Early treatment of high-risk hospitalized COVID-19 patients with a combination of interferon beta-1b and remdesivir: a phase 2 open-label randomized controlled trial.

BACKGROUND: Early antiviral therapy was effective in the treatment of COVID-19. We assessed the efficacy and safety of combined interferon beta-1b and remdesivir treatment in hospitalized COVID-19 patients. METHODS: We conducted a multicentre, prospective open-label, randomized-controlled trial involving high-risk adults hospitalized for COVID-19. Patients were randomly assigned to a 5-day interferon beta-1b 16 million units daily and remdesivir 200mg loading on day 1 followed by 100mg daily on day 2 to 5 (combination-group), or to remdesivir only of similar regimen (control-group) (1:1). The primary end-point was the time to complete alleviation of symptoms (NEWS2 = 0). RESULTS: Two-hundred and twelve patients were enrolled. The median days of starting treatment from symptom-onset was 3 days. The median age was 65 years and 159 patients (75%) had chronic disease. The baseline demographics were similar. There was no mortality. For the primary-endpoint, the combination-group was significantly quicker to NEWS2 = 0 (4 versus 6.5 days; hazard-ratio [HR],6.59; 95% confidence-interval [CI],6.1-7.09; p < 0.0001) when compared to the control-group. For the secondary endpoints, the combination-group was quicker to negative NPS VL (6 versus 8 days; HR,8.16; 95% CI,7.79-8.52; p < 0.0001) and develop seropositive IgG (8 versus 10 days; HR,10.78; 95% CI,9.98-11.58; p < 0.0001). All adverse events resolved upon follow-up. Combination group (HR,4.1 95%CI,1.9-8.6, p < 0.0001), was the most significant independent factor associated with NEWS2 = 0 on day 4. CONCLUSIONS: Early treatment with interferon beta-1b and remdesivir was safe and better than remdesivir only in alleviating symptoms, shorten viral shedding and hospitalization with earlier seropositivity in high-risk COVID-19 patients.

Air and environmental sampling for SARS-CoV-2 around hospitalized patients with coronavirus disease 2019 (COVID-19).

BACKGROUND: The role of severe respiratory coronavirus virus 2 (SARS-CoV-2)-laden aerosols in the transmission of coronavirus disease 2019 (COVID-19) remains uncertain. Discordant findings of SARS-CoV-2 RNA in air samples were noted in early reports. METHODS: Sampling of air close to 6 asymptomatic and symptomatic COVID-19 patients with and without surgical masks was performed with sampling devices using sterile gelatin filters. Frequently touched environmental surfaces near 21 patients were swabbed before daily environmental disinfection. The correlation between the viral loads of patients' clinical samples and environmental samples was analyzed. RESULTS: All air samples were negative for SARS-CoV-2 RNA in the 6 patients singly isolated inside airborne infection isolation rooms (AIIRs) with 12 air changes per hour. Of 377 environmental samples near 21 patients, 19 (5.0%) were positive by reverse-transcription polymerase chain reaction (RT-PCR) assay, with a median viral load of 9.2 × 102 copies/mL (range, 1.1 × 102 to 9.4 × 104 copies/mL). The contamination rate was highest on patients' mobile phones (6 of 77, 7.8%), followed by bed rails (4 of 74, 5.4%) and toilet door handles (4 of 76, 5.3%). We detected a significant correlation between viral load ranges in clinical samples and positivity rate of environmental samples (P < .001). CONCLUSION: SARS-CoV-2 RNA was not detectable by air samplers, which suggests that the airborne route is not the predominant mode of transmission of SARS-CoV-2. Wearing a surgical mask, appropriate hand hygiene, and thorough environmental disinfection are sufficient infection control measures for COVID-19 patients isolated singly in AIIRs. However, this conclusion may not apply during aerosol-generating procedures or in cohort wards with large numbers of COVID-19 patients.

Changes in the Intranetwork and Internetwork Connectivity of the Default Mode Network and Olfactory Network in Patients with COVID-19 and Olfactory Dysfunction.

Olfactory dysfunction (OD) is a common symptom in coronavirus disease 2019 (COVID-19) patients. Moreover, many neurological manifestations have been reported in these patients, suggesting central nervous system involvement. The default mode network (DMN) is closely associated with olfactory processing. In this study, we investigated the internetwork and intranetwork connectivity of the DMN and the olfactory network (ON) in 13 healthy controls and 22 patients presenting with COVID-19-related OD using independent component analysis and region of interest functional magnetic resonance imaging (fMRI) analysis. There was a significant correlation between the butanol threshold test (BTT) and the intranetwork connectivity in ON. Meanwhile, the COVID-19 patients with OD showed significantly higher intranetwork connectivity in the DMN, as well as higher internetwork connectivity between ON and DMN. However, no significant difference was found between groups in the intranetwork connectivity within ON. We postulate that higher intranetwork functional connectivities compensate for the deficits in olfactory processing and general well-being in COVID-19 patients. Nevertheless, the compensation process in the ON may not be obvious at this stage. Our results suggest that resting-state fMRI is a potentially valuable tool to evaluate neurosensory dysfunction in COVID-19 patients.

Early-Morning vs Spot Posterior Oropharyngeal Saliva for Diagnosis of SARS-CoV-2 Infection: Implication of Timing of Specimen Collection for Community-Wide Screening.

BACKGROUND: Posterior oropharyngeal saliva is increasingly recognized as a valid respiratory specimen for SARS-CoV-2 diagnosis. It is easy to collect and suitable for community-wide screening. The optimal timing of collection is currently unknown, and we speculate that an early-morning specimen before oral hygiene and breakfast would increase the diagnostic yield. METHODS: Posterior oropharyngeal saliva was collected at 5 different time points within the same day from 18 patients with previously confirmed SARS-CoV-2 infection by molecular testing. Cycle threshold (Ct) values were compared. RESULTS: There was an overall trend of lower Ct values from specimens collected in the early morning, with a gradual decrease of viral load towards nighttime, but reaching statistical significance only when compared with the specimens collected at bedtime. Eight out of 13 subjects had a higher viral load in the early morning than the rest of the 4 time points (before lunch, before teatime at 3 pm, before dinner, before bedtime). CONCLUSIONS: The result suggests a diurnal variation of viral shedding from the upper respiratory tract with a trend showing higher viral load in the early morning. For community screening purposes, posterior oropharyngeal saliva could be taken throughout the day, but preferably in the early morning to maximize the yield.

Neurosensory Rehabilitation and Olfactory Network Recovery in Covid-19-related Olfactory Dysfunction.

Non-conductive olfactory dysfunction (OD) is an important extra-pulmonary manifestation of coronavirus disease 2019 (COVID-19). Olfactory bulb (OB) volume loss and olfactory network functional connectivity (FC) defects were identified in two patients suffering from prolonged COVID-19-related OD. One patient received olfactory treatment (OT) by the combination of oral vitamin A and smell training via the novel electronic portable aromatic rehabilitation (EPAR) diffusers. After four-weeks of OT, clinical recuperation of smell was correlated with interval increase of bilateral OB volumes [right: 22.5 mm3 to 49.5 mm3 (120%), left: 37.5 mm3 to 42 mm3 (12%)] and improvement of mean olfactory FC [0.09 to 0.15 (66.6%)].

Lessons learned 1 year after SARS-CoV-2 emergence leading to COVID-19 pandemic.

Without modern medical management and vaccines, the severity of the Coronavirus Disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome (SARS) coronavirus 2 (SARS-CoV-2) might approach the magnitude of 1894-plague (12 million deaths) and 1918-A(H1N1) influenza (50 million deaths) pandemics. The COVID-19 pandemic was heralded by the 2003 SARS epidemic which led to the discovery of human and civet SARS-CoV-1, bat SARS-related-CoVs, Middle East respiratory syndrome (MERS)-related bat CoV HKU4 and HKU5, and other novel animal coronaviruses. The suspected animal-to-human jumping of 4 betacoronaviruses including the human coronaviruses OC43(1890), SARS-CoV-1(2003), MERS-CoV(2012), and SARS-CoV-2(2019) indicates their significant pandemic potential. The presence of a large reservoir of coronaviruses in bats and other wild mammals, culture of mixing and selling them in urban markets with suboptimal hygiene, habit of eating exotic mammals in highly populated areas, and the rapid and frequent air travels from these areas are perfect ingredients for brewing rapidly exploding epidemics. The possibility of emergence of a hypothetical SARS-CoV-3 or other novel viruses from animals or laboratories, and therefore needs for global preparedness should not be ignored. We reviewed representative publications on the epidemiology, virology, clinical manifestations, pathology, laboratory diagnostics, treatment, vaccination, and infection control of COVID-19 as of 20 January 2021, which is 1 year after person-to-person transmission of SARS-CoV-2 was announced. The difficulties of mass testing, labour-intensive contact tracing, importance of compliance to universal masking, low efficacy of antiviral treatment for severe disease, possibilities of vaccine or antiviral-resistant virus variants and SARS-CoV-2 becoming another common cold coronavirus are discussed.

A systematic review and meta-analysis protocol examining the clinical characteristics and epidemiological features of olfactory dysfunction (OD) in coronavirus disease 2019 (COVID-19).

BACKGROUND: The coronavirus disease 2019 (COVID-19) pandemic has caused recurring and major outbreaks in multiple human populations around the world. The plethora of clinical presentations of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been described extensively, of which olfactory dysfunction (OD) was established as an important and common extrapulmonary manifestation of COVID-19 infection. The aim of this protocol is to conduct a systematic review and meta-analysis on peer-reviewed articles which described clinical data of OD in COVID-19 patients. METHODS: This research protocol has been prospectively registered with the Prospective Register of Systematic Reviews (PROSPERO; CRD42020196202). CINAHL, ClinicalTrials.gov, Cochrane Central, EMBASE, MEDLINE and PubMed, as well as Chinese medical databases China National Knowledge Infrastructure (CNKI), VIP and WANFANG, will be searched using keywords including 'COVID-19', 'coronavirus disease', '2019-nCoV', 'SARS-CoV-2', 'novel coronavirus', 'anosmia', 'hyposmia', 'loss of smell', and 'olfactory dysfunction'. Systematic review and meta-analysis will be conducted according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) and the Meta-analyses Of Observational Studies in Epidemiology (MOOSE) guidelines. Articles will be screened according to pre-specified inclusion and exclusion criteria to extract studies that include new clinical data investigating the effect of COVID-19 on olfactory dysfunction. Included articles will be reviewed in full; data including patient demographics, clinical characteristics of COVID-19-related OD, methods of olfactory assessment and relevant clinical outcomes will be extracted. Statistical analyses will be performed using the Comprehensive Meta-Analysis version 3. DISCUSSION: This systematic review and meta-analysis protocol will aim to collate and synthesise all available clinical evidence regarding COVID-19-related OD as an important neurosensory dysfunction of COVID-19 infection. A comprehensive search strategy and screening process will be conducted to incorporate broad clinical data for robust statistical analyses and representation. The outcome of the systematic review and meta-analysis will aim to improve our understanding of the symptomatology and clinical characteristics of COVID-19-related OD and identify knowledge gaps in its disease process, which will guide future research in this specific neurosensory defect. SYSTEMATIC REVIEW REGISTRATION: PROSPERO registration number: CRD42020196202.

Seroprevalence of SARS-CoV-2 in Hong Kong and in residents evacuated from Hubei province, China: a multicohort study.

BACKGROUND: The role of subclinical severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections in perpetuating the COVID-19 pandemic is unknown because population seroprevalence data are absent. We aimed to establish the sensitivity and specificity of our enzyme immunoassay and microneutralisation assay, and the seroprevalence of SARS-CoV-2 in Hong Kong before and after the pandemic, as well as in Hong Kong residents evacuated from Hubei province, China. METHODS: We did a multicohort study in a hospital and university in Hong Kong. We evaluated the sensitivity of our enzyme immunoassay and microneutralisation assay with RT-PCR data from patients positive for SARS-CoV-2 and the specificity of our enzyme immunoassay and microneutralisation assay with archived serum samples collected before 2019. We compared the seropositivity of the general population of Hong Kong before and after the pandemic had begun, and determined the seropositivity of Hong Kong residents evacuated from Hubei province, China, in March, 2020. FINDINGS: Between Feb 26 and March 18, 2020, we assessed RT-PCR samples from 45 patients who had recovered from COVID-19 to establish the sensitivity of our enzyme immunoassay and microneutralisation assay. To establish the specificity of these assays, we retrieved archived serum. The sensitivity was 91·1% (41 of 45 [95% CI 78·8-97·5]) for the microneutralisation assay, 57·8% (26 of 45 [42·2-72·3]) for anti-nucleoprotein IgG, 66·7% (30 of 45 [51·1-80·0]) for anti-spike protein receptor binding domain (RBD) IgG, and 73·3% (33 of 45 [58·1-85·4]) for enzyme immunoassay (either positive for anti-nucleoprotein or anti-RBD IgG). The specificity was 100% (152 of 152 [95% CI 97·6-100·0]) for both the enzyme immunoassay and microneutralisation assay. Among the Hong Kong general population, 53 (2·7%) of 1938 were enzyme immunoassay positive, but of those who were positive, all 53 were microneutralisation negative, and no significant increase was seen in the seroprevalence between April 12, 2018, and Feb 13, 2020. Among asymptomatic Hubei returnees, 17 (4%) of 452 were seropositive with the enzyme immunoassay or the microneutralisation assay, with 15 (88%) of 17 seropositive with the microneutralisation assay, and two familial clusters were identified. INTERPRETATION: Our serological data suggest that SARS-CoV-2 is a new emerging virus. The seropositivity rate in Hubei returnees indicates that RT-PCR-confirmed patients only represent a small proportion of the total number of cases. The low seroprevalence suggests that most of the Hong Kong and Hubei population remain susceptible to COVID-19. Future waves of the outbreak are inevitable without a vaccine or antiviral prophylaxis. The role of age-related cross reactive non-neutralising antibodies in the pathogenesis of COVID-19 warrants further investigation. FUNDING: Richard and Carol Yu, May Tam Mak Mei Yin, Shaw Foundation (Hong Kong), Michael Tong, Marina Lee, and the Government Consultancy Service (see acknowledgments for full list).

Detection of SARS-CoV-2 in conjunctival secretions from patients without ocular symptoms.

PURPOSE: To evaluate the presence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in conjunctival secretions from patients without ocular symptoms. METHODS: Conjunctival swabs were prospectively collected from laboratory-confirmed Coronavirus disease 2019 (COVID-19) patients without ocular symptoms for reverse transcription-polymerase chain reaction (RT-PCR) and viral culture. RESULTS: A total of 158 conjunctival swabs were obtained from 49 laboratory-confirmed COVID-19 patients. The median duration of illness when the first conjunctival swab was obtained was 10 days (range 2-27 days). Four conjunctival swabs from four different patients (4/49, 8.2%) were positive for SARS-CoV-2 RNA by RT-PCR. The Ct values ranged from 32.7 to 37.7 (mean 35.4). Viral cultures were negative for all four RT-PCR-positive conjunctival swabs. CONCLUSION: Conjunctival secretions of a minority of COVID-19 patients without ocular symptoms may contain relatively low levels of SARS-CoV-2 RNA, but their infectiousness remains undetermined. Appropriate infection control measures should be implemented during ophthalmological assessment of COVID-19 patients to prevent potential nosocomial transmission of SARS-CoV-2.

Improved Detection of Antibodies against SARS-CoV-2 by Microsphere-Based Antibody Assay.

Currently available COVID-19 antibody tests using enzyme immunoassay (EIA) or immunochromatographic assay have variable sensitivity and specificity. Here, we developed and evaluated a novel microsphere-based antibody assay (MBA) for detecting immunoglobulin G (IgG) against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleoprotein (NP) and spike protein receptor binding domain (RBD). The seropositive cutoff value was set using a cohort of 294 anonymous serum specimens collected in 2018. The specificity was assessed using serum specimens collected from organ donors or influenza patients before 2020. Seropositive rate was determined among COVID-19 patients. Time-to-seropositivity and signal-to-cutoff (S/CO) ratio were compared between MBA and EIA. MBA had a specificity of 100% (93/93; 95% confidence interval (CI), 96-100%) for anti-NP IgG, 98.9% (92/93; 95% CI 94.2-100%) for anti-RBD IgG. The MBA seropositive rate for convalescent COVID-19 patients was 89.8% (35/39) for anti-NP IgG and 79.5% (31/39) for anti-RBD IgG. The time-to-seropositivity was shorter with MBA than EIA. MBA could better differentiate between COVID-19 patients and negative controls with higher S/CO ratio for COVID-19 patients, lower S/CO ratio with negative controls and fewer specimens in the equivocal range. MBA is robust, simple and is suitable for clinical microbiology laboratory for the accurate determination of anti-SARS-CoV-2 antibodies for diagnosis, serosurveillance, and vaccine trials.

Development and validation of risk prediction models for COVID-19 positivity in a hospital setting.

OBJECTIVES: To develop: (1) two validated risk prediction models for coronavirus disease-2019 (COVID-19) positivity using readily available parameters in a general hospital setting; (2) nomograms and probabilities to allow clinical utilisation. METHODS: Patients with and without COVID-19 were included from 4 Hong Kong hospitals. The database was randomly split into 2:1: for model development database (n = 895) and validation database (n = 435). Multivariable logistic regression was utilised for model creation and validated with the Hosmer-Lemeshow (H-L) test and calibration plot. Nomograms and probabilities set at 0.1, 0.2, 0.4 and 0.6 were calculated to determine sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV). RESULTS: A total of 1330 patients (mean age 58.2 ± 24.5 years; 50.7% males; 296 COVID-19 positive) were recruited. The first prediction model developed had age, total white blood cell count, chest x-ray appearances and contact history as significant predictors (AUC = 0.911 [CI = 0.880-0.941]). The second model developed has the same variables except contact history (AUC = 0.880 [CI = 0.844-0.916]). Both were externally validated on the H-L test (p = 0.781 and 0.155, respectively) and calibration plot. Models were converted to nomograms. Lower probabilities give higher sensitivity and NPV; higher probabilities give higher specificity and PPV. CONCLUSION: Two simple-to-use validated nomograms were developed with excellent AUCs based on readily available parameters and can be considered for clinical utilisation.

Frequency and Distribution of Chest Radiographic Findings in Patients Positive for COVID-19.

Background Current coronavirus disease 2019 (COVID-19) radiologic literature is dominated by CT, and a detailed description of chest radiography appearances in relation to the disease time course is lacking. Purpose To describe the time course and severity of findings of COVID-19 at chest radiography and correlate these with real-time reverse transcription polymerase chain reaction (RT-PCR) testing for severe acute respiratory syndrome coronavirus 2, or SARS-CoV-2, nucleic acid. Materials and Methods This is a retrospective study of patients with COVID-19 confirmed by using RT-PCR and chest radiographic examinations who were admitted across four hospitals and evaluated between January and March 2020. Baseline and serial chest radiographs (n = 255) were reviewed with RT-PCR. Correlation with concurrent CT examinations (n = 28) was performed when available. Two radiologists scored each chest radiograph in consensus for consolidation, ground-glass opacity, location, and pleural fluid. A severity index was determined for each lung. The lung scores were summed to produce the final severity score. Results The study was composed of 64 patients (26 men; mean age, 56 years ± 19 [standard deviation]). Of these, 58 patients had initial positive findings with RT-PCR (91%; 95% confidence interval: 81%, 96%), 44 patients had abnormal findings at baseline chest radiography (69%; 95% confidence interval: 56%, 80%), and 38 patients had initial positive findings with RT-PCR testing and abnormal findings at baseline chest radiography (59%; 95% confidence interval: 46%, 71%). Six patients (9%) showed abnormalities at chest radiography before eventually testing positive for COVID-19 with RT-PCR. Sensitivity of initial RT-PCR (91%; 95% confidence interval: 83%, 97%) was higher than that of baseline chest radiography (69%; 95% confidence interval: 56%, 80%) (P = .009). Radiographic recovery (mean, 6 days ± 5) and virologic recovery (mean, 8 days ± 6) were not significantly different (P = .33). Consolidation was the most common finding (30 of 64; 47%) followed by ground-glass opacities (21 of 64; 33%). Abnormalities at chest radiography had a peripheral distribution (26 of 64; 41%) and lower zone distribution (32 of 64; 50%) with bilateral involvement (32 of 64; 50%). Pleural effusion was uncommon (two of 64; 3%). The severity of findings at chest radiography peaked at 10-12 days from the date of symptom onset. Conclusion Findings at chest radiography in patients with coronavirus disease 2019 frequently showed bilateral lower zone consolidation, which peaked at 10-12 days from symptom onset. © RSNA, 2020.

A Rapid, Simple, Inexpensive, and Mobile Colorimetric Assay COVID-19-LAMP for Mass On-Site Screening of COVID-19.

To control the COVID-19 pandemic and prevent its resurgence in areas preparing for a return of economic activities, a method for a rapid, simple, and inexpensive point-of-care diagnosis and mass screening is urgently needed. We developed and evaluated a one-step colorimetric reverse-transcriptional loop-mediated isothermal amplification assay (COVID-19-LAMP) for detection of SARS-CoV-2, using SARS-CoV-2 isolate and respiratory samples from patients with COVID-19 (n = 223) and other respiratory virus infections (n = 143). The assay involves simple equipment and techniques and low cost, without the need for expensive qPCR machines, and the result, indicated by color change, is easily interpreted by naked eyes. COVID-19-LAMP can detect SARS-CoV-2 RNA with detection limit of 42 copies/reaction. Of 223 respiratory samples positive for SARS-CoV-2 by qRT-PCR, 212 and 219 were positive by COVID-19-LAMP at 60 and 90 min (sensitivities of 95.07% and 98.21%) respectively, with the highest sensitivities among nasopharyngeal swabs (96.88% and 98.96%), compared to sputum/deep throat saliva samples (94.03% and 97.02%), and throat swab samples (93.33% and 98.33%). None of the 143 samples with other respiratory viruses were positive by COVID-19-LAMP, showing 100% specificity. Samples with higher viral load showed shorter detection time, some as early as 30 min. This inexpensive, highly sensitive and specific COVID-19-LAMP assay can be useful for rapid deployment as mobile diagnostic units to resource-limiting areas for point-of-care diagnosis, and for unlimited high-throughput mass screening at borders to reduce cross-regional transmission.

SARS-CoV-2 shedding and seroconversion among passengers quarantined after disembarking a cruise ship: a case series.

BACKGROUND: A cruise ship is a closed-off environment that simulates the basic functioning of a city in terms of living conditions and interpersonal interactions. Thus, the Diamond Princess cruise ship, which was quarantined because of an onboard outbreak of COVID-19 in February, 2020, provides an opportunity to define the shedding pattern of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and patient antibody responses before and after the onset of symptoms. METHODS: We recruited adult (≥18 years) passengers from Hong Kong who had been on board the Diamond Princess cruise ship docked in Yokohama, Japan in February, 2020. All participants had been found to be negative for SARS-CoV-2 by RT-PCR 4 days before disembarking and were transferred to further quarantine in a public estate in Hong Kong, where they were recruited. Participants were prospectively screened by quantitative RT-PCR (RT-qPCR) of nasopharyngeal and throat swabs, and serum IgG and IgM against internal nucleoprotein and the surface spike receptor-binding protein (RBD) of SARS-CoV-2 at baseline (upon entering quarantine) and on days 4, 8, and 12 of quarantine. FINDINGS: On Feb 22, 2020, 215 adults were recruited, of whom nine (4%; 95% CI 2-8) were positive for SARS-CoV-2 by RT-qPCR or serology and were hospitalised. Of these nine patients, nasopharyngeal swab RT-qPCR was positive in eight patients (89%; 57-99) at baseline. All nine patients were positive for anti-RBD IgG by day 8. Eight (89%; 57-99) were simultaneously positive for nasopharyngeal swab RT-PCR and anti-RBD IgG. One patient who was positive for anti-RBD IgG and had a negative viral load had multifocal peripheral ground-glass changes on high-resolution CT that were typical of COVID-19. Five patients (56%; 27-81) with ground-glass changes on high-resolution CT were found to have higher anti-nucleoprotein-IgG OD values on day 8 and 12 and anti-RBD IgG OD value on day 12 than patients without ground-glass changes. Six (67%; 35-88) patients remained asymptomatic throughout the 14-day quarantine period. INTERPRETATION: Patients with COVID-19 can develop asymptomatic lung infection with viral shedding and those with evidence of pneumonia on imaging tend to have an increased antibody response. Positive IgG or IgM confirmed infection of COVID-19 in both symptomatic and asymptomatic patients. A combination of RT-PCR and serology should be implemented for case finding and contact tracing to facilitate early diagnosis, prompt isolation, and treatment. FUNDING: Shaw Foundation Hong Kong; Sanming-Project of Medicine (Shenzhen); High Level-Hospital Program (Guangdong Health Commission).

Olfactory Dysfunction in Coronavirus Disease 2019 Patients: Observational Cohort Study and Systematic Review.

BACKGROUND: Olfactory dysfunction (OD) has been reported in coronavirus disease 2019 (COVID-19). However, there are knowledge gaps about the severity, prevalence, etiology, and duration of OD in COVID-19 patients. METHODS: Olfactory function was assessed in all participants using questionnaires and the butanol threshold test (BTT). Patients with COVID-19 and abnormal olfaction were further evaluated using the smell identification test (SIT), sinus imaging, and nasoendoscopy. Selected patients received nasal biopsies. Systematic review was performed according to PRISMA guidelines. PubMed items from January 1, 2020 to April 23, 2020 were searched. Studies that reported clinical data on olfactory disturbances in COVID-19 patients were analyzed. RESULTS: We included 18 COVID-19 patients and 18 controls. Among COVID-19 patients, 12 of 18 (67%) reported olfactory symptoms and OD was confirmed in 6 patients by BTT and SIT. Olfactory dysfunction was the only symptom in 2 patients. Mean BTT score of patients was worse than controls (P = .004, difference in means = 1.8; 95% confidence interval, 0.6-2.9). Sinusitis and olfactory cleft obstruction were absent in most patients. Immunohistochemical analysis of nasal biopsy revealed the presence of infiltrative CD68+ macrophages harboring severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antigen in the stroma. Olfactory dysfunction persisted in 2 patients despite clinical recovery. Systematic review showed that the prevalence of olfactory disturbances in COVID-19 ranged from 5% to 98%. Most studies did not assess olfaction quantitatively. CONCLUSIONS: Olfactory dysfunction is common in COVID-19 and may be the only symptom. Coronavirus disease 2019-related OD can be severe and prolonged. Mucosal infiltration by CD68+ macrophages expressing SARS-CoV-2 viral antigen may contribute to COVID-19-related OD.

Identification of nsp1 gene as the target of SARS-CoV-2 real-time RT-PCR using nanopore whole-genome sequencing.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused the coronavirus disease 2019 (COVID-19) pandemic. Accurate detection of SARS-CoV-2 using molecular assays is critical for patient management and the control of the COVID-19 pandemic. However, there is an increasing number of SARS-CoV-2 viruses with mutations at the primer or probe binding sites, and these mutations may affect the sensitivity of currently available real-time reverse transcription-polymerase chain reaction (RT-PCR) assays targeting the nucleocapsid (N), envelope (E), and open reading frame 1a or 1b genes. Using sequence-independent single-primer amplification and nanopore whole-genome sequencing, we have found that the nonstructural protein 1 (nsp1) gene, located at the 5' end of the SARS-CoV-2 genome, was highly expressed in the nasopharyngeal or saliva specimens of 9 COVID-19 patients of different clinical severity. Based on this finding, we have developed a novel nsp1 real-time RT-PCR assay. The primers and probes are highly specific for SARS-CoV-2. Validation with 101 clinical specimens showed that our nsp1 RT-PCR assay has a sensitivity of 93.1% (95% confidence interval [CI]: 86.2%-97.2%), which was similar to those of N and E gene RT-PCR assays. The diagnostic specificity was 100% (95% CI: 92.9%-100%). The addition of nsp1 for multitarget detection of SARS-CoV-2 can avoid false-negative results due to mutations at the primers/probes binding sites of currently available RT-PCR assays.

Evaluation of the commercially available LightMix® Modular E-gene kit using clinical and proficiency testing specimens for SARS-CoV-2 detection.

BACKGROUND: Rapid and sensitive diagnostic assays for SARS-CoV-2 detection are required for prompt patient management and infection control. The analytical and clinical performances of LightMix® Modular SARS and Wuhan CoV E-gene kit, a widely used commercial assay for SARS-CoV-2 detection, have not been well studied. OBJECTIVE: To evaluate the performance characteristics of the LightMix® E-gene kit in comparison with well-validated in-house developed COVID-19 RT-PCR assays. STUDY DESIGN: Serial dilutions of SARS-CoV-2 culture isolate extracts were used for analytical sensitivity evaluation. A total of 289 clinical specimens from 186 patients with suspected COVID-19 and 8 proficiency testing (PT) samples were used to evaluate the diagnostic performance of the LightMix® E-gene kit against in-house developed COVID-19-RdRp/Hel and COVID-19-N RT-PCR assays. RESULTS: The LightMix® E-gene kit had a limit of detection of 1.8 × 10-1 TCID50/mL, which was one log10 lower than those of the two in-house RT-PCR assays. The LightMix® E-gene kit (149/289 [51.6%]) had similar sensitivity as the in-house assays (144/289 [49.8%] for RdRp/Hel and 146/289 [50.5%] for N). All three assays gave correct results for all the PT samples. Cycle threshold (Cp) values of the LightMix® E-gene kit and in-house assays showed excellent correlation. Reproducibility of the Cp values was satisfactory with intra- and inter-assay coefficient of variation values <5%. Importantly, the LightMix® E-gene kit, when used as a stand-alone assay, was equally sensitive as testing algorithms using multiple COVID-19 RT-PCR assays. CONCLUSIONS: The LightMix® E-gene kit is a rapid and sensitive assay for SARS-CoV-2 detection. It has fewer verification requirements compared to laboratory-developed tests.

Triple combination of interferon beta-1b, lopinavir-ritonavir, and ribavirin in the treatment of patients admitted to hospital with COVID-19: an open-label, randomised, phase 2 trial.

BACKGROUND: Effective antiviral therapy is important for tackling the coronavirus disease 2019 (COVID-19) pandemic. We assessed the efficacy and safety of combined interferon beta-1b, lopinavir-ritonavir, and ribavirin for treating patients with COVID-19. METHODS: This was a multicentre, prospective, open-label, randomised, phase 2 trial in adults with COVID-19 who were admitted to six hospitals in Hong Kong. Patients were randomly assigned (2:1) to a 14-day combination of lopinavir 400 mg and ritonavir 100 mg every 12 h, ribavirin 400 mg every 12 h, and three doses of 8 million international units of interferon beta-1b on alternate days (combination group) or to 14 days of lopinavir 400 mg and ritonavir 100 mg every 12 h (control group). The primary endpoint was the time to providing a nasopharyngeal swab negative for severe acute respiratory syndrome coronavirus 2 RT-PCR, and was done in the intention-to-treat population. The study is registered with ClinicalTrials.gov, NCT04276688. FINDINGS: Between Feb 10 and March 20, 2020, 127 patients were recruited; 86 were randomly assigned to the combination group and 41 were assigned to the control group. The median number of days from symptom onset to start of study treatment was 5 days (IQR 3-7). The combination group had a significantly shorter median time from start of study treatment to negative nasopharyngeal swab (7 days [IQR 5-11]) than the control group (12 days [8-15]; hazard ratio 4·37 [95% CI 1·86-10·24], p=0·0010). Adverse events included self-limited nausea and diarrhoea with no difference between the two groups. One patient in the control group discontinued lopinavir-ritonavir because of biochemical hepatitis. No patients died during the study. INTERPRETATION: Early triple antiviral therapy was safe and superior to lopinavir-ritonavir alone in alleviating symptoms and shortening the duration of viral shedding and hospital stay in patients with mild to moderate COVID-19. Future clinical study of a double antiviral therapy with interferon beta-1b as a backbone is warranted. FUNDING: The Shaw-Foundation, Richard and Carol Yu, May Tam Mak Mei Yin, and Sanming Project of Medicine.

Development of a Novel, Genome Subtraction-Derived, SARS-CoV-2-Specific COVID-19-nsp2 Real-Time RT-PCR Assay and Its Evaluation Using Clinical Specimens.

The pandemic novel coronavirus infection, Coronavirus Disease 2019 (COVID-19), has affected at least 190 countries or territories, with 465,915 confirmed cases and 21,031 deaths. In a containment-based strategy, rapid, sensitive and specific testing is important in epidemiological control and clinical management. Using 96 SARS-CoV-2 and 104 non-SARS-CoV-2 coronavirus genomes and our in-house program, GolayMetaMiner, four specific regions longer than 50 nucleotides in the SARS-CoV-2 genome were identified. Primers were designed to target the longest and previously untargeted nsp2 region and optimized as a probe-free real-time reverse transcription-polymerase chain reaction (RT-PCR) assay. The new COVID-19-nsp2 assay had a limit of detection (LOD) of 1.8 TCID50/mL and did not amplify other human-pathogenic coronaviruses and respiratory viruses. Assay reproducibility in terms of cycle threshold (Cp) values was satisfactory, with the total imprecision (% CV) values well below 5%. Evaluation of the new assay using 59 clinical specimens from 14 confirmed cases showed 100% concordance with our previously developed COVID-19-RdRp/Hel reference assay. A rapid, sensitive, SARS-CoV-2-specific real-time RT-PCR assay, COVID-19-nsp2, was developed.