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1.
Front Immunol ; 11: 618402, 2020.
Article in English | MEDLINE | ID: covidwho-1045518

ABSTRACT

Prolonged shedding of viral RNA occurs in some individuals following SARS-CoV-2 infection. We perform comprehensive immunologic evaluation of one individual with prolonged shedding. The case subject recovered from severe COVID-19 and tested positive for SARS-CoV-2 viral RNA repeatedly as many as 87 days after the first positive test, 97 days after symptom onset. The subject did not have any associated rise in anti-Spike protein antibody titers or plasma neutralization activity, arguing against re-infection. This index subject exhibited a profoundly diminished circulating CD8+ T cell population and correspondingly low SARS-CoV-2-specific CD8+ T cell responses when compared with a cohort of other recovering COVID-19 subjects. CD4+ T cell responses and neutralizing antibody responses developed as expected in this individual. Our results demonstrate that detectable viral RNA shedding in the upper airway can occur more than 3 months following infection in some individuals with COVID-19 and suggest that impaired CD8+ T cells may play a role in prolonged viral RNA shedding.


Subject(s)
CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/virology , /virology , RNA, Viral/immunology , Virus Shedding/immunology , Aged, 80 and over , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , CD4-Positive T-Lymphocytes/immunology , CD4-Positive T-Lymphocytes/virology , Humans , Male , Prospective Studies , Viral Load/methods
2.
Anal Methods ; 13(2): 169-178, 2021 01 21.
Article in English | MEDLINE | ID: covidwho-1039652

ABSTRACT

We demonstrate a loop-mediated isothermal amplification (LAMP) method to detect and amplify SARS-CoV-2 genetic sequences using a set of in-house designed initiators that target regions encoding the N protein. We were able to detect and amplify SARS-CoV-2 nucleic acids in the range of 62 to 2 × 105 DNA copies by this straightforward method. Using synthetic SARS-CoV-2 samples and RNA extracts from patients, we demonstrate that colorimetric LAMP is a quantitative method comparable in diagnostic performance to RT-qPCR (i.e., sensitivity of 92.85% and specificity of 81.25% in a set of 44 RNA extracts from patients analyzed in a hospital setting).


Subject(s)
/methods , Molecular Diagnostic Techniques/methods , Nucleic Acid Amplification Techniques/methods , RNA/analysis , Viral Load/methods , /diagnosis , Colorimetry/methods , DNA/analysis , DNA/chemistry , Fluorescent Dyes/chemistry , Humans , Intercalating Agents/chemistry , Phenolsulfonphthalein/chemistry , Phosphoproteins , RNA/chemistry
3.
Sci Rep ; 10(1): 22425, 2020 12 30.
Article in English | MEDLINE | ID: covidwho-1003311

ABSTRACT

Here we present a rapid and versatile method for capturing and concentrating SARS-CoV-2 from contrived transport medium and saliva samples using affinity-capture magnetic hydrogel particles. We demonstrate that the method concentrates virus from 1 mL samples prior to RNA extraction, substantially improving detection of virus using real-time RT-PCR across a range of viral titers (100-1,000,000 viral copies/mL) and enabling detection of virus using the 2019 nCoV CDC EUA Kit down to 100 viral copies/mL. This method is compatible with commercially available nucleic acid extraction kits (i.e., from Qiagen) and a simple heat and detergent method that extracts viral RNA directly off the particle, allowing a sample processing time of 10 min. We furthermore tested our method in transport medium diagnostic remnant samples that previously had been tested for SARS-CoV-2, showing that our method not only correctly identified all positive samples but also substantially improved detection of the virus in low viral load samples. The average improvement in cycle threshold value across all viral titers tested was 3.1. Finally, we illustrate that our method could potentially be used to enable pooled testing, as we observed considerable improvement in the detection of SARS-CoV-2 RNA from sample volumes of up to 10 mL.


Subject(s)
/methods , Hydrogels/chemistry , Nasopharynx/virology , RNA, Viral/analysis , Saliva/virology , Diagnostic Tests, Routine , Humans , Real-Time Polymerase Chain Reaction , Sensitivity and Specificity , Specimen Handling , Viral Load/methods
4.
PLoS Pathog ; 16(11): e1008949, 2020 11.
Article in English | MEDLINE | ID: covidwho-922716

ABSTRACT

The COVID-19 has emerged as an epidemic, causing severe pneumonia with a high infection rate globally. To better understand the pathogenesis caused by SARS-CoV-2, we developed a rhesus macaque model to mimic natural infection via the nasal route, resulting in the SARS-CoV-2 virus shedding in the nose and stool up to 27 days. Importantly, we observed the pathological progression of marked interstitial pneumonia in the infected animals on 5-7 dpi, with virus dissemination widely occurring in the lower respiratory tract and lymph nodes, and viral RNA was consistently detected from 5 to 21 dpi. During the infection period, the kinetics response of T cells was revealed to contribute to COVID-19 progression. Our findings implied that the antiviral response of T cells was suppressed after 3 days post infection, which might be related to increases in the Treg cell population in PBMCs. Moreover, two waves of the enhanced production of cytokines (TGF-α, IL-4, IL-6, GM-CSF, IL-10, IL-15, IL-1ß), chemokines (MCP-1/CCL2, IL-8/CXCL8, and MIP-1ß/CCL4) were detected in lung tissue. Our data collected from this model suggested that T cell response and cytokine/chemokine changes in lung should be considered as evaluation parameters for COVID-19 treatment and vaccine development, besides of observation of virus shedding and pathological analysis.


Subject(s)
Betacoronavirus/pathogenicity , Coronavirus Infections/pathology , Pneumonia, Viral/pathology , Animals , Coronavirus Infections/drug therapy , Coronavirus Infections/virology , Cytokines/immunology , Disease Models, Animal , Lung/immunology , Lung/pathology , Macaca mulatta , Pandemics , Pneumonia, Viral/virology , Viral Load/methods , Virulence , Virus Shedding
5.
Trials ; 21(1): 892, 2020 Oct 27.
Article in English | MEDLINE | ID: covidwho-895025

ABSTRACT

OBJECTIVES: The primary objectives of this study are to determine efficacy of Siddha medicine, Kabasura kudineer in reduction of SARS-CoV-2 viral load and reducing the onset of symptoms in asymptomatic COVID-19 when compared to Vitamin C and Zinc (CZ) supplementation. In addition, the trial will examine the changes in the immunological markers of the Siddha medicine against control. The secondary objectives of the trial are to evaluate the safety of the Siddha medicine and to document clinical profile of asymptomatic COVID-19 as per principles of Siddha system of Medicine. TRIAL DESIGN: A single centre, open-label, parallel group (1:1 allocation ratio), exploratory randomized controlled trial. PARTICIPANTS: Cases admitted at non-hospital settings designated as COVID Care Centre and managed by the State Government Stanley Medical College, Chennai, Tamil Nadu, India will be recruited. Eligible participants will be those tested positive for COVID-19 by Reverse Transcriptase Polymerase Chain reaction (RT-PCR) aged 18 to 55 years without any symptoms and co-morbidities like diabetes mellitus, hypertension and bronchial asthma. Those pregnant or lactating, with severe respiratory disease, already participating in COVID trials and with severe illness like malignancy will be excluded. INTERVENTION AND COMPARATOR: Adopting traditional methods, decoction of Kabasura kudineer will be prepared by boiling 5g of KSK powder in 240 ml water and reduced to one-fourth (60ml) and filtered. The KSK group will receive a dose of 60ml decoction, orally in the morning and evening after food for 14 days. The control group will receive Vitamin C (60000 IU) and Zinc tablets (100mg) orally in the morning and evening respectively for 14 days. MAIN OUTCOMES: The primary outcomes are the reduction in the SARS-CoV-2 load [as measured by cyclic threshold (CT) value of RT-PCR] from the baseline to that of seventh day of the treatment, prevention of progression of asymptomatic to symptomatic state (clinical symptoms like fever, cough and breathlessness) and changes in the immunity markers [Interleukins (IL) 6, IL10, IL2, Interferon gamma (IFNγ) and Tumor Necrosis Factor (TNF) alpha]. Clinical assessment of COVID-19 as per standard Siddha system of medicine principles and the occurrence of adverse effects will be documented as secondary outcomes. RANDOMISATION: The assignment to the study or control group will be allocated in equal numbers through randomization using random number generation in Microsoft Excel by a statistician who is not involved in the trial. The allocation scheme will be made by an independent statistician using a sealed envelope. The participants will be allocated immediately after the eligibility assessment and informed consent procedures. BLINDING (MASKING): This study is unblinded. The investigators will be blinded to data analysis, which will be carried out by a statistician who is not involved in the trial. NUMBERS TO BE RANDOMISED (SAMPLE SIZE): Sample size could not be calculated, as there is no prior trial on KSK. This trial will be a pilot trial. Hence, we intend to recruit 60 participants in total using a 1:1 allocation ratio, with 30 participants randomised into each arm. TRIAL STATUS: Protocol version 2.0 dated 16th May 2020. Recruitment is completed. The trial started recruitment on the 25th May 2020. We anticipate study including data analysis will finish on November 2020. We also stated that protocol was submitted before the end of data collection TRIAL REGISTRATION: The study protocol was registered with clinical trial registry of India (CTRI) with CTRI/2020/05/025215 on 16 May 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).


Subject(s)
Ascorbic Acid , Betacoronavirus , Coronavirus Infections , Medicine, Ayurvedic/methods , Pandemics , Pneumonia, Viral , Zinc , Adult , Ascorbic Acid/administration & dosage , Ascorbic Acid/adverse effects , Asymptomatic Infections/therapy , Betacoronavirus/drug effects , Betacoronavirus/isolation & purification , Coronavirus Infections/diagnosis , Coronavirus Infections/drug therapy , Dietary Supplements , Drug Monitoring/methods , Female , Humans , India , Male , Pneumonia, Viral/diagnosis , Pneumonia, Viral/drug therapy , Randomized Controlled Trials as Topic , Treatment Outcome , Viral Load/methods , Zinc/administration & dosage , Zinc/adverse effects
6.
Trials ; 21(1): 886, 2020 Oct 27.
Article in English | MEDLINE | ID: covidwho-895021

ABSTRACT

OBJECTIVES: We will evaluate the efficacy and safety of favipiravir and interferon beta-1a compared to lopinavir/ritonavir and interferon beta-1a in patients with confirmed COVID-19, who are moderately ill. TRIAL DESIGN: This is a phase 3, single-center, randomized, open-label, controlled trial with a parallel-group design carried out at Shahid Mohammadi Hospital, Bandar Abbas, Iran. PARTICIPANTS: All patients with age ≥ 20 years admitted at the Severe Acute Respiratory Syndrome Departments of the Shahid Mohammadi Hospital, Bandar Abbas, Iran, will be screened for the following criteria. INCLUSION CRITERIA: 1. Confirmed diagnosis of infection with SARS-CoV-2 using polymerase chain reaction and/or antibody tests. 2. Moderate COVID-19 pneumonia (via computed tomography and/or X-ray imaging), requiring hospitalization. 3. Hospitalized ≤ 48 h. 4. Signing informed consent and willingness of the participant to accept randomization to any assigned treatment arm. EXCLUSION CRITERIA: 1. Underlying conditions, including chronic hepatitis, cirrhosis, cholestatic liver diseases, cholecystitis, peptic ulcers, acute and chronic renal failure, and peptic ulcers. 2. Severe and critical COVID-19 pneumonia. 3. History of allergy to favipiravir, lopinavir/ritonavir, and interferon beta-1a. 4. Pregnancy and breastfeeding. INTERVENTION AND COMPARATOR: Intervention group: favipiravir (Zhejiang Hisun, China) with interferon beta-1a (CinnaGen, Iran). This group will receive 1600 mg favipiravir twice a day for the first day and 600 mg twice a day for the following 4 days with five doses of 44 mcg interferon beta-1a every other day. CONTROL GROUP: lopinavir/ritonavir (Heterd Company, India) with interferon beta-1a (CinnaGen, Iran). This group will receive 200/50 mg lopinavir/ritonavir twice a day for 7 days with five doses of 44 mcg interferon beta-1a every other day. Other supportive and routine care will be the same in both groups. MAIN OUTCOMES: The primary outcome of the trial is the viral load of SARS-CoV-2 in the nasopharyngeal samples assessed by RT-PCR after 7 days of randomization as well as clinical improvement of fever and O2 saturation within 7 days of randomization. The secondary outcomes are the length of hospital stay and the incidence of serious adverse drug reactions within 7 days of randomization. RANDOMIZATION: Eligible patients will be allocated to one of the study arms using block randomization in a 1:1 ratio (each block consists of 10 patients). A web-based system will be used to generate random numbers for the allocation sequence. Each number relates to one of the study arms. BLINDING (MASKING): This is an open-label trial without blinding and placebo control. NUMBERS TO BE RANDOMIZED (SAMPLE SIZE): A total of 60 patients will be randomized into two groups (30 patients in the intervention group and 30 patients in the control group). TRIAL STATUS: The trial protocol is version 1.0, 22 July 2020. Recruitment began on 25 July 2020 and is anticipated to be completed by 25 September 2020. TRIAL REGISTRATION: Iranian Registry of Clinical Trials (IRCT) IRCT20200506047323N3 . Registered on 22 July 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 the dissemination of this material, the familiar formatting has been eliminated; this letter serves as a summary of the key elements of the full protocol.


Subject(s)
Amides , Coronavirus Infections , Drug Therapy, Combination/methods , Interferons , Lopinavir , Pandemics , Pneumonia, Viral , Pyrazines , Ritonavir , Adult , Amides/administration & dosage , Amides/adverse effects , Antiviral Agents/administration & dosage , Antiviral Agents/adverse effects , Betacoronavirus/drug effects , Betacoronavirus/isolation & purification , Clinical Laboratory Techniques/methods , Coronavirus Infections/diagnosis , Coronavirus Infections/drug therapy , Drug Combinations , Drug Monitoring/methods , Female , Humans , Interferons/administration & dosage , Interferons/adverse effects , Iran , Lopinavir/administration & dosage , Lopinavir/adverse effects , Male , Pneumonia, Viral/diagnosis , Pneumonia, Viral/drug therapy , Pyrazines/administration & dosage , Pyrazines/adverse effects , Randomized Controlled Trials as Topic , Ritonavir/administration & dosage , Ritonavir/adverse effects , Severity of Illness Index , Treatment Outcome , Viral Load/methods
7.
Trials ; 21(1): 785, 2020 Sep 14.
Article in English | MEDLINE | ID: covidwho-757090

ABSTRACT

OBJECTIVES: 1- To compare the effectiveness of 1% Hydrogen peroxide, 0.2% Povidone-Iodine, 2% hypertonic saline and a novel solution Neem extract (Azardirachta indica) in reducing intra-oral viral load in COVID-19 positive patients. 2- To determine the salivary cytokine profiles of IL-2, IL-4, IL-6, IL-10, TNF-α, IFN-γ and IL- 17 among COVID-19 patients subjected to 1% Hydrogen peroxide, 0.2% Povidone-Iodine, 2% hypertonic saline or Neem extract (Azardirachta indica) based gargles. TRIAL DESIGN: This will be a parallel group, quadruple blind-randomised controlled pilot trial with an add on laboratory based study. PARTICIPANTS: A non-probability, purposive sampling technique will be followed to identify participants for this study. The clinical trial will be carried out at the Aga Khan University Hospital (AKUH), Karachi, Pakistan. The viral PCR tests will be done at main AKUH clinical laboratories whereas the immunological tests (cytokine analysis) will be done at the Juma research laboratory of AKUH. The inclusion criteria are laboratory-confirmed COVID-19 positive patients, male or female, in the age range of 18-65 years, with mild to moderate disease, already admitted to the AKUH. Subjects with low Glasgow coma score, with a history of radiotherapy or chemotherapy, who are more than 7 days past the onset of COVID- 19 symptoms, or intubated or edentulous patients will be excluded. Patients who are being treated with any form of oral or parenteral antiviral therapy will be excluded, as well as patients with known pre-existing chronic mucosal lesions such as lichen planus. INTERVENTION AND COMPARATOR: Group A (n=10) patients on 10 ml gargle and nasal lavage using 0.2% Povidone-Iodine (Betadiene® by Aviro Health Inc./ Pyodine® by Brooks Pharma Inc.) for 20-30 seconds, thrice daily for 6 days. Group B (n=10) patients will be subjected to 10 ml gargle and nasal lavage using 1% Hydrogen peroxide (HP® by Karachi Chemicals Products Inc./ ActiveOxy® by Boumatic Inc.) for 20-30 seconds, thrice daily for 6 days. Group C will comprised of (n=10) subjects on 10ml gargle and nasal lavage using Neem extract solution (Azardirachta indica) formulated by Karachi University (chemistry department laboratories) for 20-30 seconds, thrice daily for 6 days. Group D (n=10) patients will use 2% hypertonic saline (Plabottle® by Otsuka Inc.) gargle and nasal lavage for a similar time period. Group E (n=10) will serve as positive controls. These will be given simple distilled water gargles and nasal lavage for 20-30 seconds, thrice daily for six days. For nasal lavage, a special douche syringe will be provided to each participant. Its use will be thoroughly explained by the data collection officer. After each use, the patient is asked not to eat, drink, or rinse their mouth for the next 30 minutes. MAIN OUTCOMES: The primary outcome is the reduction in the intra-oral viral load confirmed with real time quantitative PCR. RANDOMISATION: The assignment to the study group/ allocation will be done using the sealed envelope method under the supervision of Clinical Trial Unit (CTU) of Aga Khan University, Karachi, Pakistan. The patients will be randomised to their respective study group (1:1:1:1:1 allocation ratio) immediately after the eligibility assessment and consent administration is done. BLINDING (MASKING): The study will be quadruple-blinded. Patients, intervention provider, outcome assessor and the data collection officer will be blinded. The groups will be labelled as A, B, C, D or E. The codes of the intervention will be kept in lock & key at the CTU and will only be revealed at the end of study or if the study is terminated prematurely. NUMBERS TO BE RANDOMISED (SAMPLE SIZE): As there is no prior work on this research question, so no assumptions for the sample size calculation could be made. The present study will serve as a pilot trial. We intend to study 50 patients in five study groups with 10 patients in each study group. For details, please refer to Fig. 1 for details. TRIAL STATUS: Protocol version is 7.0, approved by the department and institutional ethics committees and clinical trial unit of the university hospital. Recruitment is planned to start as soon as the funding is sanctioned. The total duration of the study is expected to be 6 months i.e. August 2020-January 2021. TRIAL REGISTRATION: This study protocol was registered at www.clinicaltrials.gov on 10 April 2020 NCT04341688 . 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). Fig. 1 Flow diagram of study-participants' timeline.


Subject(s)
Azadirachta , Betacoronavirus , Coronavirus Infections , Hydrogen Peroxide/administration & dosage , Pandemics , Plant Extracts/administration & dosage , Pneumonia, Viral , Povidone-Iodine/administration & dosage , Saline Solution, Hypertonic/administration & dosage , Viral Load , Adult , Anti-Infective Agents, Local/administration & dosage , Betacoronavirus/drug effects , Betacoronavirus/isolation & purification , Coronavirus Infections/diagnosis , Coronavirus Infections/immunology , Coronavirus Infections/therapy , Female , Hospitalization , Humans , Male , Monitoring, Immunologic/methods , Mouthwashes/administration & dosage , Nasal Lavage/methods , Pneumonia, Viral/diagnosis , Pneumonia, Viral/immunology , Pneumonia, Viral/therapy , Randomized Controlled Trials as Topic , Viral Load/drug effects , Viral Load/methods
8.
J Infect Dis ; 222(6): 903-909, 2020 08 17.
Article in English | MEDLINE | ID: covidwho-726096

ABSTRACT

High-throughput molecular testing for severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) may be enabled by group testing in which pools of specimens are screened, and individual specimens tested only after a pool tests positive. Several laboratories have recently published examples of pooling strategies applied to SARS-CoV-2 specimens, but overall guidance on efficient pooling strategies is lacking. Therefore we developed a model of the efficiency and accuracy of specimen pooling algorithms based on available data on SAR-CoV-2 viral dynamics. For a fixed number of tests, we estimate that programs using group testing could screen 2-20 times as many specimens compared with individual testing, increase the total number of true positive infections identified, and improve the positive predictive value of results. We compare outcomes that may be expected in different testing situations and provide general recommendations for group testing implementation. A free, publicly-available Web calculator is provided to help inform laboratory decisions on SARS-CoV-2 pooling algorithms.


Subject(s)
Betacoronavirus/isolation & purification , Clinical Laboratory Techniques/methods , Coronavirus Infections/epidemiology , Pneumonia, Viral/epidemiology , Specimen Handling/methods , Algorithms , Betacoronavirus/genetics , Coronavirus Infections/diagnosis , Humans , Incidence , Pandemics , Pneumonia, Viral/diagnosis , Predictive Value of Tests , RNA, Viral/genetics , RNA, Viral/isolation & purification , Reverse Transcriptase Polymerase Chain Reaction , Sensitivity and Specificity , Viral Load/methods
9.
J Infect Chemother ; 26(12): 1324-1327, 2020 Dec.
Article in English | MEDLINE | ID: covidwho-723195

ABSTRACT

Most patients with coronavirus disease 2019 (COVID-19) have just only mild symptoms, but about 5% are very severe. Although extracorporeal membranous oxygenation (ECMO) is sometimes used in critically patients with COVID-19, ECMO is only an adjunct, not the main treatment. If the patient's condition deteriorates and it is determined to be irreversible, it is necessary to decide to stop ECMO. A 54-year-old man was admitted on day 6 of onset with a chief complaint of high fever and cough. Computed tomography (CT) showed a ground glass opacity in both lungs, and reverse transcription-polymerase chain reaction (RT-PCR) diagnosed COVID-19. He was admitted to the hospital and started to receive oxygen and favipiravir. After that, his respiratory condition deteriorated, and he was intubated and ventilated on day 9 of onset, and ECMO was introduced on day 12. Two days after the introduction of ECMO, C-reactive protein (CRP) increased, chest X-p showed no improvement in pneumonia, and PaO2/FiO2 decreased again. As D-dimer rose and found a blood clot in the ECMO circuit, we had to decide whether to replace the circuit and continue with ECMO or stop ECMO. At this time, the viral load by RT-PCR was drastically reduced to about 1/1750. We decided to continue ECMO therapy and replaced the circuit. The patient's respiratory status subsequently improved and ECMO was stopped on day 21 of onset. In conclusion, viral load measurement by RT-PCR may be one of the indicators for promoting the treatment of severe COVID-19 patients.


Subject(s)
Betacoronavirus/isolation & purification , Coronavirus Infections/therapy , Coronavirus Infections/virology , Extracorporeal Membrane Oxygenation/methods , Pneumonia, Viral/therapy , Pneumonia, Viral/virology , Viral Load/methods , Amides/therapeutic use , Antiviral Agents/therapeutic use , Clinical Laboratory Techniques , Coronavirus Infections/diagnosis , Decision Making , Hospitalization , Humans , Lung/diagnostic imaging , Male , Middle Aged , Pandemics , Pneumonia, Viral/diagnosis , Pyrazines/therapeutic use , Reverse Transcriptase Polymerase Chain Reaction , Tomography, X-Ray Computed , Treatment Outcome
10.
Clin Infect Dis ; 71(15): 847-849, 2020 07 28.
Article in English | MEDLINE | ID: covidwho-719206

ABSTRACT

A well 6-month-old infant with coronavirus disease 2019 (COVID-19) had persistently positive nasopharyngeal swabs up to day 16 of admission. This case highlights the difficulties in establishing the true incidence of COVID-19, as asymptomatic individuals can excrete the virus. These patients may play important roles in human-to-human transmission in the community.


Subject(s)
Coronavirus Infections/transmission , Coronavirus Infections/virology , Pneumonia, Viral/transmission , Pneumonia, Viral/virology , Betacoronavirus/pathogenicity , Humans , Infant , Male , Pandemics , Serologic Tests/methods , Singapore , Viral Load/methods
12.
Viruses ; 12(6)2020 06 08.
Article in English | MEDLINE | ID: covidwho-574875

ABSTRACT

Clinical samples collected in coronavirus disease 19 (COVID-19), patients are commonly manipulated in biosafety level 2 laboratories for molecular diagnostic purposes. Here, we tested French norm NF-EN-14476+A2 derived from European standard EN-14885 to assess the risk of manipulating infectious viruses prior to RNA extraction. SARS-CoV-2 cell-culture supernatant and nasopharyngeal samples (virus-spiked samples and clinical samples collected in COVID-19 patients) were used to measure the reduction of infectivity after 10 minute contact with lysis buffer containing various detergents and chaotropic agents. A total of thirteen protocols were evaluated. Two commercially available formulations showed the ability to reduce infectivity by at least 6 log 10, whereas others proved less effective.


Subject(s)
Betacoronavirus/drug effects , Coronavirus Infections/virology , Pneumonia, Viral/virology , Virus Inactivation/drug effects , Animals , Betacoronavirus/genetics , Betacoronavirus/isolation & purification , Betacoronavirus/physiology , Cell Culture Techniques/methods , Chlorocebus aethiops , Containment of Biohazards/methods , Containment of Biohazards/standards , Humans , Nasopharynx/virology , Pandemics , RNA, Viral/isolation & purification , Specimen Handling/methods , Vero Cells , Viral Load/methods
13.
Emerg Microbes Infect ; 9(1): 1259-1268, 2020 Dec.
Article in English | MEDLINE | ID: covidwho-342833

ABSTRACT

Quantitative real time PCR (RT-PCR) is widely used as the gold standard for clinical detection of SARS-CoV-2. However, due to the low viral load specimens and the limitations of RT-PCR, significant numbers of false negative reports are inevitable, which results in failure to timely diagnose, cut off transmission, and assess discharge criteria. To improve this situation, an optimized droplet digital PCR (ddPCR) was used for detection of SARS-CoV-2, which showed that the limit of detection of ddPCR is significantly lower than that of RT-PCR. We further explored the feasibility of ddPCR to detect SARS-CoV-2 RNA from 77 patients, and compared with RT-PCR in terms of the diagnostic accuracy based on the results of follow-up survey. 26 patients of COVID-19 with negative RT-PCR reports were reported as positive by ddPCR. The sensitivity, specificity, PPV, NPV, negative likelihood ratio (NLR) and accuracy were improved from 40% (95% CI: 27-55%), 100% (95% CI: 54-100%), 100%, 16% (95% CI: 13-19%), 0.6 (95% CI: 0.48-0.75) and 47% (95% CI: 33-60%) for RT-PCR to 94% (95% CI: 83-99%), 100% (95% CI: 48-100%), 100%, 63% (95% CI: 36-83%), 0.06 (95% CI: 0.02-0.18), and 95% (95% CI: 84-99%) for ddPCR, respectively. Moreover, 6/14 (42.9%) convalescents were detected as positive by ddPCR at 5-12 days post discharge. Overall, ddPCR shows superiority for clinical diagnosis of SARS-CoV-2 to reduce the false negative reports, which could be a powerful complement to the RT-PCR.


Subject(s)
Betacoronavirus/genetics , Coronavirus Infections/diagnosis , Pneumonia, Viral/diagnosis , Real-Time Polymerase Chain Reaction/methods , False Negative Reactions , Humans , Limit of Detection , Pandemics , RNA, Viral/genetics , Viral Load/methods
14.
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
15.
Clin Infect Dis ; 71(15): 793-798, 2020 07 28.
Article in English | MEDLINE | ID: covidwho-17963

ABSTRACT

BACKGROUND: Coronavirus disease 2019 (COVID-19) has become a public health emergency. The widely used reverse transcription-polymerase chain reaction (RT-PCR) method has limitations for clinical diagnosis and treatment. METHODS: A total of 323 samples from 76 COVID-19-confirmed patients were analyzed by droplet digital PCR (ddPCR) and RT-PCR based 2 target genes (ORF1ab and N). Nasal swabs, throat swabs, sputum, blood, and urine were collected. Clinical and imaging data were obtained for clinical staging. RESULTS: In 95 samples that tested positive by both methods, the cycle threshold (Ct) of RT-PCR was highly correlated with the copy number of ddPCR (ORF1ab gene, R2 = 0.83; N gene, R2 = 0.87). Four (4/161) negative and 41 (41/67) single-gene positive samples tested by RT-PCR were positive according to ddPCR with viral loads ranging from 11.1 to 123.2 copies/test. The viral load of respiratory samples was then compared and the average viral load in sputum (17 429 ±â€…6920 copies/test) was found to be significantly higher than in throat swabs (2552 ±â€…1965 copies/test, P < .001) and nasal swabs (651 ±â€…501 copies/test, P < .001). Furthermore, the viral loads in the early and progressive stages were significantly higher than that in the recovery stage (46 800 ±â€…17 272 vs 1252 ±â€…1027, P < .001) analyzed by sputum samples. CONCLUSIONS: Quantitative monitoring of viral load in lower respiratory tract samples helps to evaluate disease progression, especially in cases of low viral load.


Subject(s)
Betacoronavirus/genetics , Coronavirus Infections/diagnosis , Coronavirus Infections/virology , Pneumonia, Viral/diagnosis , Pneumonia, Viral/virology , Adult , Diagnostic Tests, Routine/methods , False Negative Reactions , Female , Humans , Male , Middle Aged , Pandemics , Real-Time Polymerase Chain Reaction/methods , Respiratory System/virology , Serologic Tests/methods , Sputum/virology , Viral Load/methods
16.
Clin Infect Dis ; 71(15): 841-843, 2020 07 28.
Article in English | MEDLINE | ID: covidwho-724

ABSTRACT

The 2019 novel coronavirus (2019-nCoV) was detected in the self-collected saliva of 91.7% (11/12) of patients. Serial saliva viral load monitoring generally showed a declining trend. Live virus was detected in saliva by viral culture. Saliva is a promising noninvasive specimen for diagnosis, monitoring, and infection control in patients with 2019-nCoV infection.


Subject(s)
Betacoronavirus/isolation & purification , Coronavirus Infections/diagnosis , Coronavirus Infections/virology , Pneumonia, Viral/diagnosis , Pneumonia, Viral/virology , Saliva/virology , Adult , Aged , Animals , Cell Line , Chlorocebus aethiops , Female , Hong Kong , Humans , Infection Control/methods , Male , Middle Aged , Pandemics , Vero Cells , Viral Load/methods
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