A Multicenter Randomized Controlled Trial To Evaluate the Efficacy and Safety of Nelfinavir in Patients with Mild COVID-19.

Nelfinavir, an orally administered inhibitor of human immunodeficiency virus protease, inhibits the replication of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in vitro. We conducted a randomized controlled trial to evaluate the clinical efficacy and safety of nelfinavir in patients with SARS-CoV-2 infection. We included unvaccinated asymptomatic or mildly symptomatic adult patients who tested positive for SARS-CoV-2 infection within 3 days before enrollment. The patients were randomly assigned (1:1) to receive oral nelfinavir (750 mg; thrice daily for 14 days) combined with standard-of-care or standard-of-care alone. The primary endpoint was the time to viral clearance, confirmed using quantitative reverse-transcription PCR by assessors blinded to the assigned treatment. A total of 123 patients (63 in the nelfinavir group and 60 in the control group) were included. The median time to viral clearance was 8.0 (95% confidence interval [CI], 7.0 to 12.0) days in the nelfinavir group and 8.0 (95% CI, 7.0 to 10.0) days in the control group, with no significant difference between the treatment groups (hazard ratio, 0.815; 95% CI, 0.563 to 1.182; P = 0.1870). Adverse events were reported in 47 (74.6%) and 20 (33.3%) patients in the nelfinavir and control groups, respectively. The most common adverse event in the nelfinavir group was diarrhea (49.2%). Nelfinavir did not reduce the time to viral clearance in this setting. Our findings indicate that nelfinavir should not be recommended in asymptomatic or mildly symptomatic patients infected with SARS-CoV-2. The study is registered with the Japan Registry of Clinical Trials (jRCT2071200023). IMPORTANCE The anti-HIV drug nelfinavir suppresses the replication of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in vitro. However, its efficacy in patients with COVID-19 has not been studied. We conducted a multicenter, randomized controlled trial to evaluate the efficacy and safety of orally administered nelfinavir in patients with asymptomatic or mildly symptomatic COVID-19. Compared to standard-of-care alone, nelfinavir (750 mg, thrice daily) did not reduce the time to viral clearance, viral load, or the time to resolution of symptoms. More patients had adverse events in the nelfinavir group than in the control group (74.6% [47/63 patients] versus 33.3% [20/60 patients]). Our clinical study provides evidence that nelfinavir, despite its antiviral effects on SARS-CoV-2 in vitro, should not be recommended for the treatment of patients with COVID-19 having no or mild symptoms.

Incomplete antiviral treatment may induce longer durations of viral shedding during SARS-CoV-2 infection.

The duration of viral shedding is determined by a balance between de novo infection and removal of infected cells. That is, if infection is completely blocked with antiviral drugs (100% inhibition), the duration of viral shedding is minimal and is determined by the length of virus production. However, some mathematical models predict that if infected individuals are treated with antiviral drugs with efficacy below 100%, viral shedding may last longer than without treatment because further de novo infections are driven by entry of the virus into partially protected, uninfected cells at a slower rate. Using a simple mathematical model, we quantified SARS-CoV-2 infection dynamics in non-human primates and characterized the kinetics of viral shedding. We counterintuitively found that treatments initiated early, such as 0.5 d after virus inoculation, with intermediate to relatively high efficacy (30-70% inhibition of virus replication) yield a prolonged duration of viral shedding (by about 6.0 d) compared with no treatment.

Revisiting the guidelines for ending isolation for COVID-19 patients.

Since the start of the COVID-19 pandemic, two mainstream guidelines for defining when to end the isolation of SARS-CoV-2-infected individuals have been in use: the one-size-fits-all approach (i.e. patients are isolated for a fixed number of days) and the personalized approach (i.e. based on repeated testing of isolated patients). We use a mathematical framework to model within-host viral dynamics and test different criteria for ending isolation. By considering a fixed time of 10 days since symptom onset as the criterion for ending isolation, we estimated that the risk of releasing an individual who is still infectious is low (0-6.6%). However, this policy entails lengthy unnecessary isolations (4.8-8.3 days). In contrast, by using a personalized strategy, similar low risks can be reached with shorter prolonged isolations. The obtained findings provide a scientific rationale for policies on ending the isolation of SARS-CoV-2-infected individuals.

Evaluation of a novel rapid TRC assay for the detection of influenza using nasopharyngeal swabs and gargle samples.

We evaluated a novel transcription-reverse transcription concerted reaction (TRC) assay that can detect influenza A and B within 15 min using nasopharyngeal swab and gargle samples obtained from patients with influenza-like illness, between January and March 2018 and between January and March 2019. Based on the combined RT-PCR and sequencing results, in the nasal swabs, the sensitivity and specificity of TRC for detecting influenza were calculated as 1.000 and 1.000, respectively. In the gargle samples, the sensitivity and specificity of TRC were 0.946 and 1.000, respectively. The TRC assay showed comparable performance to RT-PCR in the detection of influenza viruses.

Discrepancy of SARS-CoV-2 PCR results due to the sample collection sites and possible improper sampling.

Polymerase chain reaction (PCR) testing for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is necessary for confirming a diagnosis of Coronavirus disease 2019 (COVID-19). Here we present a COVID-19 case of an elderly woman whose SARS-CoV-2 PCR tests showed false negative repeatedly by evaluating with different sampling sites and procedures. Nasopharyngeal swabs, suctioned sputum, and tongue swabs were collected for SARS-CoV-2-PCR. As for tongue swabs, we compared between two different sample conditions; one obtained with dry condition and the other obtained with moistened condition inside the oral cavity. SARS-CoV-2-PCR showed positive for an extended period with suctioned sputum samples compared with nasopharyngeal swabs and tongue swabs. No SARS-CoV-2 from a nasopharyngeal swab sample obtained on day 46 after symptoms onset was isolated despite high viral load (183740.5 copies/5µL). An adequate production of neutralizing antibody in a serum sample on day 46 was also confirmed. The number of RNA copies of the tongue swab samples was higher with moistened condition than with dry condition. The present case suggests that the difference of sampling site or sample condition can affect PCR results. High loads viral RNA detection does not always correlate with infectivity.

Detection of significant antiviral drug effects on COVID-19 with reasonable sample sizes in randomized controlled trials: A modeling study.

BACKGROUND: Development of an effective antiviral drug for Coronavirus Disease 2019 (COVID-19) is a global health priority. Although several candidate drugs have been identified through in vitro and in vivo models, consistent and compelling evidence from clinical studies is limited. The lack of evidence from clinical trials may stem in part from the imperfect design of the trials. We investigated how clinical trials for antivirals need to be designed, especially focusing on the sample size in randomized controlled trials. METHODS AND FINDINGS: A modeling study was conducted to help understand the reasons behind inconsistent clinical trial findings and to design better clinical trials. We first analyzed longitudinal viral load data for Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) without antiviral treatment by use of a within-host virus dynamics model. The fitted viral load was categorized into 3 different groups by a clustering approach. Comparison of the estimated parameters showed that the 3 distinct groups were characterized by different virus decay rates (p-value < 0.001). The mean decay rates were 1.17 d-1 (95% CI: 1.06 to 1.27 d-1), 0.777 d-1 (0.716 to 0.838 d-1), and 0.450 d-1 (0.378 to 0.522 d-1) for the 3 groups, respectively. Such heterogeneity in virus dynamics could be a confounding variable if it is associated with treatment allocation in compassionate use programs (i.e., observational studies). Subsequently, we mimicked randomized controlled trials of antivirals by simulation. An antiviral effect causing a 95% to 99% reduction in viral replication was added to the model. To be realistic, we assumed that randomization and treatment are initiated with some time lag after symptom onset. Using the duration of virus shedding as an outcome, the sample size to detect a statistically significant mean difference between the treatment and placebo groups (1:1 allocation) was 13,603 and 11,670 (when the antiviral effect was 95% and 99%, respectively) per group if all patients are enrolled regardless of timing of randomization. The sample size was reduced to 584 and 458 (when the antiviral effect was 95% and 99%, respectively) if only patients who are treated within 1 day of symptom onset are enrolled. We confirmed the sample size was similarly reduced when using cumulative viral load in log scale as an outcome. We used a conventional virus dynamics model, which may not fully reflect the detailed mechanisms of viral dynamics of SARS-CoV-2. The model needs to be calibrated in terms of both parameter settings and model structure, which would yield more reliable sample size calculation. CONCLUSIONS: In this study, we found that estimated association in observational studies can be biased due to large heterogeneity in viral dynamics among infected individuals, and statistically significant effect in randomized controlled trials may be difficult to be detected due to small sample size. The sample size can be dramatically reduced by recruiting patients immediately after developing symptoms. We believe this is the first study investigated the study design of clinical trials for antiviral treatment using the viral dynamics model.

Detection of SARS-CoV-2 using qRT-PCR in saliva obtained from asymptomatic or mild COVID-19 patients, comparative analysis with matched nasopharyngeal samples.

OBJECTIVES: The accurate detection of severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) is essential for the diagnosis of coronavirus disease 2019 (COVID-19). We compared the quantitative RT-PCR results between nasopharyngeal swabs and saliva specimens. METHODS: A COVID-19 outbreak occurred on a cruise ship at Nagasaki port, Japan. We obtained 123 nasopharyngeal swabs and saliva each from asymptomatic or mild patients in the late phase of infection. RESULTS: The intervals from the diagnosis to the sampling were 25.5 days for nasopharyngeal swabs and 28.9 days for saliva. The positive rate was 19.5% (24/123) for nasopharyngeal swabs and 38.2% (47/123) for saliva (P = 0.48). The quantified viral copies (mean ± SEM copies/5 µl) were 9.3±2.6 in nasopharyngeal swabs and 920±850 in saliva (P = 0.0006). CONCLUSIONS: The advantages of saliva specimens include positive rate improvement and accurate viral load detection. Saliva may be used as a reliable sample for SARS-CoV-2 detection.

Efficacy and safety of nelfinavir in asymptomatic and mild COVID-19 patients: a structured summary of a study protocol for a multicenter, randomized controlled trial.

OBJECTIVES: The aim of this trial is to evaluate the antiviral efficacy, clinical efficacy, and safety of nelfinavir in patients with asymptomatic and mild COVID-19. TRIAL DESIGN: The study is designed as a multicenter, open-label, blinded outcome assessment, parallel group, investigator-initiated, exploratory, randomized (1:1 ratio) controlled clinical trial. PARTICIPANTS: Asymptomatic and mild COVID-19 patients will be enrolled in 10 university and teaching hospitals in Japan. The inclusion and exclusion criteria are as follows: Inclusion criteria: (1) Japanese male or female patients aged ≥ 20 years (2) SARS-CoV-2 detected from a respiratory tract specimen (e.g., nasopharyngeal swab or saliva) using PCR, LAMP, or an antigen test within 3 days before obtaining the informed consent (3) Provide informed consent Exclusion criteria: (1) Symptoms developed ≥ 8 days prior to enrolment (2) SpO2 < 96 % (room air) (3) Any of the following screening criteria: a) ALT or AST ≥ 5 × upper limit of the reference range b) Child-Pugh class B or C c) Serum creatinine ≥ 2 × upper limit of the reference range and creatinine clearance < 30 mL/min (4) Poorly controlled diabetes (random blood glucose ≥ 200 mg/dL or HbA1c ≥ 7.0%, despite treatment) (5) Unsuitable serious complications based on the assessment of either the principal investigator or the sub-investigator (6) Hemophiliac or patients with a marked hemorrhagic tendency (7) Severe diarrhea (8) Hypersensitivity to the investigational drug (9) Breastfeeding or pregnancy (10) With childbearing potential and rejecting contraceptive methods during the study period from the initial administration of the investigational drug (11) Receiving rifampicin within the previous 2 weeks (12) Participated in other clinical trials and received drugs within the previous 12 weeks (13) Undergoing treatment for HIV infection (14) History of SARS-CoV-2 vaccination or wishes to be vaccinated against SARS-CoV-2 (15) Deemed inappropriate (for miscellaneous reasons) based on the assessment of either the principal investigator or the sub-investigator INTERVENTION AND COMPARATOR: Patients who meet the inclusion criteria and do not meet any of the exclusion criteria will be randomized to either the nelfinavir group or the symptomatic treatment group. The nelfinavir group will be administered 750 mg of nelfinavir orally, three times daily for 14 days (treatment period). However, if a participant tests negative on two consecutive PCR tests of saliva samples, administration of the investigational drug for that participant can be discontinued at the discretion of the investigators. The symptomatic treatment group will not be administered the investigational drug, but all other study procedures and conditions will be the same for both groups for the duration of the treatment period. After the treatment period of 14 days, each group will be followed up for 14 days (observational period). MAIN OUTCOMES: The primary endpoint is the time to negative conversion of SARS-CoV-2. During the study period from Day 1 to Day 28, two consecutive negative PCR results of saliva samples will be considered as the negative conversion of the virus. The secondary efficacy endpoints are as follows: For patients with both asymptomatic and mild disease: area under the curve of viral load, half decay period of viral load, body temperature at each time point, all-cause mortality, incidence rate of pneumonia, percentage of patients with newly developed pneumonia, rate of oxygen administration, and the percentage of patients who require oxygen administration. For asymptomatic patients: incidence of symptomatic COVID-19, incidence of fever (≥ 37.0 °C for two consecutive days), incidence of cough For patients with mild disease: incidence of defervescence (< 37.0 °C), incidence of recovery from clinical symptoms, incidence of improvement of each symptom The secondary safety endpoints are adverse events and clinical examinations. RANDOMIZATION: Patients will be randomized to either the nelfinavir group or the symptomatic treatment group using the electric data capture system (1:1 ratio, dynamic allocation based on severity [asymptomatic], and age [< 60 years]). BLINDING (MASKING): Only the assessors of the primary outcome will be blinded (blinded outcome assessment). NUMBERS TO BE RANDOMIZED (SAMPLE SIZE): The sample size was determined based on our power analysis to reject the null hypothesis, S (t | z =1) = S (t | z = 0) where S is a survival function, t is time to negative conversion, and z denotes randomization group, by the log-rank test with a two-sided p value of 0.05. We estimated viral dynamic parameters by fitting a nonlinear mixed-effects model to reported viral load data, and simulated our primary endpoint from viral-load time-courses that were realized from sets of viral dynamics parameters sampled from the estimated probability distribution of the parameters (sample size: 2000; 1000 each for randomization group). From this estimation of the hazard ratio between the randomization groups for the event of negative conversion using this simulation dataset, the required number of events for rejecting our null hypothesis with a power of 0.80 felled 97.345 by plugging the estimated hazard ratio, 1.79, in Freedman's equation. Therefore, we decided the required number of randomizations to be 120 after consideration of the frequency of censoring and the anticipated rate of withdrawal caused by factors such as withdrawal of consent. TRIAL STATUS: Protocol version 6.0 of February 12, 2021. Recruitment started on July 22, 2020 and is anticipated to be completed by March 31, 2022. TRIAL REGISTRATION: This trial was registered in Japan Registry of Clinical Trials (jRCT) ( jRCT2071200023 ) on 21 July 21, 2020. FULL PROTOCOL: The full protocol is attached as an additional file, accessible from the Trials website (Additional file 1). In the interest in expediting dissemination of this material, the familiar formatting has been eliminated; this Letter serves as a summary of the key elements of the full protocol. The study protocol has been reported in accordance with the Standard Protocol Items: Recommendations for Clinical Interventional Trials (SPIRIT) guidelines (Additional file 2).

Time variation in the probability of failing to detect a case of polymerase chain reaction testing for SARS-CoV-2 as estimated from a viral dynamics model.

Viral tests including polymerase chain reaction (PCR) tests are recommended to diagnose COVID-19 infection during the acute phase of infection. A test should have high sensitivity; however, the sensitivity of the PCR test is highly influenced by viral load, which changes over time. Because it is difficult to collect data before the onset of symptoms, the current literature on the sensitivity of the PCR test before symptom onset is limited. In this study, we used a viral dynamics model to track the probability of failing to detect a case of PCR testing over time, including the presymptomatic period. The model was parametrized by using longitudinal viral load data collected from 30 hospitalized patients. The probability of failing to detect a case decreased toward symptom onset, and the lowest probability was observed 2 days after symptom onset and increased afterwards. The probability on the day of symptom onset was 1.0% (95% CI: 0.5 to 1.9) and that 2 days before symptom onset was 60.2% (95% CI: 57.1 to 63.2). Our study suggests that the diagnosis of COVID-19 by PCR testing should be done carefully, especially when the test is performed before or way after symptom onset. Further study is needed of patient groups with potentially different viral dynamics, such as asymptomatic cases.

Evaluation of four commercial severe acute respiratory coronavirus 2 antibody tests.

INTRODUCTION: Numerous severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) serological tests exists commercially; however, their performance using clinical samples is limited. Although insufficient to detect SARS-CoV-2 in the early phase of infection, antibody assays can be of great use for surveillance studies or for some coronavirus disease 2019 (COVID-19) patients presenting late to the hospital. METHODS: This study evaluated the sensitivity and specificity of four commercial SARS-CoV-2 lateral flow antibody tests using 213 serum specimens from 90 PCR-positive confirmed COVID-19 patients. Of 59 negative control sera, 50 were obtained from patients with other respiratory infectious diseases before COVID-19 pandemic began while nine were from patients infected with other respiratory viruses, including two seasonal coronaviruses. RESULTS: The varied sensitivities for the four commercial kits were 70.9%, 65.3%, 45.1%, and 65.7% for BioMedomics, Autobio Diagnostics, Genbody, and KURABO, respectively, between sick days 1 and 155 in COVID-19 patients. The sensitivities of the four tests gradually increased over time after infection before sick day 5 (15.0%, 12.5%, 15.0%, and 20.0%); from sick day 11-15 (95.7%, 87.2%, 53.2%, and 89.4%); and after sick day 20 (100%, 100%, 68.6%, and 96.1%), respectively. For severe illness, the sensitivities were quite high in the late phase after sick day 15. The specificities were over 96% for all four tests. No cross-reaction due to other pathogens, including seasonal coronaviruses, was observed. CONCLUSIONS: Our results demonstrated the large differences in the antibody test performances. This ought to be considered when performing surveillance analysis.

Impact of Corticosteroid Administration within 7 Days of the Hospitalization for Influenza Pneumonia with Respiratory Failure: A Propensity Score Analysis Using a Nationwide Administrative Database.

Influenza pneumonia, which causes acute respiratory distress syndrome and multiple organ failure, has no established management protocol. Recently, corticosteroid therapy was used to treat coronavirus disease 2019 with respiratory failure; however, its effectiveness as a treatment for influenza pneumonia remains controversial. To investigate the impact of corticosteroid therapy for the early phase of severe influenza pneumonia, we compared influenza pneumonia patients with respiratory failure treated with or without corticosteroids within 7 days after hospital admission using a Japanese nationwide administrative database. The primary endpoint was the mortality rate. The secondary endpoints were duration of intensive-care unit management, invasive mechanical ventilation, and hospital stay. The inverse probability weighting method with estimated propensity scores was used to minimize the data collection bias. We included 3519 patients with influenza pneumonia with respiratory failure. Of these, 875 were treated with corticosteroids. There was no significant difference between the groups regarding 30-day and 90-day mortality, duration of intensive-care unit management, invasive mechanical ventilation, and hospital stay. However, the in-hospital mortality rate was higher in the corticosteroid group. The use of systematic corticosteroid therapy in patients with influenza pneumonia was associated with a higher in-hospital mortality rate.

Evaluation of Nelfinavir for asymptomatic and mild COVID-19