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1.
Front Cell Infect Microbiol ; 12: 960938, 2022.
Article in English | MEDLINE | ID: covidwho-2154694

ABSTRACT

Coronavirus disease 2019 (COVID-19) is an extremely contagious illness caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Early disease recognition of COVID-19 is crucial not only for prompt diagnosis and treatment of the patients, but also for effective public health surveillance and response. The reverse transcription-polymerase chain reaction (RT-PCR) is the most common method for the detection of SARS-CoV-2 viral mRNA and is regarded as the gold standard test for COVID-19. However, this test and those for antibodies (IgM and IgG) and antigens have certain limitations (e.g., by yielding false-negative and false-positive results). We have developed an RNA fluorescence in situ hybridization (FISH) method for high-sensitivity detection of SARS-CoV-2 mRNAs in HEK 293T cell cultures as a model. After transfection of HEK 293T cells with plasmids, Spike (S)/envelope (E) proteins and their mRNAs were clearly detected inside the cells. In addition, hybridization time could be reduced to 2 hours for faster detection when probe concentration was increased. Our approach might thus significantly improve the sensitivity and specificity of SARS-CoV-2 detection and be widely applied for the high-sensitivity single-molecular detection of other RNA viruses (e.g., Middle East respiratory syndrome coronavirus (MERS-CoV), Hepatitis A virus, all influenza viruses, and human immunodeficiency virus (HIV)) in various types of samples including tissue, body fluid, blood, and water. RNA FISH can also be utilized for the detection of DNA viruses (e.g., Monkeypox virus, human papillomavirus (HPV), and cytomegalovirus (CMV)) by detection of their mRNAs inside cells or body fluid.


Subject(s)
COVID-19 , SARS-CoV-2 , Humans , SARS-CoV-2/genetics , COVID-19 Testing , Clinical Laboratory Techniques/methods , RNA, Messenger/genetics , In Situ Hybridization, Fluorescence , HEK293 Cells , Immunoglobulin M , Immunoglobulin G , Water
2.
N Z Med J ; 135(1559): 53-58, 2022 Aug 05.
Article in English | MEDLINE | ID: covidwho-2147482

ABSTRACT

AIM: To compare detection of SARS-CoV-2 from paired nasopharyngeal swabs (NPS) and saliva using molecular methods in common use for testing swabs in New Zealand. METHOD: Samples from individuals testing positive for SARS-CoV-2 in Auckland, Wellington and Dunedin were tested at the local laboratories using methods previously established for these sample types. RESULTS: One hundred and ninety-six paired samples from unique individuals were tested, with 46 (23%) positive from either sample type, of which 43/46 (93%) tested positive from NPS, and 42/46 (91%) from saliva, indicating no significant difference in performance between sample types (p=0.69). The average Δ Ct between saliva and nasopharyngeal swabs overall across the sample set was 0.22 cycles, indicating excellent concordance; however, the difference between NPS and saliva collected from the same individual was quite variable with up to 19 cycles difference between the sample types. CONCLUSION: We found that saliva is an equivalent sample type to nasopharyngeal swab for the detection of SARS-CoV-2 in our laboratories using multiple assay combinations and is suitable for use as a diagnostic and surveillance test for selected groups of individuals.


Subject(s)
COVID-19 , Nucleic Acids , COVID-19/diagnosis , Clinical Laboratory Techniques/methods , Humans , Nasopharynx , New Zealand , SARS-CoV-2/genetics , Saliva , Specimen Handling/methods
3.
Elife ; 92020 08 17.
Article in English | MEDLINE | ID: covidwho-2155739

ABSTRACT

Temporal inference from laboratory testing results and triangulation with clinical outcomes extracted from unstructured electronic health record (EHR) provider notes is integral to advancing precision medicine. Here, we studied 246 SARS-CoV-2 PCR-positive (COVIDpos) patients and propensity-matched 2460 SARS-CoV-2 PCR-negative (COVIDneg) patients subjected to around 700,000 lab tests cumulatively across 194 assays. Compared to COVIDneg patients at the time of diagnostic testing, COVIDpos patients tended to have higher plasma fibrinogen levels and lower platelet counts. However, as the infection evolves, COVIDpos patients distinctively show declining fibrinogen, increasing platelet counts, and lower white blood cell counts. Augmented curation of EHRs suggests that only a minority of COVIDpos patients develop thromboembolism, and rarely, disseminated intravascular coagulopathy (DIC), with patients generally not displaying platelet reductions typical of consumptive coagulopathies. These temporal trends provide fine-grained resolution into COVID-19 associated coagulopathy (CAC) and set the stage for personalizing thromboprophylaxis.


Subject(s)
Betacoronavirus/isolation & purification , Blood Coagulation Disorders/diagnosis , Blood Coagulation Tests , Blood Coagulation , Clinical Laboratory Techniques , Coronavirus Infections/diagnosis , Pneumonia, Viral/diagnosis , Aged , Betacoronavirus/pathogenicity , Biomarkers/blood , Blood Coagulation Disorders/blood , Blood Coagulation Disorders/virology , COVID-19 , COVID-19 Testing , Coronavirus Infections/blood , Coronavirus Infections/virology , Disease Progression , Female , Fibrinogen/metabolism , Host Microbial Interactions , Humans , Leukocyte Count , Longitudinal Studies , Male , Middle Aged , Pandemics , Platelet Count , Pneumonia, Viral/blood , Pneumonia, Viral/virology , Predictive Value of Tests , Reproducibility of Results , Retrospective Studies , SARS-CoV-2 , Time Factors
4.
Anal Chim Acta ; 1234: 340533, 2022 Nov 22.
Article in English | MEDLINE | ID: covidwho-2129675

ABSTRACT

The emerging pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) critically challenges early and accurate virus diagnoses. However, the current gold standard for SARS-CoV-2 detection, reverse transcription-quantitative polymerase chain reaction (RT-qPCR), has reportedly failed to detect low-viral loads. One compound, graphene oxide (GO), which adsorbs single-stranded DNA (ssDNA), has been widely applied in molecular pathogen detection. This study presents a highly sensitive GO-multiplex qPCR method for simultaneous detection of two SARS-CoV-2 genes (RdRP and E) and one reference gene (RNase P). In a GO-multiplex qPCR system, GO pre-absorbs each forward primer to form specific GO-forward primer composites before entering the amplification system. Target gene amplification is confined within the primer-enriched composites, thus, improving the sensitivity of the assay. Compared to conventional multiplex qPCR, GO-multiplex qPCR reduces the limit of detection by 10-fold to 10 copies/reaction. Hence, the GO-multiplex qPCR assay can be effectively used for SARS-CoV-2 detection.


Subject(s)
COVID-19 , SARS-CoV-2 , Humans , SARS-CoV-2/genetics , COVID-19 Testing , Clinical Laboratory Techniques/methods , COVID-19/diagnosis , RNA, Viral/genetics , RNA, Viral/analysis , Sensitivity and Specificity
5.
Arch. argent. pediatr ; 120(5): 336-339, oct. 2022. tab
Article in English, Spanish | WHO COVID, LILACS (Americas) | ID: covidwho-2144796

ABSTRACT

Frenar la propagación de la enfermedad por el coronavirus 2019 (COVID-19, por su sigla en inglés) es fundamental, y se puede realizar mediante técnicas de detección rápidas y efectivas. El objetivo fue comparar la precisión diagnóstica de un test rápido de antígeno (TRAg,) con la reacción en cadena de polimerasa con retrotranscripción (RT-qPCR, por su sigla en inglés) y describir los umbrales de amplificación (Ct, por su sigla en inglés). Participaron niños de 1 mes a 11 años que tuvieran menos de 7 días de síntomas, sin resultado detectable en los últimos 90 días, e inmunocompetentes. Se incluyeron 1855 pacientes con una prevalencia de COVID-19 del 4,7 %. La sensibilidad global del TRAg fue del 60,2 % y su especificidad, del 99,8 %; en niños mayores de 5 años los valores fueron de 69,8 % y 99,8 %, respectivamente. Los valores de Ct de las muestras discordantes fueron más altos. En conclusión, la precisión diagnóstica muestra que TRAg tiene una especificidad similar a la RT-qPCR, pero una sensibilidad considerablemente menor, sobre todo en niños de menos de 5 años.


Stopping the spread of coronavirus disease 2019 (COVID-19) is critical and can be achieved through rapid and effective detection techniques. Our objective was to compare the diagnostic accuracy of rapid antigen tests (RAgT) and reverse transcription quantitative polymerase chain reaction (RT-qPCR) and to describe amplification cycle thresholds (Cts). Participants were children aged 1 month to 11 years with symptoms for less than 7 days, who did not have a detectable result in the past 90 days, and were immunocompetent. A total of 1855 patients were included; the prevalence of COVID-19 was 4.7%. For the RAgT, overall sensitivity was 60.2% and specificity, 99.8%; in children older than 5 years, values were 69.8% and 99.8%, respectively. Ct values for discordant samples were higher. To conclude, the diagnostic accuracy indicated that the specificity of RAgT is similar to that of RT-qPCR, but its sensitivity is notably lower,especially in children younger than 5 years.


Subject(s)
Humans , Infant , Child, Preschool , Child , SARS-CoV-2 , COVID-19/diagnosis , Cross-Sectional Studies , Sensitivity and Specificity , Clinical Laboratory Techniques/methods , Real-Time Polymerase Chain Reaction , COVID-19 Testing
6.
Ann Intern Med ; 173(10): JC57, 2020 11 17.
Article in English | MEDLINE | ID: covidwho-2110754

ABSTRACT

SOURCE CITATION: Deeks JJ, Dinnes J, Takwoingi Y, et al. Antibody tests for identification of current and past infection with SARS-CoV-2. Cochrane Database Syst Rev. 2020;6:CD013652. 32584464.


Subject(s)
Coronavirus Infections , Pandemics , Pneumonia, Viral , Antibodies, Viral , Betacoronavirus , COVID-19 , COVID-19 Testing , Clinical Laboratory Techniques , Coronavirus Infections/diagnosis , Coronavirus Infections/epidemiology , Humans , Pneumonia, Viral/epidemiology , SARS-CoV-2 , Sensitivity and Specificity
11.
Infect Control Hosp Epidemiol ; 41(11): 1328-1330, 2020 Nov.
Article in English | MEDLINE | ID: covidwho-2096354

ABSTRACT

Environmental surface testing was performed to search for evidence of severe acute respiratory coronavirus virus 2 (SARS-CoV-2) environmental contamination by an asymptomatic SARS-CoV-2 carrier with persistently high viral loads under isolation. No evidence of environmental contamination was found. Further studies are needed to measure environmental contamination by SARS-CoV-2 carriers and to determine reasonable isolation periods.


Subject(s)
Asymptomatic Infections , Betacoronavirus/isolation & purification , Clinical Laboratory Techniques , Coronavirus Infections/diagnosis , Fomites/virology , Pneumonia, Viral/diagnosis , Quarantine/methods , Viral Load , Adult , COVID-19 , COVID-19 Testing , Child , Coronavirus Infections/prevention & control , Coronavirus Infections/transmission , Coronavirus Infections/virology , Female , Humans , Pandemics/prevention & control , Patients' Rooms , Pneumonia, Viral/prevention & control , Pneumonia, Viral/transmission , Pneumonia, Viral/virology , Quarantine/standards , SARS-CoV-2
20.
PLoS Comput Biol ; 18(10): e1010629, 2022 10.
Article in English | MEDLINE | ID: covidwho-2089313

ABSTRACT

The rapid spread of SARS-CoV-2 has placed a significant burden on public health systems to provide swift and accurate diagnostic testing highlighting the critical need for innovative testing approaches for future pandemics. In this study, we present a novel sample pooling procedure based on compressed sensing theory to accurately identify virally infected patients at high prevalence rates utilizing an innovative viral RNA extraction process to minimize sample dilution. At prevalence rates ranging from 0-14.3%, the number of tests required to identify the infection status of all patients was reduced by 69.26% as compared to conventional testing in primary human SARS-CoV-2 nasopharyngeal swabs and a coronavirus model system. Our method provided quantification of individual sample viral load within a pool as well as a binary positive-negative result. Additionally, our modified pooling and RNA extraction process minimized sample dilution which remained constant as pool sizes increased. Compressed sensing can be adapted to a wide variety of diagnostic testing applications to increase throughput for routine laboratory testing as well as a means to increase testing capacity to combat future pandemics.


Subject(s)
COVID-19 , Humans , COVID-19/diagnosis , SARS-CoV-2 , COVID-19 Testing , Clinical Laboratory Techniques/methods , Pandemics , Sensitivity and Specificity
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