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1.
Genet Test Mol Biomarkers ; 25(2): 85-101, 2021 Feb.
Article in English | MEDLINE | ID: covidwho-1091280

ABSTRACT

Coronavirus disease 2019 (COVID-19) displays a broad spectrum of clinical presentations ranging from lack of symptoms to severe multiorgan system complications and death. Various laboratory assays have been employed in the diagnosis of COVID-19, including: nucleic acid-based tests; antigen tests; and serum testing for anti-severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) antibodies. The disease can also be diagnosed based on suggestive clinical features and radiological findings. Until now, remdesivir is the only medication approved for the treatment of COVID-19 by the U.S. Food and Drug Administration (FDA); however, it is anticipated that several anti-SARS-CoV-2 monoclonal antibodies will gain soon approval. Other methods of treatment include supportive care directed toward treating the symptoms. Nevertheless, many studies have recently emerged, showing controversial preliminary results with the off-label medication hydroxychloroquine. Given that all results are still preliminary, including those seen by remdesivir, additional evidence and research are required to identify effective medications that are broadly effective and well tolerated. Importantly, two RNA-based vaccines have recently gained approval from Pfizer and Moderna, with many others still in clinical trials. This article reviews various aspects of COVID-19, including its epidemiology; its evolution and mutational spectrum; and its clinical dynamics, symptoms and complications, diagnosis, and treatment.


Subject(s)
Global Burden of Disease/statistics & numerical data , Pandemics/statistics & numerical data , /pathogenicity , Adenosine Monophosphate/analogs & derivatives , Adenosine Monophosphate/therapeutic use , Alanine/analogs & derivatives , Alanine/therapeutic use , Antiviral Agents/therapeutic use , /drug therapy , /therapy , /methods , Clinical Trials as Topic , Evolution, Molecular , Humans , Hydroxychloroquine/therapeutic use , Mutation , Off-Label Use , Pandemics/prevention & control , RNA, Viral/genetics , RNA, Viral/isolation & purification , /immunology , Severity of Illness Index
2.
Sci Rep ; 11(1): 3459, 2021 02 10.
Article in English | MEDLINE | ID: covidwho-1078606

ABSTRACT

During the COVID-19 pandemic it is essential to test as many people as possible, in order to detect early outbreaks of the infection. Present testing solutions are based on the extraction of RNA from patients using oropharyngeal and nasopharyngeal swabs, and then testing with real-time PCR for the presence of specific RNA filaments identifying the virus. This approach is limited by the availability of reactants, trained technicians and laboratories. One of the ways to speed up the testing procedures is a group testing, where the swabs of multiple patients are grouped together and tested. In this paper we propose to use the group testing technique in conjunction with an advanced replication scheme in which each patient is allocated in two or more groups to reduce the total numbers of tests and to allow testing of even larger numbers of people. Under mild assumptions, a 13 ×  average reduction of tests can be achieved compared to individual testing without delay in time.


Subject(s)
/methods , RNA, Viral/isolation & purification , Real-Time Polymerase Chain Reaction/methods , /isolation & purification , Humans , Pandemics
3.
mBio ; 12(1)2021 02 09.
Article in English | MEDLINE | ID: covidwho-1075939

ABSTRACT

Whether mother-to-infant SARS-CoV-2 transmission can occur during breastfeeding and, if so, whether the benefits of breastfeeding outweigh this risk during maternal COVID-19 illness remain important questions. Using RT-qPCR, we did not detect SARS-CoV-2 RNA in any milk sample (n = 37) collected from 18 women following COVID-19 diagnosis. Although we detected evidence of viral RNA on 8 out of 70 breast skin swabs, only one was considered a conclusive positive result. In contrast, 76% of the milk samples collected from women with COVID-19 contained SARS-CoV-2-specific IgA, and 80% had SARS-CoV-2-specific IgG. In addition, 62% of the milk samples were able to neutralize SARS-CoV-2 infectivity in vitro, whereas milk samples collected prior to the COVID-19 pandemic were unable to do so. Taken together, our data do not support mother-to-infant transmission of SARS-CoV-2 via milk. Importantly, milk produced by infected mothers is a beneficial source of anti-SARS-CoV-2 IgA and IgG and neutralizes SARS-CoV-2 activity. These results support recommendations to continue breastfeeding during mild-to-moderate maternal COVID-19 illness.IMPORTANCE Results from prior studies assaying human milk for the presence of SARS-CoV-2, the causative virus of COVID-19, have suggested milk may act as a potential vehicle for mother-to-child transmission. Most previous studies are limited because they followed only a few participants, were cross-sectional, and/or failed to report how milk was collected and/or analyzed. As such, considerable uncertainty remains regarding whether human milk is capable of transmitting SARS-CoV-2 from mother to child. Here, we report that repeated milk samples collected from 18 women following COVID-19 diagnosis did not contain SARS-CoV-2 RNA; however, risk of transmission via breast skin should be further evaluated. Importantly, we found that milk produced by infected mothers is a source of anti-SARS-CoV-2 IgA and IgG and neutralizes SARS-CoV-2 activity. These results support recommendations to continue breastfeeding during mild-to-moderate maternal COVID-19 illness as milk likely provides specific immunologic benefits to infants.


Subject(s)
Antibodies, Neutralizing/metabolism , Antibodies, Viral/metabolism , Milk, Human/immunology , Pregnancy Complications, Infectious/immunology , /immunology , Adult , Breast/virology , Breast Feeding , /virology , Female , Humans , Infant , Infectious Disease Transmission, Vertical , Male , Milk, Human/virology , Mothers , Pregnancy , Pregnancy Complications, Infectious/virology , RNA, Viral/isolation & purification , /isolation & purification
5.
PLoS One ; 15(12): e0243959, 2020.
Article in English | MEDLINE | ID: covidwho-1067398

ABSTRACT

There has been significant concern regarding fertility and reproductive outcomes during the SARS-CoV2 pandemic. Recent data suggests a high concentration of SARS-Cov2 receptors, ACE2 or TMPRSS2, in nasal epithelium and cornea, which explains person-to-person transmission. We investigated the prevalence of SARS-CoV2 receptors among reproductive tissues by exploring the single-cell sequencing datasets from uterus, myometrium, ovary, fallopian tube, and breast epithelium. We did not detect significant expression of either ACE2 or TMPRSS2 in the normal human myometrium, uterus, ovaries, fallopian tube, or breast. Furthermore, none of the cell types in the female reproductive organs we investigated, showed the co-expression of ACE2 with proteases, TMPRSS2, Cathepsin B (CTSB), and Cathepsin L (CTSL) known to facilitate the entry of SARS2-CoV2 into the host cell. These results suggest that myometrium, uterus, ovaries, fallopian tube, and breast are unlikely to be susceptible to infection by SARS-CoV2.


Subject(s)
/genetics , Cathepsin B/genetics , Cathepsin L/genetics , Serine Endopeptidases/genetics , /metabolism , Breast/metabolism , Breast/virology , /transmission , Epithelium/metabolism , Epithelium/virology , Fallopian Tubes/metabolism , Fallopian Tubes/virology , Female , Fertility/genetics , High-Throughput Nucleotide Sequencing , Humans , Myometrium/metabolism , Myometrium/virology , Ovary/metabolism , Ovary/virology , RNA, Viral/genetics , RNA, Viral/isolation & purification , Reproductive Tract Infections/genetics , Reproductive Tract Infections/virology , Serine Endopeptidases/metabolism , Single-Cell Analysis , Uterus/metabolism , Uterus/virology
6.
BMJ Case Rep ; 14(2)2021 Feb 04.
Article in English | MEDLINE | ID: covidwho-1066843

ABSTRACT

We present a case of a patient who had a history of severe coronavirus disease (COVID-19) 4 months prior to this current presentation and, after a long asymptomatic period, subsequently tested positive for severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) by a RNA PCR assay, after several interval negative SARS-CoV-2 RNA tests. We present this potential case of SARS-CoV-2 reinfection in order to incite discussion around differentiating persistent infection with intermittent viral shedding and reinfection, as well as to discuss evolving knowledge and approaches to the clinical management, follow-up molecular testing and treatment of COVID-19 reinfection.


Subject(s)
/diagnosis , /virology , Virus Shedding , Adult , Antibodies, Monoclonal, Humanized/therapeutic use , /virology , Humans , Intensive Care Units , Male , RNA, Viral/isolation & purification , Radiography/methods , Treatment Outcome
7.
Sci Rep ; 11(1): 3134, 2021 02 04.
Article in English | MEDLINE | ID: covidwho-1065962

ABSTRACT

We aimed to test the sensitivity of naso-oropharyngeal saliva and self-administered nasal (SN) swab compared to nasopharyngeal (NP) swab for COVID-19 testing in a large cohort of migrant workers in Singapore. We also tested the utility of next-generation sequencing (NGS) for diagnosis of COVID-19. Saliva, NP and SN swabs were collected from subjects who presented with acute respiratory infection, their asymptomatic roommates, and prior confirmed cases who were undergoing isolation at a community care facility in June 2020. All samples were tested using RT-PCR. SARS-CoV-2 amplicon-based NGS with phylogenetic analysis was done for 30 samples. We recruited 200 subjects, of which 91 and 46 were tested twice and thrice respectively. In total, 62.0%, 44.5%, and 37.7% of saliva, NP and SN samples were positive. Cycle threshold (Ct) values were lower during the earlier period of infection across all sample types. The percentage of test-positive saliva was higher than NP and SN swabs. We found a strong correlation between viral genome coverage by NGS and Ct values for SARS-CoV-2. Phylogenetic analyses revealed Clade O and lineage B.6 known to be circulating in Singapore. We found saliva to be a sensitive and viable sample for COVID-19 diagnosis.


Subject(s)
/diagnosis , Nasal Mucosa/virology , RNA, Viral/isolation & purification , Saliva/virology , Specimen Handling , Adult , Cohort Studies , Female , Humans , Male , Nasopharynx/virology , /isolation & purification , Sensitivity and Specificity , Singapore/epidemiology
8.
Sci Rep ; 11(1): 3122, 2021 02 04.
Article in English | MEDLINE | ID: covidwho-1065961

ABSTRACT

Sample pooling strategy was intended to determine the optimal parameters for group testing of pooled specimens for the detection of SARS-CoV-2 and process them without significant loss of test usability. Standard molecular diagnostic laboratory equipment, and commercially available centrifugal filters, RNA isolation kits and SARS Cov2 PCR tests were used. The basic idea was to combine and concentrate several samples to the maximal volume, which can be extracted with the single extraction column. Out of 16 tested pools, 12 were positive with cycle threshold (Ct) values within 0.5 and 3.01 Ct of the original individual specimens. The analysis of 112 specimens determined that 12 pools were positive, followed by identification of 6 positive individual specimens among the 112 tested. This testing was accomplished with the use of 16 extractions/PCR tests, resulting in saving of 96 reactions but adding the 40 centrifugal filters. The present study demonstrated that pool testing could detect even up to a single positive sample with Ct value as high as 34. According to the standard protocols, reagents and equipment, this pooling method can be applied easily in current clinical testing laboratories.


Subject(s)
/methods , RNA, Viral/isolation & purification , Specimen Handling/methods , Humans , /isolation & purification , Sensitivity and Specificity
9.
PLoS One ; 16(2): e0246647, 2021.
Article in English | MEDLINE | ID: covidwho-1060986

ABSTRACT

Re-opening of communities in the midst of the ongoing COVID-19 pandemic has ignited new waves of infections in many places around the world. Mitigating the risk of reopening will require widespread SARS-CoV-2 testing, which would be greatly facilitated by simple, rapid, and inexpensive testing methods. This study evaluates several protocols for RNA extraction and RT-qPCR that are simpler and less expensive than prevailing methods. First, isopropanol precipitation is shown to provide an effective means of RNA extraction from nasopharyngeal (NP) swab samples. Second, direct addition of NP swab samples to RT-qPCRs is evaluated without an RNA extraction step. A simple, inexpensive swab collection solution suitable for direct addition is validated using contrived swab samples. Third, an open-source master mix for RT-qPCR is described that permits detection of viral RNA in NP swab samples with a limit of detection of approximately 50 RNA copies per reaction. Quantification cycle (Cq) values for purified RNA from 30 known positive clinical samples showed a strong correlation (r2 = 0.98) between this homemade master mix and commercial TaqPath master mix. Lastly, end-point fluorescence imaging is found to provide an accurate diagnostic readout without requiring a qPCR thermocycler. Adoption of these simple, open-source methods has the potential to reduce the time and expense of COVID-19 testing.


Subject(s)
/diagnosis , RNA, Viral/analysis , Real-Time Polymerase Chain Reaction/methods , /genetics , /virology , Chemical Precipitation , Humans , Limit of Detection , Nasopharynx/virology , Phosphoproteins/genetics , RNA, Viral/isolation & purification , RNA, Viral/metabolism , /isolation & purification
10.
J Prim Care Community Health ; 12: 2150132720987711, 2021.
Article in English | MEDLINE | ID: covidwho-1060256

ABSTRACT

SARS-CoV-2 initially emerged in Wuhan, China in late 2019. It has since been recognized as a pandemic and has led to great social and economic disruption globally. The Reverse Transcriptase Real-Time Polymerase Chain Reaction (rtRT-PCR) has become the primary method for COVID-19 testing worldwide. The method requires a specialized laboratory set up. Long-term persistence of SARS-CoV-2 RNA in nasopharyngeal secretion after full clinical recovery of the patient is regularly observed nowadays. This forces the patients to spend a longer period in isolation and test repeatedly to obtain evidence of viral clearance. Repeated COVID-19 testing in asymptomatic or mildly symptomatic cases often leads to extra workload for laboratories that are already struggling with a high specimen turnover. Here, we present 5 purposively selected cases with different patterns of clinical presentations in which nasopharyngeal shedding of SARS-CoV-2 RNA was observed in patients for a long time. From these case studies, we emphasized the adoption of a symptom-based approach for discontinuing transmission-based precautions over a test-based strategy to reduce the time spent by asymptomatic and mildly symptomatic COVID-19 patients in isolation. A symptom-based approach will also help reduce laboratory burden for COVID-19 testing as well as conserve valuable resources and supplies utilized for rtRT-PCR testing in an emerging lower-middle-income setting. Most importantly, it will also make room for critically ill COVID-19 patients to visit or avail COVID-19 testing at their convenience.


Subject(s)
/methods , Health Care Rationing/methods , Symptom Assessment , Adult , /statistics & numerical data , Developing Countries , Female , Humans , Laboratories/statistics & numerical data , Male , Patient Isolation/statistics & numerical data , RNA, Viral/isolation & purification , Reverse Transcriptase Polymerase Chain Reaction , /isolation & purification , Young Adult
11.
Sci Rep ; 11(1): 2402, 2021 01 28.
Article in English | MEDLINE | ID: covidwho-1054048

ABSTRACT

The COVID-19 pandemic has resulted in an urgent need for a rapid, point of care diagnostic testing that could be rapidly scaled on a worldwide level. We developed and tested a highly sensitive and robust assay based on reverse transcription loop mediated isothermal amplification (RT-LAMP) that uses readily available reagents and a simple heat block using contrived spike-in and actual clinical samples. RT-LAMP testing on RNA-spiked samples showed a limit of detection (LoD) of 2.5 copies/µl of viral transport media. RT-LAMP testing directly on clinical nasopharyngeal swab samples in viral transport media had an 85% positive percentage agreement (PPA) (17/20), and 100% negative percentage agreement (NPV) and delivered results in 30 min. Our optimized RT-LAMP based testing method is a scalable system that is sufficiently sensitive and robust to test for SARS-CoV-2 directly on clinical nasopharyngeal swab samples in viral transport media in 30 min at the point of care without the need for specialized or proprietary equipment or reagents. This cost-effective and efficient one-step testing method can be readily available for COVID-19 testing world-wide, especially in resource poor settings.


Subject(s)
/methods , Molecular Diagnostic Techniques/methods , Nucleic Acid Amplification Techniques/methods , RNA, Viral/isolation & purification , /genetics , Clinical Laboratory Techniques/methods , Diagnostic Techniques and Procedures , Diagnostic Tests, Routine , Humans , Limit of Detection , Point-of-Care Testing , RNA, Viral/genetics , Reverse Transcription , /metabolism , Sensitivity and Specificity
12.
Parasit Vectors ; 14(1): 76, 2021 Jan 22.
Article in English | MEDLINE | ID: covidwho-1041990

ABSTRACT

BACKGROUND: On 11 March 2020, the World Health Organisation (WHO) declared the coronavirus disease 2019 (COVID-19) outbreak to be a pandemic. As the mosquito season progressed, the understandable concern that mosquitoes could transmit the virus began to increase among the general public and public health organisations. We have investigated the vector competence of Culex pipiens and Aedes albopictus, the two most common species of vector mosquitoes in Europe, for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Due to the very unusual feeding behaviour of Ae. albopictus, we also evaluated the role of this mosquito in a potential mechanical transmission of the virus. METHODS: For the vector competence study, mosquitoes were allowed to take several infectious blood meals. The mosquitoes were then collected and analysed at 0, 3, 7 and 10 days post-feeding. For the mechanical transmission test, Ae. albopictus females were allowed to feed for a short time on a feeder containing infectious blood and then on a feeder containing virus-free blood. Both mosquitoes and blood were tested for viral presence. RESULTS: Culex pipiens and Ae. albopictus were found not be competent vectors for SARS-CoV-2, and Ae. albopictus was unable to mechanically transmit the virus. CONCLUSIONS: This is the first study to show that the most common species of vector mosquitoes in Europe do not transmit SARS-CoV-2 and that Ae. albopictus is unable to mechanically transmit the virus from a positive host to a healthy host through host-feeding.


Subject(s)
Aedes/virology , Culex/virology , Mosquito Vectors/virology , /physiology , Animals , Blood/virology , Europe , Female , RNA, Viral/analysis , RNA, Viral/isolation & purification , Real-Time Polymerase Chain Reaction , Sheep/blood
13.
PLoS One ; 15(12): e0243703, 2020.
Article in English | MEDLINE | ID: covidwho-1027759

ABSTRACT

BACKGROUND: Since the first cases reported in Wuhan, China, in December 2019, the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has spread worldwide. In Indonesia, the first case was reported in early March 2020, and the numbers of confirmed infections have been increasing until now. Efforts to contain the virus globally and in Indonesia are ongoing. This is the very first manuscript using a spatial-temporal model to describe the SARS-CoV-2 transmission in Indonesia, as well as providing a patient profile for all confirmed COVID-19 cases. METHOD: Data was collected from the official website of the Indonesia National Task Force for the Acceleration of COVID-19, from the period of 02 March 2020-02 August 2020. The data from RT-PCR confirmed, SARS-CoV-2 positive patients was categorized according to demographics, symptoms and comorbidities based on case categorization (confirmed, recovered, dead). The data collected provides granular and thorough information on time and geographical location for all 34 Provinces across Indonesia. RESULTS: A cumulative total of 111,450 confirmed cases of were reported in Indonesia during the study period. Of those confirmed cases 67.79% (75,551/111,450) were shown as recovered and 4.83% (5,382/111,450) of them as died. Patients were mostly male (50.52%; 56,300/111,450) and adults aged 31 to 45 years old (29.73%; 33,132/111,450). Overall patient presentation symptoms of cough and fever, as well as chronic disease comorbidities were in line with previously published data from elsewhere in South-East Asia. The data reported here, shows that from the detection of the first confirmed case and within a short time period of 40 days, all the provinces of Indonesia were affected by COVID-19. CONCLUSIONS: This study is the first to provide detailed characteristics of the confirmed SARS-CoV-2 patients in Indonesia, including their demographic profile and COVID-19 presentation history. It used a spatial-temporal analysis to present the epidemic spread from the very beginning of the outbreak throughout all provinces in the country. The increase of new confirmed cases has been consistent during this time period for all provinces, with some demonstrating a sharp increase, in part due to the surge in national diagnostic capacity. This information delivers a ready resource that can be used for prediction modelling, and is utilized continuously by the current Indonesian Task Force in order to advise on potential implementation or removal of public distancing measures, and on potential availability of healthcare capacity in their efforts to ultimately manage the outbreak.


Subject(s)
/epidemiology , Disease Outbreaks , RNA, Viral/isolation & purification , /pathogenicity , /diagnosis , /virology , Female , Fever/diagnosis , Fever/epidemiology , Fever/virology , Humans , Indonesia/epidemiology , Male , Middle Aged , RNA, Viral/genetics
14.
PLoS One ; 16(1): e0243712, 2021.
Article in English | MEDLINE | ID: covidwho-1024413

ABSTRACT

To respond to the urgent need for COVID-19 testing, countries perform nucleic acid amplification tests (NAAT) for the detection of SARS-CoV-2 in centralized laboratories. Real-time RT-PCR (Reverse transcription-Polymerase Chain Reaction), used to amplify and detect the viral RNA., is considered, as the current gold standard for diagnostics. It is an efficient process, but the complex engineering required for automated RNA extraction and temperature cycling makes it incompatible for use in point of care settings [1]. In the present work, by harnessing progress made in the past two decades in isothermal amplification and paper microfluidics, we created a portable test, in which SARS-CoV-2 RNA is extracted, amplified isothermally by RT-LAMP (Loop-mediated Isothermal Amplification), and detected using intercalating dyes or fluorescent probes. Depending on the viral load in the tested samples, the detection takes between twenty minutes and one hour. Using a set of 16 pools of naso-pharyngal swab eluates, we estimated a limit of detection comparable to real-time RT-PCR (i.e. 1 genome copies per microliter of clinical sample) and no cross-reaction with eight major respiratory viruses currently circulating in Europe. We designed and fabricated an easy-to-use portable device called "COVIDISC" to carry out the test at the point of care. The low cost of the materials along with the absence of complex equipment will expedite the widespread dissemination of this device. What is proposed here is a new efficient tool to help managing the pandemics.


Subject(s)
/instrumentation , Molecular Diagnostic Techniques/instrumentation , Nucleic Acid Amplification Techniques/instrumentation , Point-of-Care Testing , RNA, Viral/genetics , /genetics , /economics , Equipment Design , Humans , Limit of Detection , Molecular Diagnostic Techniques/economics , Nucleic Acid Amplification Techniques/economics , Point-of-Care Testing/economics , RNA, Viral/isolation & purification , Time Factors
15.
JCO Glob Oncol ; 7: 46-55, 2021 01.
Article in English | MEDLINE | ID: covidwho-1024378

ABSTRACT

PURPOSE: The COVID-19 pandemic remains a public health emergency of global concern. Determinants of mortality in the general population are now clear, but specific data on patients with cancer remain limited, particularly in Latin America. MATERIALS AND METHODS: A longitudinal multicenter cohort study of patients with cancer and confirmed COVID-19 from Oncoclínicas community oncology practice in Brazil was conducted. The primary end point was all-cause mortality after isolation of the SARS-CoV-2 by Real-Time Polymerase Chain Reaction (RT-PCR) in patients initially diagnosed in an outpatient environment. We performed univariate and multivariable logistic regression analysis and recursive partitioning modeling to define the baseline clinical determinants of death in the overall population. RESULTS: From March 29 to July 4, 2020, 198 patients with COVID-19 were prospectively registered in the database, of which 167 (84%) had solid tumors and 31 (16%) had hematologic malignancies. Most patients were on active systemic therapy or radiotherapy (77%), largely for advanced or metastatic disease (64%). The overall mortality rate was 16.7% (95% CI, 11.9 to 22.7). In univariate models, factors associated with death after COVID-19 diagnosis were age ≥ 60 years, current or former smoking, coexisting comorbidities, respiratory tract cancer, and management in a noncurative setting (P < .05). In multivariable logistic regression and recursive partitioning modeling, only age, smoking history, and noncurative disease setting remained significant determinants of mortality, ranging from 1% in cancer survivors under surveillance or (neo)adjuvant therapy to 60% in elderly smokers with advanced or metastatic disease. CONCLUSION: Mortality after COVID-19 in patients with cancer is influenced by prognostic factors that also affect outcomes of the general population. Fragile patients and smokers are entitled to active preventive measures to reduce the risk of SARS-CoV-2 infection and close monitoring in the case of exposure or COVID-19-related symptoms.


Subject(s)
/mortality , Cancer Survivors/statistics & numerical data , Neoplasms/mortality , /isolation & purification , Adult , Aged , Aged, 80 and over , Brazil/epidemiology , /virology , Cause of Death , Databases, Factual/statistics & numerical data , Female , Frailty/epidemiology , Humans , Longitudinal Studies , Male , Medical Oncology/statistics & numerical data , Middle Aged , Neoplasms/complications , Prognosis , Prospective Studies , RNA, Viral/isolation & purification , Risk Assessment/statistics & numerical data , Risk Factors , Smoking/epidemiology , Young Adult
16.
Dis Markers ; 2020: 8869424, 2020.
Article in English | MEDLINE | ID: covidwho-1024275

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has rapidly spread worldwide from the beginning of 2020. The presence of viral RNA in samples by nucleic acid (NA) molecular analysis is the only method available to diagnose COVID-19 disease and to assess patients' viral load. Since the demand for laboratory reagents has increased, there has been a worldwide shortage of RNA extraction kits. We, therefore, developed a fast and cost-effective viral genome isolation method that, combined with quantitative RT-PCR assay, detects SARS-CoV-2 RNA in patient samples. The method relies on the addition of Proteinase K followed by a controlled heat-shock incubation and, then, E gene evaluation by RT-qPCR. It was validated for sensitivity, specificity, linearity, reproducibility, and precision. It detects as low as 10 viral copies/sample, is rapid, and has been characterized in 60 COVID-19-infected patients. Compared to automated extraction methods, our pretreatment guarantees the same positivity rate with the advantage of shortening the time of the analysis and reducing its cost. This is a rapid workflow meant to aid the healthcare system in the rapid identification of infected patients, such as during a pathogen-related outbreak. For its intrinsic characteristics, this workflow is suitable for large-scale screenings.


Subject(s)
/methods , RNA, Viral/isolation & purification , Reverse Transcriptase Polymerase Chain Reaction/methods , /genetics , /genetics , Humans , Limit of Detection , Nasopharynx/virology , Sensitivity and Specificity , Workflow
17.
Forensic Sci Int ; 319: 110653, 2021 Feb.
Article in English | MEDLINE | ID: covidwho-1023567

ABSTRACT

Post-mortem swabs for Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) RNA detection have been recommended by several Scientific Committees and Institutions as a standard procedure for post-mortem assessment of potential Coronavirus Disease-19 (COVID-19) related deaths. To date there is no data about the SARS-CoV-2 RNA detectability period in human bodies after death. The present case documents the persistence of SARS-CoV-2 RNA in the upper respiratory tract 35-days after death. Post-mortem swabs could be used as a valuable tool in preventive evaluation of the risks-benefits ratio associated with autopsy execution. SARS-CoV-2 RNA post-mortem detection could have a key diagnostic role in deaths lacking medical assistance, unattended deaths, and patients with multiple comorbidities. Based on the present report, staged post-mortem swabs should be performed even after a long post-mortem interval.


Subject(s)
Cadaver , Nasal Cavity/virology , Oropharynx/virology , RNA, Viral/isolation & purification , /genetics , Humans , Male , Middle Aged , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , Specimen Handling , Time Factors
18.
Diagn Microbiol Infect Dis ; 99(1): 115206, 2021 Jan.
Article in English | MEDLINE | ID: covidwho-1023529

ABSTRACT

The diagnosis of coronavirus disease-19 (COVID-19) relies on the detection of severe acute respiratory syndrome coronavirus 2 (SARS CoV-2) RNA by real-time reverse-transcription polymerase chain reaction in respiratory samples. Rapid increase in the COVID-19 cases across the world requires fast and efficient testing as testing capacity is a bottleneck in diagnosis. In this context, pooling strategy can be opted for rapid testing in a cost-effective manner. In this study, the authors have optimized and compared the effect of pooling (5 and 10 samples) before and after nucleic acid extraction. It was concluded that there was no significant difference in the SARS CoV-2 RNA detection in the pools prepared at sample or RNA level. Even after pooling, 10-fold dilution was detectable with 3-cycle threshold value change in both type of pools when compared with individual samples. Hence, sample pool size of 10 can be used in low-prevalent areas, and testing capacity can be substantially increased.


Subject(s)
/methods , /isolation & purification , Specimen Handling/methods , /standards , Genes, Viral/genetics , Humans , India/epidemiology , Nasopharynx/virology , Pharynx/virology , RNA, Viral/genetics , RNA, Viral/isolation & purification , Sensitivity and Specificity , Specimen Handling/standards , Tertiary Care Centers
19.
Zhonghua Er Ke Za Zhi ; 58(4): 275-278, 2020 Apr 02.
Article in Chinese | MEDLINE | ID: covidwho-1024679

ABSTRACT

Objective: To explore imaging characteristics of children with 2019 novel coronavirus (2019-nCoV) infection. Methods: A retrospective analysis was performed on clinical data and chest CT images of 15 children diagnosed with 2019-nCoV infection. They were admitted to the Third People's Hospital of Shenzhen from January 16 to February 6, 2020. The distribution and morphology of pulmonary lesions on chest CT images were analyzed. Results: Among the 15 children, 5 were males and 10 females, aged from 4 to 14 years. Five of the 15 children were febrile and 10 were asymptomatic on the first visit. The first nasal or pharyngeal swab samples in all the 15 cases were positive for 2019-nCoV nucleic acid. For their first chest CT images, 6 patients had no lesions, while 9 patients had pulmonary inflammatory lesions. Seven cases had small nodular ground glass opacities and 2 cases had speckled ground glass opacities. After 3 to 5 days of treatment, 2019-nCoV nucleic acid in a second respiratory sample turned negative in 6 cases. Among them, chest CT images showed less lesions in 2 cases, no lesion in 3 cases, and no improvement in 1 case. The remaining 9 cases were still positive in a second nucleic acid test. Six patients showed similar chest CT inflammation, while 3 patients had new lesions, which were all small nodular ground glass opacities. Conclusions: The early chest CT images of children with 2019-nCoV infection are mostly small nodular ground glass opacities. The clinical symptoms of children with 2019-nCoV infection are nonspecific. Dynamic reexamination of chest CT and nucleic acid are important.


Subject(s)
Betacoronavirus , Coronavirus Infections/diagnostic imaging , Lung/diagnostic imaging , Pneumonia, Viral/diagnostic imaging , Tomography, X-Ray Computed , Adolescent , Child , Child, Preschool , China , Clinical Laboratory Techniques , Coronavirus Infections/diagnosis , Female , Humans , Lung/pathology , Male , Pandemics , RNA, Viral/isolation & purification , Radiography, Thoracic , Retrospective Studies
20.
Clin Chem ; 66(4): 549-555, 2020 04 01.
Article in English | MEDLINE | ID: covidwho-1024088

ABSTRACT

BACKGROUND: A novel coronavirus of zoonotic origin (2019-nCoV) has recently been identified in patients with acute respiratory disease. This virus is genetically similar to SARS coronavirus and bat SARS-like coronaviruses. The outbreak was initially detected in Wuhan, a major city of China, but has subsequently been detected in other provinces of China. Travel-associated cases have also been reported in a few other countries. Outbreaks in health care workers indicate human-to-human transmission. Molecular tests for rapid detection of this virus are urgently needed for early identification of infected patients. METHODS: We developed two 1-step quantitative real-time reverse-transcription PCR assays to detect two different regions (ORF1b and N) of the viral genome. The primer and probe sets were designed to react with this novel coronavirus and its closely related viruses, such as SARS coronavirus. These assays were evaluated using a panel of positive and negative controls. In addition, respiratory specimens from two 2019-nCoV-infected patients were tested. RESULTS: Using RNA extracted from cells infected by SARS coronavirus as a positive control, these assays were shown to have a dynamic range of at least seven orders of magnitude (2x10-4-2000 TCID50/reaction). Using DNA plasmids as positive standards, the detection limits of these assays were found to be below 10 copies per reaction. All negative control samples were negative in the assays. Samples from two 2019-nCoV-infected patients were positive in the tests. CONCLUSIONS: The established assays can achieve a rapid detection of 2019n-CoV in human samples, thereby allowing early identification of patients.


Subject(s)
Betacoronavirus/genetics , Clinical Laboratory Techniques , Coronavirus Infections/diagnosis , Pneumonia, Viral/diagnosis , Coronavirus Infections/epidemiology , Coronavirus Infections/virology , Disease Outbreaks , Humans , Pandemics , Phylogeny , Pneumonia, Viral/epidemiology , Pneumonia, Viral/virology , RNA, Viral/genetics , RNA, Viral/isolation & purification , Real-Time Polymerase Chain Reaction
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