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1.
Infection ; 2020 Sep 17.
Article in English | MEDLINE | ID: covidwho-774008

ABSTRACT

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.

2.
Int J Mol Sci ; 21(18)2020 Sep 09.
Article in English | MEDLINE | ID: covidwho-760933

ABSTRACT

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.

3.
Clin Infect Dis ; 2020 Aug 25.
Article in English | MEDLINE | ID: covidwho-729112

ABSTRACT

BACKGROUND: Waning immunity occurs in patients who have recovered from COVID-19. However, it remains unclear whether true re-infection occurs. METHODS: Whole genome sequencing was performed directly on respiratory specimens collected during two episodes of COVID-19 in a patient. Comparative genome analysis was conducted to differentiate re-infection from persistent viral shedding. Laboratory results, including RT-PCR Ct values and serum SARS-CoV-2 IgG, were analyzed. RESULTS: The second episode of asymptomatic infection occurred 142 days after the first symptomatic episode in an apparently immunocompetent patient. During the second episode, there was serological evidence of elevated C-reactive protein and SARS-CoV-2 IgG seroconversion. Viral genomes from first and second episodes belong to different clades/lineages. Compared to viral genomes in GISAID, the first virus genome has a stop codon at position 64 of orf8 leading to a truncation of 58 amino acids, and was phylogenetically closely related to strains collected in March/April 2020, while the second virus genome was closely related to strains collected in July/August 2020. Another 23 nucleotide and 13 amino acid differences located in 9 different proteins, including positions of B and T cell epitopes, were found between viruses from the first and second episodes. CONCLUSIONS: Epidemiological, clinical, serological and genomic analyses confirmed that the patient had re-infection instead of persistent viral shedding from first infection. Our results suggest SARS-CoV-2 may continue to circulate among the human populations despite herd immunity due to natural infection or vaccination. Further studies of patients with re-infection will shed light on protective correlates important for vaccine design.

4.
Diagn Microbiol Infect Dis ; 98(3): 115141, 2020 Jul 17.
Article in English | MEDLINE | ID: covidwho-714791

ABSTRACT

BACKGROUND: Kawasaki disease (KD) is an acute febrile and eruptive disease with systemic vasculitis predominantly affecting young East Asian children. Recent reports showed that children with KD-like disease from KD low prevalence regions had positive SARS-CoV-2 serology despite a negative SARS-CoV-2 polymerase chain reaction (PCR) in respiratory samples. OBJECTIVES: To describe 3 pediatric Kawasaki Disease patients with false positive SARS-CoV-2 serology. STUDY DESIGN: We retrospectively recruited children with KD diagnosed during the COVID-19 outbreak in Hong Kong. Clinical characteristics and laboratory test results including SARS-CoV-2 PCR results were retrieved. We performed a microparticle-based immunoassay for the detection of IgG against nucleoprotein (NP) and spike protein receptor binding domain (RBD), and a microneutralization assay for the detection of neutralizing antibodies. RESULTS: Three Chinese children with typical KD were identified. They had no epidemiological links with COVID-19 patients and tested negative for SARS-CoV-2 NPA PCR. They were treated with IVIG and aspirin, and were discharged without complications. Subsequently 2 of them were tested positive against anti-RBD and anti-NP antibodies and 1 was tested positive against anti- RBD antibodies. However, microneutralization assay showed that neutralizing antibodies were absent, suggesting a false-positive IgG result. CONCLUSION: Detection of neutralizing antibodies is recommended to confirm previous SARS-CoV-2 infection in IgG-positive but PCR-negative patients.

5.
Int J Mol Sci ; 21(16)2020 Aug 07.
Article in English | MEDLINE | ID: covidwho-714483

ABSTRACT

Sensitive molecular assays are critical for coronavirus disease 2019 (COVID-19) diagnosis. Here, we designed and evaluated two single-tube nested (STN) real-time RT-PCR assays, targeting SARS-CoV-2 RdRp/Hel and N genes. Both STN assays had a low limit of detection and did not cross react with other human coronaviruses and respiratory viruses. Using 213 initial respiratory specimens from suspected COVID-19 patients, the sensitivity of both the STN COVID-19-RdRp/Hel and the STN COVID-19-N assays was 100% (99/99), while that of the comparator non-nested N assay was 95% (94/99). Among 108 follow-up specimens from confirmed COVID-19 patients who tested negative by the non-nested COVID-19-RdRp/Hel assay, 28 (25.9%) were positive for SARS-CoV-2 by the STN COVID-19-RdRp/Hel or the STN COVID-19-N assay. To evaluate the performance of our novel STN assays in pooled specimens, we created four sample pools, with each pool consisting of one low positive specimen and 49 negative specimens. While the non-nested COVID-19-RdRp/Hel assay was positive in only one of four sample pools (25%), both of the STN assays were positive in two of four samples pools (50%). In conclusion, the STN assays are highly sensitive and specific for SARS-CoV-2 detection. Their boosted sensitivity offers advantages in non-traditional COVID-19 testing algorithms such as saliva screening and pooled sample screening during massive screening.


Subject(s)
Betacoronavirus/genetics , Coronavirus Infections/virology , Molecular Diagnostic Techniques/methods , Pneumonia, Viral/virology , Real-Time Polymerase Chain Reaction/methods , Betacoronavirus/pathogenicity , Coronavirus Infections/diagnosis , Humans , Molecular Diagnostic Techniques/standards , Nucleocapsid Proteins/genetics , Pandemics , Pneumonia, Viral/diagnosis , RNA Replicase/genetics , Real-Time Polymerase Chain Reaction/standards , Respiratory Mucosa/virology , Sensitivity and Specificity , Viral Nonstructural Proteins/genetics
6.
Clin Infect Dis ; 2020 Aug 05.
Article in English | MEDLINE | ID: covidwho-694735

ABSTRACT

After two months of relative quiescence, a large COVID-19 outbreak occurred in Hong Kong in July 2020 after gradual relaxation of social distancing policy. Two unique SARS-CoV-2 phylogenetic clusters have been identified among locally-acquired cases, with most genomes belonging to cluster HK1 which is phylogenetically related to SARS-CoV-2 reported overseas.

7.
J Hosp Infect ; 2020 Jul 08.
Article in English | MEDLINE | ID: covidwho-635357

ABSTRACT

BACKGROUND: In late 2019, a novel human coronavirus, SARS-CoV-2, emerged in Wuhan, China. This virus has caused a global pandemic involving more than 200 countries. SARS-CoV-2 is highly adapted to humans and readily transmits from person-to-person. AIM: The aim of this study was to investigate the infectivity of SARS-CoV-2 under various environmental factors, disinfectants and different pH conditions. The efficacy of a variety of laboratory virus inactivation methods and home disinfectants against SARS-CoV-2 were investigated. METHODS: The residual virus in dried form or in solution was titrated on Vero E6 cell line at day 0, 1, 3, 5, and 7 after incubation at different temperatures. The viability of virus was determined after treatment with different disinfectants and pH solutions at room temperature (20∼25oC). FINDINGS: SARS-CoV-2 was able to retain viability for 3-5 days in dried form or 7 days in solution at room temperature. SARS-CoV-2 could be detected under a wide range of pH conditions from pH4 to pH11 for several days and 1 to 2 days in stool at room temperature but lost 5 logs of infectivity. A variety of commonly used disinfectants and laboratory inactivation procedures were found to reduce viral viability effectively. CONCLUSION: This study demonstrates the stability of SARS-CoV-2 on environmental surfaces and raises the possibility of faecal-oral transmission. Commonly used fixatives, nucleic acid extraction methods and heat inactivation were found to significantly reduce viral infectivity that could ensure hospital and laboratory safety during the COVID-19 pandemic.

8.
Emerg Microbes Infect ; 9(1): 1664-1670, 2020 Dec.
Article in English | MEDLINE | ID: covidwho-630769

ABSTRACT

Coronavirus disease 2019 (COVID-19) has a wide spectrum of disease severity from mild upper respiratory symptoms to respiratory failure. The role of neutralizing antibody (NAb) response in disease progression remains elusive. This study determined the seroprevalence of 733 non-COVID-19 individuals from April 2018 to February 2020 in the Hong Kong Special Administrative Region and compared the neutralizing antibody (NAb) responses of eight COVID-19 patients admitted to the intensive care unit (ICU) with those of 42 patients not admitted to the ICU. We found that NAb against SARS-CoV-2 was not detectable in any of the anonymous serum specimens from the 733 non-COVID-19 individuals. The peak serum geometric mean NAb titer was significantly higher among the eight ICU patients than the 42 non-ICU patients (7280 [95% confidence interval (CI) 1468-36099]) vs (671 [95% CI, 368-1223]). Furthermore, NAb titer increased significantly at earlier infection stages among ICU patients than among non-ICU patients. The median number of days to reach the peak Nab titers after symptoms onset was shorter among the ICU patients (17.6) than that of the non-ICU patients (20.1). Multivariate analysis showed that oxygen requirement and fever during admission were the only clinical factors independently associated with higher NAb titers. Our data suggested that SARS-CoV-2 was unlikely to have silently spread before the COVID-19 emergence in Hong Kong. ICU patients had an accelerated and augmented NAb response compared to non-ICU patients, which was associated with disease severity. Further studies are required to understand the relationship between high NAb response and disease severity.


Subject(s)
Antibodies, Neutralizing/blood , Antibodies, Viral/blood , Betacoronavirus/immunology , Coronavirus Infections/immunology , Pneumonia, Viral/immunology , Adult , Aged , Cells, Cultured , Female , Humans , Intensive Care Units , Male , Middle Aged , Pandemics
10.
Lancet Infect Dis ; 20(9): 1051-1060, 2020 09.
Article in English | MEDLINE | ID: covidwho-597077

ABSTRACT

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).


Subject(s)
Betacoronavirus/physiology , Coronavirus Infections/virology , Disease Outbreaks , Pneumonia, Viral/virology , Seroconversion , Virus Shedding , Adult , Aged , Betacoronavirus/genetics , Betacoronavirus/immunology , Contact Tracing , Coronavirus Infections/diagnostic imaging , Coronavirus Infections/epidemiology , Coronavirus Infections/prevention & control , Female , Hong Kong , Humans , Japan/epidemiology , Male , Middle Aged , Pandemics/prevention & control , Pneumonia, Viral/diagnostic imaging , Pneumonia, Viral/epidemiology , Pneumonia, Viral/prevention & control , Quarantine , Ships , Thorax/diagnostic imaging , Viral Load , Young Adult
11.
Open Forum Infect. Dis. ; 6(7)20200605.
Article in English | ELSEVIER | ID: covidwho-592170

ABSTRACT

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.

12.
Infect Control Hosp Epidemiol ; : 1-8, 2020 Jun 08.
Article in English | MEDLINE | ID: covidwho-547446

ABSTRACT

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.

13.
J Med Virol ; 2020 Jun 05.
Article in English | MEDLINE | ID: covidwho-530466

ABSTRACT

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.

14.
Open Forum Infect. Dis. ; 6(7)20200605.
Article in English | ELSEVIER | ID: covidwho-548236

ABSTRACT

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.

15.
The Lancet Microbe ; 2020.
Article | WHO COVID | ID: covidwho-505614

ABSTRACT

Summary 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)

16.
J Clin Microbiol ; 58(8)2020 Jul 23.
Article in English | MEDLINE | ID: covidwho-457502

ABSTRACT

In December 2019, the coronavirus disease 2019 (COVID-19) pandemic caused by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) was first reported in the Hubei province of China and later spread all over the world. There was an urgent need of a high-throughput molecular test for screening the COVID-19 patients in the community. The Luminex NxTAG CoV extended panel is a high-throughput FDA emergency use-authorized molecular diagnostic assay for SARS-CoV-2 detection. This system targets three genes (ORF1ab, N, and E genes) of SARS-CoV-2, the ORF1ab region of SARS-CoV, and the ORF5 region of MERS-CoV. In this study, we evaluated the diagnostic performance of this system with nasopharyngeal swab specimens of 214 suspected COVID-19 patients in Hong Kong. The results were compared with our routine COVID-19 reverse transcription-PCR (RT-PCR) protocol with a LightMix SarbecoV E-gene kit and an in-house RdRp/Hel RT-PCR assay. The NxTAG CoV extended panel demonstrated 97.8% sensitivity and 100% specificity to SARS-CoV-2 in nasopharyngeal specimens. On low-viral load specimens, the sensitivity of the NxTAG panel could still maintain at 85.71%. Strong agreement was observed between the NxTAG panel and the routine COVID-19 RT-PCR protocol (kappa value = 0.98). Overall, the E gene target of the NxTAG panel demonstrated the highest sensitivity among the three SARS-CoV-2 targets, while the N gene targets demonstrated the least. In conclusion, the NxTAG CoV extended panel is simple to use, and it has high diagnostic sensitivity and specificity to SARS-CoV-2 in nasopharyngeal specimens. We recommend this diagnostic system for high-throughput COVID-19 screening in the community.


Subject(s)
Betacoronavirus/isolation & purification , Clinical Laboratory Techniques/methods , Coronavirus Infections/diagnosis , High-Throughput Nucleotide Sequencing/methods , Molecular Diagnostic Techniques/methods , Pneumonia, Viral/diagnosis , Adult , Aged , Betacoronavirus/genetics , Coronavirus Infections/virology , Female , Hong Kong , Humans , Male , Middle Aged , Nasopharynx/virology , Pandemics , Pneumonia, Viral/virology , Sensitivity and Specificity
17.
Clin Infect Dis ; 2020 May 30.
Article in English | MEDLINE | ID: covidwho-423113

ABSTRACT

BACKGROUND: Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is believed to be mostly transmitted by medium-to-large sized respiratory droplets although airborne transmission is theoretically possible in healthcare settings involving aerosol-generating procedures. Exposure to respiratory droplets can theoretically be reduced by surgical mask usage. However, there is a lack of experimental evidence supporting surgical mask usage for prevention of COVID-19. METHODS: We used a well-established golden Syrian hamster SARS-CoV-2 model. We placed SARS-CoV-2-challenged index hamsters and naïve hamsters into closed system units each comprising two different cages separated by a polyvinyl chloride air porous partition with unidirectional airflow within the isolator. The effect of a surgical mask partition placed in between the cages was investigated. Besides clinical scoring, hamster specimens were tested for viral load, histopathology, and viral nucleocapsid antigen expression. RESULTS: Non-contact transmission was found in 66.7% (10/15) of exposed naïve hamsters. Surgical mask partition for challenged index or naïve hamsters significantly reduced transmission to 25% (6/24, P=0.018). Surgical mask partition for challenged index hamsters significantly reduced transmission to only 16.7% (2/12, P=0.019) of exposed naïve hamsters. Unlike the severe COVID-19 manifestations of challenged hamsters, infected naïve hamsters had lower clinical scores, milder histopathological changes, and lower viral nucleocapsid antigen expression in respiratory tract tissues. CONCLUSIONS: SARS-CoV-2 could be transmitted by respiratory droplets or airborne droplet nuclei in the hamster model. Such transmission could be reduced by surgical mask usage, especially when masks were worn by infected individuals.

18.
J Clin Virol ; 129: 104476, 2020 08.
Article in English | MEDLINE | ID: covidwho-401826

ABSTRACT

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.


Subject(s)
Betacoronavirus/isolation & purification , Clinical Laboratory Techniques/methods , Coronavirus Infections/diagnosis , Molecular Diagnostic Techniques/methods , Pneumonia, Viral/diagnosis , RNA, Viral/analysis , Adolescent , Adult , Aged , Aged, 80 and over , Betacoronavirus/genetics , Female , Humans , Limit of Detection , Male , Middle Aged , Pandemics , RNA, Viral/genetics , Reference Standards , Reproducibility of Results , Sensitivity and Specificity , Time Factors , Young Adult
19.
Emerg Microbes Infect ; 9(1): 1356-1359, 2020 Dec.
Article in English | MEDLINE | ID: covidwho-381795

ABSTRACT

During the Coronavirus disease 2019 (COVID-19) pandemic, logistic problems associated with specimen collection limited the SARS-CoV-2 testing, especially in the community. In this study, we assessed the use of posterior oropharyngeal saliva as specimens for the detection of SARS-CoV-2 in an automated point-of-care molecular assay. Archived nasopharyngeal swab (NPS) and posterior oropharyngeal saliva specimens of 58 COVID-19 patients were tested with the Xpert® Xpress SARS-CoV-2 assay. SARS-CoV-2 was detected in either NPS or saliva specimens of all patients. Among them, 84.5% (49/58) tested positive in both NPS and saliva, 10.3% (6/58) tested positive in NPS only, and 5.2% (3/58) tested positive in saliva only. No significant difference in the detection rate was observed between NPS and saliva (McNemar's test p = 0.5078). The detection rate was slightly higher for N2 (NPS 94.8% and Saliva 93.1%) than that of the E gene target (Saliva: 89.7% vs 82.8%) on both specimen types. Significantly earlier median Ct value was observed for NPS comparing to that of saliva on both E (26.8 vs 29.7, p = 0.0002) and N2 gene target (29.3 vs 32.3, p = 0.0002). The median Ct value of E gene target was significantly earlier than that of the N2 gene target for both NPS (26.8 vs 29.3, p < 0.0001) and saliva (29.7 vs 32.3, p < 0.0001). In conclusion, posterior oropharyngeal saliva and NPS were found to have similar detection rates in the point-of-care test for SARS-CoV-2 detection. Since posterior oropharyngeal saliva can be collected easily, the use of saliva as an alternative specimen type for SARS-CoV-2 detection is recommended.


Subject(s)
Betacoronavirus/isolation & purification , Biological Assay , Clinical Laboratory Techniques/methods , Coronavirus Infections/diagnosis , Pandemics , Pneumonia, Viral/diagnosis , Point-of-Care Systems/standards , Viral Envelope Proteins/isolation & purification , Adult , Betacoronavirus/pathogenicity , Clinical Laboratory Techniques/instrumentation , Coronavirus Infections/epidemiology , Coronavirus Infections/virology , Female , Hong Kong/epidemiology , Humans , Male , Middle Aged , Oropharynx/virology , Pneumonia, Viral/epidemiology , Pneumonia, Viral/virology , Saliva/virology , Specimen Handling/methods
20.
Nat Med ; 26(7): 1077-1083, 2020 07.
Article in English | MEDLINE | ID: covidwho-260261

ABSTRACT

A novel coronavirus-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-emerged in humans in Wuhan, China, in December 2019 and has since disseminated globally1,2. As of April 16, 2020, the confirmed case count of coronavirus disease 2019 (COVID-19) had surpassed 2 million. Based on full-genome sequence analysis, SARS-CoV-2 shows high homology to SARS-related coronaviruses identified in horseshoe bats1,2. Here we show the establishment and characterization of expandable intestinal organoids derived from horseshoe bats of the Rhinolophus sinicus species that can recapitulate bat intestinal epithelium. These bat enteroids are fully susceptible to SARS-CoV-2 infection and sustain robust viral replication. Development of gastrointestinal symptoms in some patients with COVID-19 and detection of viral RNA in fecal specimens suggest that SARS-CoV-2 might cause enteric, in addition to respiratory, infection3,4. Here we demonstrate active replication of SARS-CoV-2 in human intestinal organoids and isolation of infectious virus from the stool specimen of a patient with diarrheal COVID-19. Collectively, we established the first expandable organoid culture system of bat intestinal epithelium and present evidence that SARS-CoV-2 can infect bat intestinal cells. The robust SARS-CoV-2 replication in human intestinal organoids suggests that the human intestinal tract might be a transmission route of SARS-CoV-2.


Subject(s)
Betacoronavirus/pathogenicity , Coronavirus Infections/pathology , Coronavirus Infections/transmission , Intestines/virology , Organoids/virology , Pneumonia, Viral/pathology , Pneumonia, Viral/transmission , Animals , Cell Differentiation , Cells, Cultured , Child, Preschool , Chiroptera/virology , Chlorocebus aethiops , Coronavirus Infections/virology , Enterocytes/pathology , Enterocytes/physiology , Enterocytes/virology , Female , Humans , Infant , Intestinal Mucosa/pathology , Intestinal Mucosa/virology , Intestines/pathology , Male , Organoids/pathology , Pandemics , Pneumonia, Viral/virology , Reverse Transcriptase Polymerase Chain Reaction , Vero Cells , Viral Load/genetics , Viral Load/methods , Viral Tropism/physiology
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