV-2 infection or symptomatic Covid-19 in healthy persons exposed to a PCR-positive case patient. (Funded by the crowdfunding campaign YoMeCorono and others; BCN-PEP-CoV2 ClinicalTrials.gov number, NCT04304053.).

Targeting HIV-1 RNase H: N' -(2-Hydroxy-benzylidene)-3,4,5-Trihydroxybenzoylhydrazone as Selective Inhibitor Active against NNRTIs-Resistant Variants

HIV-1 infection requires life-long treatment and with 2.1 million new infections/year, faces the challenge of an increased rate of transmitted drug-resistant mutations. Therefore, a constant and timely effort is needed to identify new HIV-1 inhibitors active against drug-resistant variants. The ribonuclease H (RNase H) activity of HIV-1 reverse transcriptase (RT) is a very promising target, but to date, still lacks an efficient inhibitor. Here, we characterize the mode of action of N'-(2-hydroxy-benzylidene)-3,4,5-trihydroxybenzoylhydrazone (compound 13), an N-acylhydrazone derivative that inhibited viral replication (EC50 = 10 µM), while retaining full potency against the NNRTI-resistant double mutant K103N-Y181C virus. Time-of-addition and biochemical assays showed that compound 13 targeted the reverse-transcription step in cell-based assays and inhibited the RT-associated RNase H function, being >20-fold less potent against the RT polymerase activity. Docking calculations revealed that compound 13 binds within the RNase H domain in a position different from other selective RNase H inhibitors;site-directed mutagenesis studies revealed interactions with conserved amino acid within the RNase H domain, suggesting that compound 13 can be taken as starting point to generate a new series of more potent RNase H selective inhibitors active against circulating drug-resistant variants.