Your browser doesn't support javascript.
Show: 20 | 50 | 100
Results 1 - 20 de 47
Filter
1.
Int J Mol Med ; 46(3): 957-964, 2020 Sep.
Article in English | MEDLINE | ID: covidwho-676117

ABSTRACT

Reverse transcription­quantitative polymerase chain reaction (RT­qPCR) is the gold standard method for the diagnosis of COVID­19 infection. Due to pre­analytical and technical limitations, samples with low viral load are often misdiagnosed as false­negative samples. Therefore, it is important to evaluate other strategies able to overcome the limits of RT­qPCR. Blinded swab samples from two individuals diagnosed positive and negative for COVID­19 were analyzed by droplet digital PCR (ddPCR) and RT­qPCR in order to assess the sensitivity of both methods. Intercalation chemistries and a World Health Organization (WHO)/Center for Disease Control and Prevention (CDC)­approved probe for the SARS­CoV­2 N gene were used. SYBR­Green RT­qPCR is not able to diagnose as positive samples with low viral load, while, TaqMan Probe RT­qPCR gave positive signals at very late Ct values. On the contrary, ddPCR showed higher sensitivity rate compared to RT­qPCR and both EvaGreen and probe ddPCR were able to recognize the sample with low viral load as positive even at 10­fold diluted concentration. In conclusion, ddPCR shows higher sensitivity and specificity compared to RT­qPCR for the diagnosis of COVID­19 infection in false­negative samples with low viral load. Therefore, ddPCR is strongly recommended in clinical practice for the diagnosis of COVID­19 and the follow­up of positive patients until complete remission.


Subject(s)
Betacoronavirus/genetics , Coronavirus Infections/diagnosis , Pneumonia, Viral/diagnosis , RNA, Viral/analysis , Real-Time Polymerase Chain Reaction/methods , Reverse Transcriptase Polymerase Chain Reaction/methods , Humans , Nucleocapsid Proteins/genetics , Pandemics , Sensitivity and Specificity , Spike Glycoprotein, Coronavirus/genetics , Viral Proteins/genetics
2.
J Clin Microbiol ; 58(8)2020 Jul 23.
Article in English | MEDLINE | ID: covidwho-670471

ABSTRACT

In early March 2020, the University of Washington Medical Center clinical virology laboratory became one of the first clinical laboratories to offer testing for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). When we first began test development in mid-January, neither of us believed there would be more than 2 million confirmed SARS-CoV-2 infections nationwide or that we would have performed more than 150,000 real-time PCR (RT-PCR) tests, with many more to come. This article will be a chronological summary of how we rapidly validated tests for SARS-CoV-2, increased our testing capacity, and addressed the many problems that came up along the way.


Subject(s)
Betacoronavirus/isolation & purification , Clinical Laboratory Techniques/methods , Coronavirus Infections/diagnosis , Molecular Diagnostic Techniques/methods , Pneumonia, Viral/diagnosis , Real-Time Polymerase Chain Reaction/methods , Academic Medical Centers , Capacity Building , Diagnostic Services/organization & administration , Humans , Pandemics , Universities , Washington
3.
Front Cell Infect Microbiol ; 10: 356, 2020.
Article in English | MEDLINE | ID: covidwho-642075

ABSTRACT

CDC and WHO guidelines for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) diagnosis only recommend synthetic fiber swabs for nasopharyngeal (NP) sampling. We show that cotton-tipped plastic swabs do not inhibit PCR and have equivalent performance to rayon swabs. Cotton-tipped plastic swabs are massively produced worldwide and would prevent swab supply shortages under the current high SARS-CoV-2 testing demands, particularly in developing countries.


Subject(s)
Betacoronavirus/isolation & purification , Clinical Laboratory Techniques/instrumentation , Coronavirus Infections/diagnosis , Diagnostic Equipment/supply & distribution , Disposable Equipment/supply & distribution , Pneumonia, Viral/diagnosis , Real-Time Polymerase Chain Reaction/instrumentation , Betacoronavirus/genetics , Cellulose/supply & distribution , Clinical Laboratory Techniques/methods , Coronavirus Infections/virology , Cotton Fiber/supply & distribution , Humans , Nasopharynx , Pandemics , Plastics/supply & distribution , Pneumonia, Viral/virology , RNA, Viral/analysis , Real-Time Polymerase Chain Reaction/methods , Specimen Handling/instrumentation , Specimen Handling/methods
4.
Theranostics ; 10(16): 7150-7162, 2020.
Article in English | MEDLINE | ID: covidwho-639991

ABSTRACT

In December 2019, a new coronavirus disease (COVID-19) outbreak occurred in Wuhan, China. Severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2), which is the seventh coronavirus known to infect humans, is highly contagious and has rapidly expanded worldwide since its discovery. Quantitative nucleic acid testing has become the gold standard for diagnosis and guiding clinical decisions regarding the use of antiviral therapy. However, the RT-qPCR assays targeting SARS-CoV-2 have a number of challenges, especially in terms of primer design. Primers are the pivotal components of a RT-qPCR assay. Once virus mutation and recombination occur, it is difficult to effectively diagnose viral infection by existing RT-qPCR primers. Some primers and probes have also been made available on the WHO website for reference. However, no previous review has systematically compared the previously reported primers and probes and described how to design new primers in the event of a new coronavirus infection. This review focuses on how primers and probes can be designed methodically and rationally, and how the sensitivity and specificity of the detection process can be improved. This brief review will be useful for the accurate diagnosis and timely treatment of the new coronavirus pneumonia.


Subject(s)
Betacoronavirus/genetics , Coronavirus Infections/diagnosis , Pneumonia, Viral/diagnosis , RNA, Viral/genetics , RNA/genetics , Real-Time Polymerase Chain Reaction/methods , Base Sequence , Coronavirus Infections/epidemiology , Coronavirus Infections/virology , Drug Design , Genes, Viral , Humans , Nucleic Acid Conformation , Pandemics , Pneumonia, Viral/epidemiology , Pneumonia, Viral/virology , RNA/chemistry , RNA Probes/genetics , RNA, Viral/chemistry , Real-Time Polymerase Chain Reaction/statistics & numerical data , Sensitivity and Specificity , Theranostic Nanomedicine
5.
Eur Radiol Exp ; 4(1): 39, 2020 06 26.
Article in English | MEDLINE | ID: covidwho-615378

ABSTRACT

BACKGROUND: Computed tomography (CT) enables quantification of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, helping in outcome prediction. METHODS: From 1 to 22 March 2020, patients with pneumonia symptoms, positive lung CT scan, and confirmed SARS-CoV-2 on reverse transcription-polymerase chain reaction (RT-PCR) were consecutively enrolled. Clinical data was collected. Outcome was defined as favourable or adverse (i.e., need for mechanical ventilation or death) and registered over a period of 10 days following CT. Volume of disease (VoD) on CT was calculated semi-automatically. Multiple linear regression was used to predict VoD by clinical/laboratory data. To predict outcome, important features were selected using a priori analysis and subsequently used to train 4 different models. RESULTS: A total of 106 consecutive patients were enrolled (median age 63.5 years, range 26-95 years; 41/106 women, 38.7%). Median duration of symptoms and C-reactive protein (CRP) was 5 days (range 1-30) and 4.94 mg/L (range 0.1-28.3), respectively. Median VoD was 249.5 cm3 (range 9.9-1505) and was predicted by lymphocyte percentage (p = 0.008) and CRP (p < 0.001). Important variables for outcome prediction included CRP (area under the curve [AUC] 0.77), VoD (AUC 0.75), age (AUC 0.72), lymphocyte percentage (AUC 0.70), coronary calcification (AUC 0.68), and presence of comorbidities (AUC 0.66). Support vector machine had the best performance in outcome prediction, yielding an AUC of 0.92. CONCLUSIONS: Measuring the VoD using a simple CT post-processing tool estimates SARS-CoV-2 burden. CT and clinical data together enable accurate prediction of short-term clinical outcome.


Subject(s)
Betacoronavirus , Coronavirus Infections/diagnosis , Lung/diagnostic imaging , Pneumonia, Viral/diagnosis , Real-Time Polymerase Chain Reaction/methods , Tomography, X-Ray Computed/methods , Adult , Aged , Aged, 80 and over , Coronavirus Infections/diagnostic imaging , Evaluation Studies as Topic , Female , Humans , Male , Middle Aged , Pandemics , Patient Outcome Assessment , Pneumonia, Viral/diagnostic imaging , Predictive Value of Tests , Prognosis , Reproducibility of Results
6.
Viruses ; 12(6)2020 06 25.
Article in English | MEDLINE | ID: covidwho-613542

ABSTRACT

The recent emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) worldwide has highlighted the importance of reliable and rapid diagnostic testing to prevent and control virus circulation. Dozens of monoplex in-house RT-qPCR assays are already available; however, the development of dual-target assays is suited to avoid false-negative results caused by polymorphisms or point mutations, that can compromise the accuracy of diagnostic and screening tests. In this study, two mono-target assays recommended by WHO (E-Sarbeco (enveloppe gene, Charite University, Berlin, Germany) and RdRp-IP4 (RdRp, Institut Pasteur, Paris, France)) were selected and combined in a unique robust test; the resulting duo SARS-CoV-2 RT-qPCR assay was compared to the two parental monoplex tests. The duo SARS-CoV-2 assay performed equally, or better, in terms of sensitivity, specificity, linearity and signal intensity. We demonstrated that combining two single systems into a dual-target assay (with or without an MS2-based internal control) did not impair performances, providing a potent tool adapted for routine molecular diagnosis in clinical microbiology laboratories.


Subject(s)
Betacoronavirus/isolation & purification , Coronavirus Infections/diagnosis , Pneumonia, Viral/diagnosis , RNA Replicase/genetics , Real-Time Polymerase Chain Reaction/methods , Viral Envelope Proteins/genetics , Viral Nonstructural Proteins/genetics , Betacoronavirus/genetics , Coronavirus Infections/virology , Humans , Pandemics , Pneumonia, Viral/virology , RNA, Viral/analysis , Sensitivity and Specificity , World Health Organization
7.
Emerg Microbes Infect ; 9(1): 1393-1396, 2020 Dec.
Article in English | MEDLINE | ID: covidwho-607850

ABSTRACT

The RNA purification is the gold standard for the detection of SARS-CoV-2 in swab samples, but it is dependent on the availability of chemical reagents. In this study, we evaluated the heat treatment method without RNA extraction as a reliable option to nucleic acid purification.


Subject(s)
Betacoronavirus/genetics , Clinical Laboratory Techniques/methods , Coronavirus Infections/diagnosis , Pneumonia, Viral/diagnosis , Real-Time Polymerase Chain Reaction/methods , Betacoronavirus/isolation & purification , Coronavirus Infections/virology , Hot Temperature , Humans , Pandemics , Pneumonia, Viral/virology , RNA, Viral/chemistry , RNA, Viral/genetics , Specimen Handling , Viral Proteins/genetics
9.
Euro Surveill ; 25(24)2020 06.
Article in English | MEDLINE | ID: covidwho-605372

ABSTRACT

Containment strategies and clinical management of coronavirus disease (COVID-19) patients during the current pandemic depend on reliable diagnostic PCR assays for the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here, we compare 11 different RT-PCR test systems used in seven diagnostic laboratories in Germany in March 2020. While most assays performed well, we identified detection problems in a commonly used assay that may have resulted in false-negative test results during the first weeks of the pandemic.


Subject(s)
Betacoronavirus/genetics , Clinical Laboratory Techniques/methods , Coronavirus Infections/diagnosis , Diagnostic Equipment , Pneumonia, Viral/diagnosis , Clinical Laboratory Techniques/instrumentation , Feces/virology , Germany , Humans , Laboratories , Multiplex Polymerase Chain Reaction/instrumentation , Multiplex Polymerase Chain Reaction/methods , Pandemics , Real-Time Polymerase Chain Reaction/instrumentation , Real-Time Polymerase Chain Reaction/methods , Reverse Transcriptase Polymerase Chain Reaction/instrumentation , Reverse Transcriptase Polymerase Chain Reaction/methods , Sensitivity and Specificity
10.
Gac Med Mex ; 156(3): 208-216, 2020.
Article in English | MEDLINE | ID: covidwho-601295

ABSTRACT

Introduction: As of March 23, 2020, suspension of non-essential activities was declared in Mexico throughout the country in order to mitigate the spread of the COVID-19 pandemic. Objective: To analyze data on the first 1,510 laboratory-confirmed cases of COVID-19 in Mexico, and to describe the geographical distribution of the disease and its transmission dynamics. Method: Description of the first COVID-19 cases with real-time RT-PCR-positive test, as well as evaluation of epidemiological measures, cumulative incidence, rate of transmission, and mortality and lethality rates during the first month of the epidemic. Results: Average age was 43 years, and 58 % were males; 44 % of initial cases were imported. Lethality in the population during the first month went from 1.08 to 3.97 per 100 cases; however, the trend is linear and similar to that observed in Europe. Conclusions: In Mexico, social distancing is being applied, but studies are still required on the dynamics of the epidemic, person-to-person transmission, incidence of subclinical infections, and patient survival.


Subject(s)
Coronavirus Infections/epidemiology , Pneumonia, Viral/epidemiology , Real-Time Polymerase Chain Reaction/methods , Social Isolation , Adolescent , Adult , Aged , Aged, 80 and over , Child , Child, Preschool , Coronavirus Infections/prevention & control , Coronavirus Infections/transmission , Female , Humans , Incidence , Infant , Infant, Newborn , Male , Mexico/epidemiology , Middle Aged , Pandemics/prevention & control , Pneumonia, Viral/prevention & control , Pneumonia, Viral/transmission , Survival , Young Adult
11.
Exp Mol Med ; 52(6): 963-977, 2020 06.
Article in English | MEDLINE | ID: covidwho-601243

ABSTRACT

SARS-CoV-2 is very contagious and has rapidly spread globally. Due to various symptomatic and asymptomatic cases and the possibility of asymptomatic transmission, there is a pressing need for a fast and sensitive detection protocol to diagnose asymptomatic people. Various SARS-CoV-2 diagnostic kits are already available from many companies and national health agencies. However, publicly available information on these diagnostic kits is lacking. In response to the growing need and the lack of information, we developed and made available a low-cost, easy-access, real-time PCR-based protocol for the early detection of the virus in a previous study. During the development of the detection protocol, we found that unoptimized primer sets could inadvertently show false-positive results, raising the possibility that commercially available diagnostic kits might also contain primer sets that produce false-positive results. Here, we provide three-step guidelines for the design and optimization of specific primer sets. The three steps include (1) the selection of primer sets for target genes (RdRP, N, E, and S) in the genome of interest (SARS-CoV-2), (2) the in silico validation of primer and amplicon sequences, and (3) the optimization of PCR conditions (i.e., primer concentrations and annealing temperatures) for specific hybridization between the primers and target genes, and the elimination of spurious primer dimers. Furthermore, we have expanded the previously developed real-time PCR-based protocol to more conventional PCR-based protocols and applied a multiplex PCR-based protocol that allows the simultaneous testing of primer sets for RdRP, N, E, and S all in one reaction. Our newly optimized protocol should be helpful for the large-scale, high-fidelity screening of asymptomatic people, even without any high-specification equipment, for the further prevention of transmission, and to achieve early intervention and treatment for the rapidly propagating virus.


Subject(s)
Betacoronavirus/genetics , Clinical Laboratory Techniques/methods , Coronavirus Infections/virology , DNA Primers , Pneumonia, Viral/virology , Polymerase Chain Reaction/methods , Coronavirus Infections/diagnosis , HEK293 Cells , Humans , Multiplex Polymerase Chain Reaction/methods , Pandemics , Pharynx/virology , Pneumonia, Viral/diagnosis , RNA Replicase/genetics , Real-Time Polymerase Chain Reaction/methods , SARS Virus/genetics , Spike Glycoprotein, Coronavirus/genetics , Viral Nonstructural Proteins/genetics , Viral Proteins/genetics
12.
Emerg Microbes Infect ; 9(1): 1489-1496, 2020 Dec.
Article in English | MEDLINE | ID: covidwho-599991

ABSTRACT

In December 2019, Wuhan, China suffered a serious outbreak of a novel coronavirus infectious disease (COVID) caused by novel severe acute respiratory syndrome-related coronavirus (SARS-CoV 2). To quickly identify the pathogen, we designed and screened primer sets, and established a sensitive and specific qRT-PCR assay for SARS-CoV 2; the lower limit of detection (LOD) was 14.8 (95% CI: 9.8-21) copies per reaction. We combined this qRT-PCR assay with an automatic integration system for nucleic acid extraction and amplification, thereby establishing an automatic integrated gene detection system (AIGS) for SARS-CoV 2. Cross reactive analysis performed in 20 other respiratory viruses and 37 nasopharyngeal swabs confirmed a 100% specificity of the assay. Using two fold diluted SARS-CoV 2 culture, the LOD of AIGS was confirmed to be 365 copies/ml (95% CI: 351-375), which was Comparable to that of conventional q RT-PCR (740 copies/ml, 95% CI: 689-750). Clinical performances of AIGS assay were assessed in 266 suspected COVID-19 clinical respiratory tract samples tested in parallel with a commercial kit. The clinical sensitivity of the AIGS test was 97.62% (95% CI: 0.9320-0.9951) based on the commercial kit test result, and concordance analysis showed a high agreement in SARS-CoV-2 detection between the two assays, Pearson R was 0.9623 (95% CI: 0.9523-0.9703). The results indicated that this AIGS could be used for rapid detection of SARS-CoV 2. With the advantage of simple operation and less time consuming, AIGS could be suitable for SARS-CoV2 detection in primary medical institutions, thus would do a great help to improve detection efficiency and control the spread of COVID-19.


Subject(s)
Betacoronavirus/isolation & purification , Coronavirus Infections/diagnosis , Pneumonia, Viral/diagnosis , Real-Time Polymerase Chain Reaction/methods , Automation, Laboratory , China , DNA Primers , Humans , Limit of Detection , Pandemics , RNA, Viral/analysis , Sensitivity and Specificity , Virus Cultivation
13.
J Virol Methods ; 283: 113919, 2020 09.
Article in English | MEDLINE | ID: covidwho-598595

ABSTRACT

The purpose of this study was to investigate the feasibility of serological total antibody tests combined with RT-PCR for detection of SARS-COV-2. We conducted a retrospective study in which 375 patients were enrolled during the outbreak of SARS-COV-2 from 25th January to 16th March 2020. Patients were divided into a COVID-19 group (n = 141) and a control group (n = 234). Serum samples and throat swabs were collected from 375 patients for total antibody testing against SARS-COV-2 and RT-PCR analysis, respectively. The results indicated that diagnostic sensitivity and specificity were 95.7 % and 98.7 %, 92.2 % and 100 % by total antibody tests and RT-PCR, respectively. The sensitivity and specificity of total antibody tests combined with RT-PCR were 98.6 % and 98.7 %. The sensitivity of the combined method was significantly higher than RT-PCR (X2 = 5.16, P < 0.05), and similar to that of total antibody tests (X2 = 1.15, P> 0.05). This study supported the advantage of the combined method for detection of SARS-COV-2 with a high degree of sensitivity and specificity, as a useful tool for accurate diagnosis and timely treatment of suspected patients, epidemiological investigation, as well as monitoring ongoing outbreaks of infections with SARS-COV-2.


Subject(s)
Antibodies, Viral/blood , Betacoronavirus/immunology , Coronavirus Infections/diagnosis , Pneumonia, Viral/diagnosis , Real-Time Polymerase Chain Reaction/methods , Serologic Tests/methods , Adolescent , Adult , Aged , Antibodies, Viral/immunology , Case-Control Studies , China , Coronavirus Infections/immunology , Female , Humans , Immunoassay/methods , Luminescent Measurements/methods , Male , Middle Aged , Pandemics , Pneumonia, Viral/immunology , Retrospective Studies , Sensitivity and Specificity , Young Adult
14.
PLoS One ; 15(6): e0234682, 2020.
Article in English | MEDLINE | ID: covidwho-595220

ABSTRACT

Novel Corona virus/Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2 or 2019-nCoV), and the subsequent disease caused by the virus (coronavirus disease 2019 or COVID-19), is an emerging global health concern that requires a rapid diagnostic test. Quantitative reverse transcription PCR (qRT-PCR) is currently the standard for SARS-CoV-2 detection; however, Reverse Transcription Loop-Mediated Isothermal Amplification (RT-LAMP) may allow for faster and cheaper field based testing at point-of-risk. The objective of this study was to develop a rapid screening diagnostic test that could be completed in 30-45 minutes. Simulated patient samples were generated by spiking serum, urine, saliva, oropharyngeal swabs, and nasopharyngeal swabs with a portion of the SARS-CoV-2 nucleic sequence. RNA isolated from nasopharyngeal swabs collected from actual COVID-19 patients was also tested. The samples were tested using RT-LAMP as well as by conventional qRT-PCR. Specificity of the RT-LAMP was evaluated by also testing against other related coronaviruses. RT-LAMP specifically detected SARS-CoV-2 in both simulated patient samples and clinical specimens. This test was performed in 30-45 minutes. This approach could be used for monitoring of exposed individuals or potentially aid with screening efforts in the field and potential ports of entry.


Subject(s)
Betacoronavirus/isolation & purification , Coronavirus Infections/diagnosis , Molecular Diagnostic Techniques/methods , Nucleic Acid Amplification Techniques/methods , Pneumonia, Viral/diagnosis , Point-of-Care Testing , Real-Time Polymerase Chain Reaction/methods , Betacoronavirus/genetics , Coronavirus Infections/virology , DNA Primers , Humans , Molecular Diagnostic Techniques/economics , Molecular Diagnostic Techniques/instrumentation , Nucleic Acid Amplification Techniques/economics , Nucleic Acid Amplification Techniques/instrumentation , Pandemics , Pneumonia, Viral/virology , Real-Time Polymerase Chain Reaction/economics , Real-Time Polymerase Chain Reaction/instrumentation , Sensitivity and Specificity , Time Factors
15.
Respir Res ; 21(1): 146, 2020 Jun 11.
Article in English | MEDLINE | ID: covidwho-593639

ABSTRACT

BACKGROUND: Older age and elevated d-dimer are reported risk factors for coronavirus disease 2019 (COVID-19). However, whether early radiographic change is a predictor of fatality remains unknown. METHODS: We retrospectively reviewed records of all laboratory-confirmed patients admitted to a quarantine unit at Tongji Hospital, a large regional hospital in Wuhan, China, between January 31 and March 5, 2020. Confirmed cases were defined by positive RT-PCR detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in throat-swab specimens. Chest CT images were reviewed independently by two radiologists. The Tongji Hospital ethics committee approved this study. RESULTS: A total of 102 patients were confirmed to have SARS-CoV-2 infection. As of March 25, 85 confirmed patients were discharged, 15 died, and 2 remained hospitalized. When compared with survivors, non-survivors were older (median age, 69 [interquartile range, 58-77] vs. 55 [44-66], p = 0.003), and more likely to have decreased lymphocyte count (0.5 vs. 0.9 ×  109/L, p = 0.006), elevated lactate dehydrogenase (LDH) (569.0 vs. 272.0 U/L, p < 0.001), elevated d-dimer (> 1 µg/mL, 86% vs. 37%, p = 0.002) on admission. Older age and elevated LDH were independent risk factors for fatality in a multivariate regression model included the above variables. In a subset of patients with CT images within the first week, higher total severity score, and more involved lung lobes (5 involved lobes) in CT images within the first week were significantly associated with fatality. Moreover, in this subset of patients, higher total severity score was the only independent risk factor in a multivariate analysis incorporating the above mentioned variables. CONCLUSIONS: Older age, elevated LDH on admission, and higher severity score of CT images within the first week are potential predictors of fatality in adults with COVID-19. These predictors may help clinicians identify patients with a poor prognosis at an early stage.


Subject(s)
Clinical Laboratory Techniques/methods , Coronavirus Infections/diagnostic imaging , Coronavirus Infections/mortality , Hospital Mortality/trends , Pandemics/statistics & numerical data , Pneumonia, Viral/diagnostic imaging , Pneumonia, Viral/mortality , Radiography, Thoracic/methods , Adult , Aged , Aged, 80 and over , Analysis of Variance , China , Coronavirus Infections/diagnosis , Coronavirus Infections/prevention & control , Coronavirus Infections/therapy , Databases, Factual , Disease Progression , Female , Hospitalization/statistics & numerical data , Hospitals, Public , Humans , Intensive Care Units , Logistic Models , Male , Middle Aged , Pandemics/prevention & control , Pneumonia, Viral/prevention & control , Pneumonia, Viral/therapy , Predictive Value of Tests , Real-Time Polymerase Chain Reaction/methods , Retrospective Studies , Severity of Illness Index , Survival Analysis
16.
J Am Med Dir Assoc ; 21(7): 933-936, 2020 Jul.
Article in English | MEDLINE | ID: covidwho-593325

ABSTRACT

OBJECTIVE: To assess the American Testing Guidance for Nursing Homes (NHs)-updated May 19, 2020-with a new COVID-19 case. DESIGN: Case investigation. SETTING AND SUBJECTS: All 79 residents and 34 health care personnel (HCP) of an NH. METHODS: Seven days after identification of a COVID-19 resident, all residents and HCP underwent real-time reverse-transcriptase polymerase chain reaction (rRT-PCR) testing for SARS-CoV-2 with nasopharyngeal swabs. This was repeated weekly in all previously negative subjects until the testing identified no new cases, and in all positive subjects until the testing was negative. COVID-19 infection prevention and control (IPC) measures were implemented in all residents and HCP with positive testing or with COVID-19 symptoms. Standard IPC was also implemented in all HCP. Six weeks after initial testing, all residents underwent testing for enzyme-linked immunosorbent assay-based IgG antibodies directed against the SARS-CoV-2. Symptoms were serially recorded in residents and HCP. RESULTS: A total of 36 residents had a positive rRT-PCR at baseline and 2 at day 7. Six HCP had a positive rRT-PCR at baseline and 2 at day 7. No new COVID-19 cases were diagnosed later. Among the SARS-CoV-2-positive cases, 6 residents (16%) and 3 HCP (37%) were asymptomatic during the 14 days before testing. Twenty-five residents (92.3%) and all 8 HCP (100%) with a positive rRT-PCR developed IgG antibodies against SARS-CoV-2. Among the residents and HCP always having tested negative, 2 (5%) and 5 (11.5%), respectively, developed IgG antibodies against SARS-CoV-2. These 2 residents had typical COVID-19 symptoms before and after testing and 2/5 HCP were asymptomatic before and after testing. CONCLUSIONS AND IMPLICATIONS: This study shows the validity of the updated American Testing Guidance for Nursing Homes (NHs). It suggests implementing COVID-19 IPC in both residents and HCP with positive testing or COVID-19 symptoms and warns that asymptomatic HCP with repeated negative rRT-PCR testing can develop antibodies against SARS-CoV-2.


Subject(s)
Clinical Laboratory Techniques/methods , Contact Tracing/statistics & numerical data , Disease Outbreaks/prevention & control , Health Personnel/statistics & numerical data , Infectious Disease Transmission, Vertical/prevention & control , Nursing Homes/organization & administration , Antibodies, Viral/analysis , Clinical Laboratory Techniques/statistics & numerical data , Contact Tracing/methods , Coronavirus Infections/diagnosis , Coronavirus Infections/epidemiology , DNA, Viral/analysis , Female , Humans , Male , Occupational Health/statistics & numerical data , Outcome Assessment, Health Care , Pandemics , Patient Safety/statistics & numerical data , Pneumonia, Viral/diagnosis , Pneumonia, Viral/epidemiology , Real-Time Polymerase Chain Reaction/methods , Skilled Nursing Facilities/organization & administration , United States/epidemiology
17.
J Clin Microbiol ; 58(8)2020 07 23.
Article in English | MEDLINE | ID: covidwho-592313

ABSTRACT

Real-time reverse transcription-PCR (RT-PCR) is currently the most sensitive method to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that causes coronavirus disease 2019 (COVID-19). However, the correlation between detectable viral RNA and culturable virus in clinical specimens remains unclear. Here, we performed virus culture for 60 specimens that were confirmed to be positive for SARS-CoV-2 RNA by real-time RT-PCR. The virus could be successfully isolated from 12 throat and nine nasopharyngeal swabs and two sputum specimens. The lowest copy number required for virus isolation was determined to be 5.4, 6.0, and 5.7 log10 genome copies/ml sample for detecting the nsp12, E, and N genes, respectively. We further examined the correlation of genome copy number and virus isolation in different regions of the viral genome, demonstrating that culturable specimens are characterized by high copy numbers with a linear correlation observed between copy numbers of amplicons targeting structural and nonstructural regions. Overall, these results indicate that in addition to the copy number, the integrity of the viral genome should be considered when evaluating the infectivity of clinical SARS-CoV-2 specimens.


Subject(s)
Betacoronavirus/growth & development , Betacoronavirus/isolation & purification , Clinical Laboratory Techniques/methods , Coronavirus Infections/diagnosis , Coronavirus Infections/virology , Pneumonia, Viral/diagnosis , Pneumonia, Viral/virology , Virus Cultivation/methods , Betacoronavirus/genetics , Correlation of Data , Humans , Nasopharynx/virology , Pandemics , Pharynx/virology , Real-Time Polymerase Chain Reaction/methods , Reverse Transcriptase Polymerase Chain Reaction/methods
18.
J Infect Public Health ; 13(7): 901-905, 2020 Jul.
Article in English | MEDLINE | ID: covidwho-548405

ABSTRACT

At present the whole world is facing pandemic of the Coronavirus disease (COVID-19); caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This disease has rapidly spreads across the world from its origin of Wuhan, China and affected millions people worldwide and make them to remain in their homes. The knowledge of available laboratory methods is essential for early and correct diagnosis of COVID-19 to identify new cases as well as monitoring treatment of confirmed cases. In this review we aim to provide the updated information about selection of specimens and availability of various diagnostic methods and their utility with current findings for the laboratory diagnosis of SARS-CoV-2 infection. This will guide the healthcare professionals and government organizations to make strategy for establishing diagnostic facilities for SARS-CoV-2 infections.


Subject(s)
Betacoronavirus/isolation & purification , Coronavirus Infections/diagnosis , Immunoassay/methods , Pneumonia, Viral/diagnosis , Real-Time Polymerase Chain Reaction/methods , Virus Cultivation/methods , Coronavirus Infections/prevention & control , Coronavirus Infections/virology , Humans , Nasopharynx/virology , Pandemics/prevention & control , Pneumonia, Viral/prevention & control , Pneumonia, Viral/virology , Reverse Transcriptase Polymerase Chain Reaction/methods , Specimen Handling
19.
J Chin Med Assoc ; 83(6): 524-526, 2020 06.
Article in English | MEDLINE | ID: covidwho-542049

ABSTRACT

The rapid spread of coronavirus disease 2019 (COVID-19) in many countries causes citizens of daily inconvenience and even life-threat for elderly population. The invasion of the main pathogen, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2; 2019 novel coronavirus [2019-nCoV]), into human body causes different levels of impact to various patients. One of the most important issues for COVID-19 is how to defend this virus with the ability to foresee the infected targets. Thus, we maintain the quarantined essentially as for as others saved from COVID-19. So far, the routine laboratory test to confirm whether infected by SARS-CoV-2/2019-nCoV or not is through real-time reverse transcription polymerase chain reaction (rRT-PCR; quantitative polymerase chain reaction [qPCR]) with certain sequence regions that recognize SARS-CoV-2/2019-nCoV RNA genome. The heavy loading of rRT-PCR (qPCR) machine and handling labor have tight-packed the instruments as well as the manpower almost in every country. Therefore, the alternative approaches are eagerly waiting to be developed. In this review article, we sort out some state-of-the-art novel approaches that might be applied for a fast, sensitive, and precise detection of SARS-CoV-2/2019-nCoV not only to help the routine laboratory testing but also to improve effective quarantine.


Subject(s)
Betacoronavirus/isolation & purification , Coronavirus Infections/diagnosis , Pneumonia, Viral/diagnosis , Clinical Laboratory Techniques , Humans , Pandemics , RNA, Viral/analysis , Real-Time Polymerase Chain Reaction/methods
20.
J Clin Virol ; 128: 104454, 2020 07.
Article in English | MEDLINE | ID: covidwho-480203

ABSTRACT

BACKGROUND: Several qPCR kits are available for SARS-CoV-2 diagnosis, mostly lacking of evaluation due to covid19 emergency. OBJECTIVE: We evaluated nCoV-QS (MiCo BioMed) kit using CDC kit as gold standard. RESULTS: We found limitations for nCoV-QS: 1) lower sensitivity 2) lack of RNA quality control probe. CONCLUSIONS: Validation studies should be implemented for any SARS-CoV-2 RT-qPCR commercial kit to prevent unreliable diagnosis.


Subject(s)
Betacoronavirus/isolation & purification , Coronavirus Infections/diagnosis , Pneumonia, Viral/diagnosis , Real-Time Polymerase Chain Reaction/methods , Betacoronavirus/genetics , Coronavirus Infections/virology , Humans , Nasopharynx/virology , Pandemics , Pneumonia, Viral/virology , Reagent Kits, Diagnostic , Sensitivity and Specificity
SELECTION OF CITATIONS
SEARCH DETAIL