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1.
Front Immunol ; 12: 732756, 2021.
Article in English | MEDLINE | ID: covidwho-1597480

ABSTRACT

Coronavirus disease 2019 (COVID-19), which started out as an outbreak of pneumonia, has now turned into a pandemic due to its rapid transmission. Besides developing a vaccine, rapid, accurate, and cost-effective diagnosis is essential for monitoring and combating the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its related variants on time with precision and accuracy. Currently, the gold standard for detection of SARS-CoV-2 is Reverse Transcription Polymerase Chain Reaction (RT-PCR), but it lacks accuracy, is time-consuming and cumbersome, and fails to detect multi-variant forms of the virus. Herein, we have summarized conventional diagnostic methods such as Chest-CT (Computed Tomography), RT-PCR, Loop Mediated Isothermal Amplification (LAMP), Reverse Transcription-LAMP (RT-LAMP), as well new modern diagnostics such as CRISPR-Cas-based assays, Surface Enhanced Raman Spectroscopy (SERS), Lateral Flow Assays (LFA), Graphene-Field Effect Transistor (GraFET), electrochemical sensors, immunosensors, antisense oligonucleotides (ASOs)-based assays, and microarrays for SARS-CoV-2 detection. This review will also provide an insight into an ongoing research and the possibility of developing more economical tools to tackle the COVID-19 pandemic.


Subject(s)
COVID-19 Testing/methods , COVID-19/diagnosis , Clinical Laboratory Techniques/methods , Molecular Diagnostic Techniques/methods , SARS-CoV-2/genetics , COVID-19/epidemiology , COVID-19/virology , Humans , Immunoassay/methods , Nucleic Acid Amplification Techniques/methods , Oligonucleotide Probes/genetics , Pandemics , RNA, Viral/genetics , Reproducibility of Results , Reverse Transcriptase Polymerase Chain Reaction/methods , SARS-CoV-2/physiology , Sensitivity and Specificity
2.
Eur J Med Res ; 26(1): 147, 2021 Dec 17.
Article in English | MEDLINE | ID: covidwho-1582004

ABSTRACT

BACKGROUND: The outbreak of novel coronavirus disease 2019 (COVID-19) has become a public health emergency of international concern. Quantitative testing of SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) virus is demanded in evaluating the efficacy of antiviral drugs and vaccines and RT-PCR can be widely deployed in the clinical assay of viral loads. Here, we developed a quantitative RT-PCR method for SARS-CoV-2 virus detection in this study. METHODS: RT-PCR kits targeting E (envelope) gene, N (nucleocapsid) gene and RdRP (RNA-dependent RNA polymerase) gene of SARS-CoV-2 from Roche Diagnostics were evaluated and E gene kit was employed for quantitative detection of COVID-19 virus using Cobas Z480. Viral load was calculated according to the standard curve established by series dilution of an E-gene RNA standard provided by Tib-Molbiol (a division of Roche Diagnostics). Assay performance was evaluated. RESULTS: The performance of the assay is acceptable with limit of detection (LOD) below 10E1 copies/µL and lower limit of quantification (LLOQ) as 10E2 copies/µL. CONCLUSION: A quantitative detection of the COVID-19 virus based on RT-PCR was established.


Subject(s)
COVID-19/diagnosis , Coronavirus Envelope Proteins/genetics , Coronavirus Nucleocapsid Proteins/genetics , Coronavirus RNA-Dependent RNA Polymerase/genetics , Reverse Transcriptase Polymerase Chain Reaction/methods , Humans , Limit of Detection , Phosphoproteins/genetics , SARS-CoV-2/genetics , SARS-CoV-2/isolation & purification , Viral Load/methods
3.
Lab Med ; 52(6): e147-e153, 2021 Nov 02.
Article in English | MEDLINE | ID: covidwho-1574316

ABSTRACT

OBJECTIVE: In this study, the performance of 2 commercially available SARS-CoV-2 antibody assays is evaluated. METHODS: The Siemens SARS-CoV-2 Total (COV2T) and IgG (COV2G) antibody tests were evaluated on a Siemens Atellica IM1300 analyzer. Imprecision was assessed with the CLSI EP15 protocol using positive controls. Ninety control group specimens were analyzed for specificity, and 175 specimens from 58 patients with polymerase chain reaction-confirmed SARS-CoV-2 were measured for the sensitivity and kinetics of the antibody response. RESULTS: Within-run and total imprecision were acceptable for both assays. Both tests showed a specificity of 100%. Sensitivity earlier in the disease state was greater for the COV2T assay than for the COV2G assay, but sensitivity >14 days after onset of symptoms approached 100% for both. For all patients, antibody titers remained above the seroconversion cutoff for all follow-up specimens. CONCLUSION: This study shows acceptable performance for both the Siemens COV2T and COV2G test, although seroconversion occurs earlier with the COV2T test.


Subject(s)
Antibodies, Viral/blood , COVID-19 Serological Testing/standards , COVID-19/diagnosis , Immunoglobulin G/blood , SARS-CoV-2/immunology , Aged , Aged, 80 and over , Automation, Laboratory , COVID-19/blood , COVID-19/immunology , COVID-19/virology , COVID-19 Serological Testing/methods , Enzyme-Linked Immunosorbent Assay/methods , Female , Humans , Male , Reagent Kits, Diagnostic , Retrospective Studies , Reverse Transcriptase Polymerase Chain Reaction/methods , Sensitivity and Specificity
4.
Lab Med ; 52(6): e154-e158, 2021 Nov 02.
Article in English | MEDLINE | ID: covidwho-1559980

ABSTRACT

OBJECTIVE: This study aims to evaluate the performance of an antigen-based rapid diagnostic test (RDT) for the detection of the SARS-CoV-2 virus. METHODS: A cross-sectional study was conducted on 677 patients. Two nasopharyngeal swabs and 1 oropharyngeal swab were collected from patients. The RDT was performed onsite by a commercially available immune-chromatographic assay on the nasopharyngeal swab. The nasopharyngeal and oropharyngeal swabs were examined for SARS-CoV-2 RNA by real-time reverse-transcription quantitative polymerase chain reaction (RT-qPCR) assay. RESULTS: The overall sensitivity of the SARS-CoV-2 RDT was 34.5% and the specificity was 99.8%. The positive predictive value and negative predictive value of the test were 96.6% and 91.5%, respectively. The detection rate of RDT in RT-qPCR positive results was high (45%) for cycle threshold values <25. CONCLUSION: The utility of RDT is in diagnosing symptomatic patients and may not be particularly suited as a screening tool for patients with low viral load. The low sensitivity of RDT does not qualify its use as a single test in patients who test negative; RT-qPCR continues to be the gold standard test.


Subject(s)
Antigens, Viral/genetics , COVID-19 Serological Testing/standards , COVID-19/diagnosis , Chromatography, Affinity/methods , RNA, Viral/genetics , SARS-CoV-2/genetics , Adolescent , Aged , Aged, 80 and over , Automation, Laboratory , COVID-19/immunology , COVID-19/virology , COVID-19 Serological Testing/methods , Cross-Sectional Studies , Female , Humans , Male , Middle Aged , Nasopharynx/virology , Oropharynx/virology , Reagent Kits, Diagnostic , Reverse Transcriptase Polymerase Chain Reaction/methods , SARS-CoV-2/immunology , Sensitivity and Specificity , Viral Load/genetics
5.
J Med Virol ; 93(12): 6693-6695, 2021 Dec.
Article in English | MEDLINE | ID: covidwho-1544321

ABSTRACT

We aimed to compare reverse transcription-polymerase chain reaction (RT-PCR) results of nasopharyngeal aspiration (NA) and nasopharyngeal swab (NS) samples in the diagnosis of coronavirus disease 2019. NS was obtained with a dacron swab and NA was performed by aspiration cannula. The sampling was performed by an otolaryngologist to ensure standardized correct sampling from the nasopharynx. RT-PCR was performed for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The level of agreement between the result of NA and NS samples for each patient was analyzed. The Ct values were compared. Thirty-three patients were enrolled in the study with a mean age of 56.3 years. Thirteen subjects resulted negative with both NS and NA; 20 subjects resulted positive with NA and 18 subjects resulted positive with NS. The mean values of Ct for NA samples and NS samples were 24.6 ± 5.9 and 24 ± 6.7, respectively. There was no statistical difference between Ct values of NA and NS samples (p = 0.48). RT-PCR for SARS-Cov2 performed with NA sample and NS sample showed a strong correlation regarding the positivity/negativity and the Ct values.


Subject(s)
COVID-19 Testing/methods , Nasopharynx/virology , Reverse Transcriptase Polymerase Chain Reaction/methods , SARS-CoV-2 , Specimen Handling/methods , Adult , Aged , Female , Humans , Male , Middle Aged , Reproducibility of Results , Sensitivity and Specificity
6.
J Med Virol ; 93(12): 6808-6812, 2021 Dec.
Article in English | MEDLINE | ID: covidwho-1544312

ABSTRACT

Real-time polymerase chain reaction (PCR) for SARS-CoV-2 is the mainstay of COVID-19 diagnosis, yet there are conflicting reports on its diagnostic performance. Wide ranges of false-negative PCR tests have been reported depending on clinical presentation, the timing of testing, specimens tested, testing method, and reference standard used. We aimed to estimate the frequency of discordance between initial nasopharyngeal (NP) PCR and repeat NP sampling PCR and serology in acutely ill patients admitted to the hospital. Panel diagnosis of COVID-19 infection is further utilized in discordance analysis. Included in the study were 160 patients initially tested by NP PCR with repeat NP sampling PCR and/or serology performed. The percent agreement between initial and repeat PCR was 96.7%, while the percent agreement between initial PCR and serology was 98.9%. There were 5 (3.1%) cases with discordance on repeat testing. After discordance analysis, 2 (1.4%) true cases tested negative on initial PCR. Using available diagnostic methods, discordance on repeat NP sampling PCR and/or serology is a rare occurrence.


Subject(s)
COVID-19/diagnosis , COVID-19/virology , Nasopharynx/virology , SARS-CoV-2/genetics , Adult , COVID-19 Testing/methods , Female , Humans , Male , Real-Time Polymerase Chain Reaction/methods , Reference Standards , Reverse Transcriptase Polymerase Chain Reaction/methods , Sensitivity and Specificity , Specimen Handling/methods
7.
J Med Virol ; 93(12): 6582-6587, 2021 Dec.
Article in English | MEDLINE | ID: covidwho-1544305

ABSTRACT

The purpose of this study was to evaluate the SARS-CoV-2 immunoglobulin M/immunoglobulin G (IgM/IgG) rapid antibody test results in symptomatic patients with COVID-19 and their chest computed tomography (CT) data. A total of 320 patients admitted to our hospital for different durations due to COVID-19 were included in the study. Serum samples were obtained within 0-7 days from COVID-19 patients confirmed by reverse-transcription polymerase chain reaction (RT-PCR) and chest CT scan. According to the SARS-CoV-2 RT-PCR results, the patients included in the study were divided into two groups: PCR positive group (n = 46) and PCR negative group (n = 274). The relationship between chest CT and rapid antibody test results were compared statistically. Of the 320 COVID-19 serum samples, IgM, IgG, and IgM/IgG were detected in 8.4%, 0.3%, and 11.6% within 1 week, respectively. IgG/IgM antibodies were not detected in 79.7% of the patients. In the study, 249 (77.8%) of 320 patients had positive chest CT scans. Four (5.6%) of 71 patients with negative chest CT scans had IgM and two (2.8%) were both IgM/IgG positive. IgM was detected in 23 (9.2%), IgG in one (0.4%), and IgM/IgG in 35 (14%) of chest CT scan positive patients. The rate of CT findings in patients with antibody positivity was found to be significantly higher than those with antibody negativity. The results of the present study show the accurate and equivalent performance of serological antibody assays and chest CT in detecting SARS-CoV-2 within 0-7 days from the onset of COVID19 symptoms. When RT-PCR is not available, we believe that the combination of immunochromatographic test and chest CT scan can increase diagnostic sensitivity for COVID-19.


Subject(s)
Antibodies, Viral/blood , COVID-19 Testing/methods , COVID-19/diagnosis , Reverse Transcriptase Polymerase Chain Reaction/methods , Adolescent , Adult , Aged , Aged, 80 and over , Antibodies, Viral/immunology , COVID-19/diagnostic imaging , Female , Humans , Immunoglobulin G/blood , Immunoglobulin G/immunology , Immunoglobulin M/blood , Immunoglobulin M/immunology , Male , Middle Aged , Radiography, Thoracic , Retrospective Studies , Tomography, X-Ray Computed , Young Adult
8.
J Med Virol ; 93(12): 6794-6797, 2021 12.
Article in English | MEDLINE | ID: covidwho-1544304

ABSTRACT

Severe acute respiratory syndrome coronavirus (SARS-CoV-2) has affected all inhabited continents, and India is currently experiencing a devastating second wave of coronavirus disease-2019 (COVID-19). Here, we examined the duration of clearance of SARS-CoV-2 in respiratory samples from 207 infected cases by real-time reverse-transcription polymerase chain reaction (RT-PCR). A substantial proportion of COVID-19 positive cases with cycle threshold (Ct) values more than or equal to 31 (45.7%) were subsequently tested negative for SARS-CoV-2 RNA within 7 days of initial detection of the viral load. A total of 60% of all the patients with COVID-19, irrespective of their Ct values, cleared SARS-CoV-2 RNA within 14 days of the initial detection. Longitudinal assessment of RT-PCR test results in individuals requiring 15-30 days to clear SARS-CoV-2 RNA showed a significant reduction of the viral load in samples with high or intermediate viral loads (Ct values ≤ 25 and between 26 and 30, respectively) but the follow-up group with low viral RNA (Ct values ≥ 31) exhibited a stable viral load. Together, these results suggest that COVID-19 positive cases with Ct values more than or equal to 31 require reduced duration to clear SARS-CoV-2, and thus, a shorter isolation period for this group might be considered to facilitate adequate space in the COVID Care Centres and reduce the burden on healthcare infrastructure.


Subject(s)
COVID-19/virology , SARS-CoV-2/genetics , Viral Load/genetics , Adult , Aged , COVID-19 Testing/methods , Diagnostic Tests, Routine/methods , Female , Humans , India , Longitudinal Studies , Male , Middle Aged , RNA, Viral/genetics , Real-Time Polymerase Chain Reaction/methods , Reverse Transcriptase Polymerase Chain Reaction/methods , Sensitivity and Specificity , Serologic Tests/methods , Young Adult
9.
Front Biosci (Landmark Ed) ; 26(10): 740-751, 2021 10 30.
Article in English | MEDLINE | ID: covidwho-1498507

ABSTRACT

Objectives: To quantify the integrated levels of ACE2 and TMPRSS2, the two well-recognized severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) entry-related genes, and to further identify key factors contributing to SARS-CoV-2 susceptibility in head and neck squamous cell carcinoma (HNSC). Methods: We developed a metric of the potential for tissue infected with SARS-CoV-2 ("TPSI") based on ACE2 and TMPRSS2 transcript levels and compared TPSI levels between tumor and matched normal tissues across 11 tumor types. For further analysis of HNSC, weighted gene co-expression network analysis (WGCNA), functional analysis, and single sample gene set enrichment analysis (ssGSEA) were conducted to investigate TPSI-relevant biological processes and their relationship with the immune landscape. TPSI-related factors were identified from clinical and mutational domains, followed by lasso regression to determine their relative effects on TPSI levels. Results: TPSI levels in tumors were generally lower than in the normal tissues. In HNSC, the genes highly associated with TPSI were enriched in viral entry-related processes, and TPSI levels were positively correlated with both eosinophils and T helper 17 (Th17) cell infiltration. Furthermore, the site of onset, human papillomaviruses (HPV) status, and nuclear receptor binding SET domain protein 1 (NSD1) mutations were identified as the most important factors shaping TPSI levels. Conclusions: This study identified the infection risk of SARS-CoV-2 between tumor and normal tissues, and provided evidence for the risk stratification of HNSC.


Subject(s)
Angiotensin-Converting Enzyme 2/genetics , COVID-19/genetics , Carcinoma, Squamous Cell/genetics , Head and Neck Neoplasms/genetics , Serine Endopeptidases/genetics , Angiotensin-Converting Enzyme 2/metabolism , COVID-19/metabolism , COVID-19/virology , Carcinoma, Squamous Cell/metabolism , Carcinoma, Squamous Cell/virology , Gene Expression Profiling/methods , Gene Expression Regulation, Neoplastic , Gene Regulatory Networks , Head and Neck Neoplasms/metabolism , Head and Neck Neoplasms/virology , Humans , Reverse Transcriptase Polymerase Chain Reaction/methods , SARS-CoV-2/metabolism , SARS-CoV-2/physiology , Serine Endopeptidases/metabolism , Virus Internalization
10.
Sci Rep ; 11(1): 21368, 2021 11 01.
Article in English | MEDLINE | ID: covidwho-1493221

ABSTRACT

There is a need for wastewater based epidemiological (WBE) methods that integrate multiple, variously sized surveillance sites across geographic areas. We developed a novel indexing method, Melvin's Index, that provides a normalized and standardized metric of wastewater pathogen load for qPCR assays that is resilient to surveillance site variation. To demonstrate the utility of Melvin's Index, we used qRT-PCR to measure SARS-CoV-2 genomic RNA levels in influent wastewater from 19 municipal wastewater treatment facilities (WWTF's) of varying sizes and served populations across the state of Minnesota during the Summer of 2020. SARS-CoV-2 RNA was detected at each WWTF during the 20-week sampling period at a mean concentration of 8.5 × 104 genome copies/L (range 3.2 × 102-1.2 × 109 genome copies/L). Lag analysis of trends in Melvin's Index values and clinical COVID-19 cases showed that increases in indexed wastewater SARS-CoV-2 levels precede new clinical cases by 15-17 days at the statewide level and by up to 25 days at the regional/county level. Melvin's Index is a reliable WBE method and can be applied to both WWTFs that serve a wide range of population sizes and to large regions that are served by multiple WWTFs.


Subject(s)
COVID-19/epidemiology , SARS-CoV-2/genetics , Suburban Population , Urban Population , Waste Disposal Facilities , Waste Water/virology , Wastewater-Based Epidemiological Monitoring , Water Purification , COVID-19/virology , Genome, Viral , Humans , Minnesota/epidemiology , Prevalence , Prognosis , RNA, Viral/genetics , Reverse Transcriptase Polymerase Chain Reaction/methods , Risk Factors
11.
Sci Rep ; 11(1): 21385, 2021 11 01.
Article in English | MEDLINE | ID: covidwho-1493218

ABSTRACT

Shortages of reverse transcriptase (RT)-polymerase chain reaction (PCR) reagents and related equipment during the COVID-19 pandemic have demonstrated the need for alternative, high-throughput methods for severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2)-mass screening in clinical diagnostic laboratories. A robust, SARS-CoV-2 RT-loop-mediated isothermal amplification (RT-LAMP) assay with high-throughput and short turnaround times in a clinical laboratory setting was established and compared to two conventional RT-PCR protocols using 323 samples of individuals with suspected SARS-CoV-2 infection. Limit of detection (LoD) and reproducibility of the isolation-free SARS-CoV-2 RT-LAMP test were determined. An almost perfect agreement (Cohen's kappa > 0.8) between the novel test and two classical RT-PCR protocols with no systematic difference (McNemar's test, P > 0.05) was observed. Sensitivity and specificity were in the range of 89.5 to 100% and 96.2 to 100% dependent on the reaction condition and the RT-PCR method used as reference. The isolation-free RT-LAMP assay showed high reproducibility (Tt intra-run coefficient of variation [CV] = 0.4%, Tt inter-run CV = 2.1%) with a LoD of 95 SARS-CoV-2 genome copies per reaction. The established SARS-CoV-2 RT-LAMP assay is a flexible and efficient alternative to conventional RT-PCR protocols, suitable for SARS-CoV-2 mass screening using existing laboratory infrastructure in clinical diagnostic laboratories.


Subject(s)
COVID-19 Nucleic Acid Testing/methods , COVID-19/diagnosis , COVID-19/epidemiology , Molecular Diagnostic Techniques/methods , Nucleic Acid Amplification Techniques/methods , Pandemics , Reverse Transcriptase Polymerase Chain Reaction/methods , SARS-CoV-2/genetics , COVID-19/virology , Genome, Viral , Humans , Infection Control/methods , Limit of Detection , Mass Screening/methods , RNA, Viral/genetics , RNA, Viral/isolation & purification , RNA-Directed DNA Polymerase/genetics , Reproducibility of Results , Reverse Transcription/genetics , Sensitivity and Specificity
12.
Molecules ; 26(21)2021 Oct 31.
Article in English | MEDLINE | ID: covidwho-1488680

ABSTRACT

Early diagnosis with rapid detection of the virus plays a key role in preventing the spread of infection and in treating patients effectively. In order to address the need for a straightforward detection of SARS-CoV-2 infection and assessment of viral spread, we developed rapid, sensitive, extraction-free one-step reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and reverse transcription loop-mediated isothermal amplification (RT-LAMP) tests for detecting SARS-CoV-2 in saliva. We analyzed over 700 matched pairs of saliva and nasopharyngeal swab (NSB) specimens from asymptomatic and symptomatic individuals. Saliva, as either an oral cavity swab or passive drool, was collected in an RNA stabilization buffer. The stabilized saliva specimens were heat-treated and directly analyzed without RNA extraction. The diagnostic sensitivity of saliva-based RT-qPCR was at least 95% in individuals with subclinical infection and outperformed RT-LAMP, which had at least 70% sensitivity when compared to NSBs analyzed with a clinical RT-qPCR test. The diagnostic sensitivity for passive drool saliva was higher than that of oral cavity swab specimens (95% and 87%, respectively). A rapid, sensitive one-step extraction-free RT-qPCR test for detecting SARS-CoV-2 in passive drool saliva is operationally simple and can be easily implemented using existing testing sites, thus allowing high-throughput, rapid, and repeated testing of large populations. Furthermore, saliva testing is adequate to detect individuals in an asymptomatic screening program and can help improve voluntary screening compliance for those individuals averse to various forms of nasal collections.


Subject(s)
COVID-19/diagnosis , COVID-19/genetics , Reverse Transcriptase Polymerase Chain Reaction/methods , COVID-19 Testing/methods , Humans , Mass Screening/methods , Nucleic Acid Amplification Techniques/methods , RNA/isolation & purification , RNA, Viral/genetics , SARS-CoV-2/genetics , SARS-CoV-2/pathogenicity , Saliva/chemistry , Sensitivity and Specificity , Specimen Handling/methods
13.
Anal Bioanal Chem ; 413(29): 7195-7204, 2021 Dec.
Article in English | MEDLINE | ID: covidwho-1482198

ABSTRACT

The pandemic of the novel coronavirus disease 2019 (COVID-19) has caused severe harm to the health of people all around the world. Molecular detection of the pathogen, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), played a crucial role in the control of the disease. Reverse transcription digital PCR (RT-dPCR) has been developed and used in the detection of SARS-CoV-2 RNA as an absolute quantification method. Here, an interlaboratory assessment of quantification of SARS-CoV-2 RNA was organized by the National Institute of Metrology, China (NIMC), using in vitro transcribed RNA samples, among ten laboratories on six different dPCR platforms. Copy number concentrations of three genes of SARS-CoV-2 were measured by all participants. Consistent results were obtained with dispersion within 2.2-fold and CV% below 23% among different dPCR platforms and laboratories, and Z' scores of all the reported results being satisfactory. Possible reasons for the dispersion included PCR assays, partition volume, and reverse transcription conditions. This study demonstrated the comparability and applicability of RT-dPCR method for quantification of SARS-CoV-2 RNA and showed the capability of the participating laboratories at SARS-CoV-2 test by RT-dPCR platform.


Subject(s)
Laboratories/organization & administration , RNA, Viral/analysis , Reverse Transcriptase Polymerase Chain Reaction/methods , SARS-CoV-2/genetics , COVID-19/virology , Humans , Limit of Detection
14.
Microbiol Spectr ; 9(2): e0084621, 2021 10 31.
Article in English | MEDLINE | ID: covidwho-1476400

ABSTRACT

Isothermal amplification-based tests have been introduced as rapid, low-cost, and simple alternatives to real-time reverse transcriptase PCR (RT-PCR) tests for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection. The clinical performance of two isothermal amplification-based tests (Atila Biosystems iAMP coronavirus disease of 2019 [COVID-19] detection test and OptiGene COVID-19 direct plus RT-loop-mediated isothermal amplification [LAMP] test) was compared with that of clinical RT-PCR assays using different sampling strategies. A total of 1,378 participants were tested across 4 study sites. Compared with standard of care RT-PCR testing, the overall sensitivity and specificity of the Atila iAMP test for detection of SARS-CoV-2 were 76.2% and 94.9%, respectively, and increased to 88.8% and 89.5%, respectively, after exclusion of an outlier study site. Sensitivity varied based on the anatomic site from which the sample was collected. Sensitivity for nasopharyngeal sampling was 65.4% (range across study sites, 52.8% to 79.8%), for midturbinate was 88.2%, for saliva was 55.1% (range across study sites, 42.9% to 77.8%), and for anterior nares was 66.7% (range across study sites, 63.6% to 76.5%). The specificity for these anatomic collection sites ranged from 96.7% to 100%. Sensitivity improved in symptomatic patients (overall, 82.7%) and those with a higher viral load (overall, 92.4% for cycle threshold [CT] of ≤25). Sensitivity and specificity of the OptiGene direct plus RT-LAMP test, which was conducted at a single study site, were 25.5% and 100%, respectively. The Atila iAMP COVID test with midturbinate sampling is a rapid, low-cost assay for detecting SARS-CoV-2, especially in symptomatic patients and those with a high viral load, and could be used to reduce the risk of SARS-CoV-2 transmission in clinical settings. Variation of performance between study sites highlights the need for site-specific clinical validation of these assays before clinical adoption. IMPORTANCE Numerous SARS-CoV-2 detection assays have been developed and introduced into the market under emergency use authorizations (EUAs). EUAs are granted primarily based on small studies of analytic sensitivity and specificity with limited clinical validations. A thorough clinical performance evaluation of SARS-CoV-2 assays is important to understand the strengths, limitations, and specific applications of these assays. In this first large-scale multicentric study, we evaluated the clinical performance and operational characteristics of two isothermal amplification-based SARS-CoV-2 tests, namely, (i) iAMP COVID-19 detection test (Atila BioSystems, USA) and (ii) COVID-19 direct plus RT-LAMP test (OptiGene Ltd., UK), compared with those of clinical RT-PCR tests using different sampling strategies (i.e., nasopharyngeal, self-sampled anterior nares, self-sampled midturbinate, and saliva). An important specific use for these isothermal amplification-based, rapid, low-cost, and easy-to-perform SARS-CoV-2 assays is to allow for a safer return to preventive clinical encounters, such as cancer screening, particularly in low- and middle-income countries that have low SARS-CoV-2 vaccination rates.


Subject(s)
COVID-19 Nucleic Acid Testing/methods , COVID-19/diagnosis , Molecular Diagnostic Techniques/methods , Nucleic Acid Amplification Techniques/methods , SARS-CoV-2/genetics , SARS-CoV-2/isolation & purification , Humans , Limit of Detection , Mass Screening , Nasopharynx/virology , Point-of-Care Systems , RNA, Viral/analysis , Reverse Transcriptase Polymerase Chain Reaction/methods , Specimen Handling , Viral Load
15.
PLoS One ; 16(10): e0257834, 2021.
Article in English | MEDLINE | ID: covidwho-1468159

ABSTRACT

COVID-19 testing is required before admission of a patient in the hospitals, invasive procedures, major and minor surgeries etc. Real Time Polymerase chain reaction is the gold standard test for the diagnosis, but requires well equipped biosafety laboratory along with trained manpower. In this study we have evaluated the diagnostic accuracy of novel TrueNat molecular assay for detecting SARS CoV-2. TrueNat is a chip-based real time PCR test and works on portable, light weight, battery powered equipment and can be used in remote areas with poor infrastructure. In this study 1807 patients samples were collected for both TrueNat and RTPCR COVID-19 testing during study period. Of these 174 (9.7%) and 174 (15%) were positive by RTPCR and TrueNat respectively and taking results of RTPCR as gold standard TrueNat test showed a sensitivity, specificity and diagnostic accuracy of 69.5, 90.9% and 89.2% respectively. It can be concluded that TrueNat is a simple, easy to use, good rapid molecular diagnostic test for diagnosis of COVID-19 especially in resource limited settings and will prove to be a game changer of molecular diagnostics in future.


Subject(s)
COVID-19/diagnosis , Reverse Transcriptase Polymerase Chain Reaction/methods , SARS-CoV-2/genetics , COVID-19/virology , COVID-19 Testing , Humans , Point-of-Care Systems , Real-Time Polymerase Chain Reaction/instrumentation , Retrospective Studies , Reverse Transcriptase Polymerase Chain Reaction/instrumentation , SARS-CoV-2/isolation & purification , Sensitivity and Specificity
16.
Pan Afr Med J ; 39: 214, 2021.
Article in English | MEDLINE | ID: covidwho-1449268

ABSTRACT

Introduction: COVID-19 equation in Cameroon is yet to be resolved. There is an urgent need for a rapid response strategy to the increasing demand of polymerase chain reaction (PCR) test results for both patients, travelers and competitors to various games. We assessed the diagnostic performance of the AmpliQuick® SARS-CoV-2 against the classic Reverse transcription polymerase chain reaction (RT-PCR). Methods: a cross-sectional and comparative study was conducted from April 27th to May 29th, 2021 in the city of Douala, Cameroon. The samples consisted of any nasopharyngeal sample received at the Douala Gynaeco-Obstetrics and Pediatric Hospital molecular biology laboratory, regardless of its origin. Sociodemographic parameters (age, profession (footballers, travelers, other), matrimonial status, nationality), comorbidity and known status of COVID-19, were recorded at collection sites. The main collection sites were the Deido Health District and the Douala Gynaeco-Obstetric and Pediatric Hospital. We performed testing using AmpliQuick® SARS-CoV-2 and the classic RT-PCR (Da An Gene Co.Ltd) on each sample during the one month period. Analytical performance parameters were determined. To determine the sensitivity of both methods, the Bayesian latent class model was performed on the median with 95% confidence interval, with p≤0.05 as significant level, as well as Kappa (κ) agreement between tests. An ethical clearance was sought and obtained from the University of Douala Institutional Ethics Committee. Results: a total of 1813 participants were enrolled, with the predominance of male (68.68%) and the age group 31 to 40 years old (31.33%). Most participants were married (53.46%) with only few with known COVID-19 status (5.47%). One thousand eight hundred and ten (1810) tests were performed by AMPLIQUICK® SARS-CoV-2 while only 1107 could be achieved with the classic RT-PCR. Over the study period, it was noted a drastic reduction in the time necessary to render results with the AMPLIQUICK® SARS-CoV-2 from 24 hours to 3 hours. The AMPLIQUICK® SARS-CoV-2 reduced technician hands-on time and its practicability was noticed based on the prefilled and ready-to-use microplates. A prevalence of 1.93% and 1.45% were obtained for AMPLIQUICK® SARS-CoV-2 and the classic RT-PCR respectively. This difference in the prevalence showed that AMPLIQUICK® SARS-CoV-2 (Sensitivity 83.5% [CI=64.6-95.2]) was more accurate than the classic RT-PCR (67.8% [CI=46.6-84.9]). Conclusion: it is time for a change of attitude to scale up the COVID-19 testing ability in Cameroon and the AMPLIQUICK® SARS-CoV-2 is an alternative diagnosis strategy which should help resolve the situation of timely and reliable results.


Subject(s)
COVID-19 Testing/methods , COVID-19/diagnosis , Reverse Transcriptase Polymerase Chain Reaction/methods , Adolescent , Adult , Aged , Cameroon , Child , Child, Preschool , Cross-Sectional Studies , Female , Humans , Infant , Infant, Newborn , Male , Middle Aged , Reproducibility of Results , Sensitivity and Specificity , Time Factors , Young Adult
17.
Pediatr Infect Dis J ; 40(11): e413-e417, 2021 11 01.
Article in English | MEDLINE | ID: covidwho-1447654

ABSTRACT

BACKGROUND: The viral dynamics and the role of children in the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are not completely understood. Our aim was to evaluate reverse transcription polymerase chain reaction (RT-PCR) cycle threshold (Ct) values among children with confirmed SARS-CoV-2 compared with that of adult subjects. METHODS: Patients (from 2 months to ≤18 years of age and adults) with signs and symptoms of acute SARS-CoV-2 infection for less than 7 days were prospectively enrolled in the study from May to November 2020. All participants performed RT-PCR assay for SARS-CoV-2 detection; Ct values of ORF1ab, N and S gene targets and the average of all the 3 probes were used as surrogates of viral load. RESULTS: There were 21 infants (2 months to <2 years), 40 children (≥2 to <12 years), 22 adolescents (≥12 to <18 years) and 293 adults of 376 participants with confirmed SARS-CoV-2 infections. RT-PCR Ct values from all participants less than 18 years of age, as well as from all childhood subgroups, were not significantly different from adults, comparing ORF1ab, N, S and all the gene targets together (P = 0.453). CONCLUSIONS: Ct values for children were comparable with that of adults. Although viral load is not the only determinant of SARS-CoV-2 transmission, children may play a role in the spread of coronavirus disease 2019 in the community.


Subject(s)
COVID-19 Testing , COVID-19/diagnosis , COVID-19/virology , Reverse Transcriptase Polymerase Chain Reaction , SARS-CoV-2/genetics , Adolescent , Adult , Age Factors , Brazil , Child , Cross-Sectional Studies , Humans , Infant , RNA, Viral , Reverse Transcriptase Polymerase Chain Reaction/methods , Reverse Transcriptase Polymerase Chain Reaction/standards , Viral Load
18.
Sci Rep ; 11(1): 18955, 2021 09 23.
Article in English | MEDLINE | ID: covidwho-1437688

ABSTRACT

The world is facing an exceptional pandemic caused by SARS-CoV-2. To allow the diagnosis of COVID-19 infections, several assays based on the real-time PCR technique have been proposed. The requests for diagnosis are such that it was immediately clear that the choice of the most suitable method for each microbiology laboratory had to be based, on the one hand, on the availability of materials, and on the other hand, on the personnel and training priorities for this activity. Unfortunately, due to high demand, the shortage of commercial diagnostic kits has also become a major problem. To overcome these critical issues, we have developed a new qualitative RT-PCR probe. Our system detects three genes-RNA-dependent RNA polymerase (RdRp), envelope (E) and nucleocapsid (N)-and uses the ß-actin gene as an endogenous internal control. The results from our assay are in complete agreement with the results obtained using a commercially available kit, except for two samples that did not pass the endogenous internal control. The coincidence rate was 0.96. The LoD of our assay was 140 cp/reaction for N and 14 cp/reaction for RdRp and E. Our kit was designed to be open, either for the nucleic acid extraction step or for the RT-PCR assay, and to be carried out on several instruments. Therefore, it is free from the industrial production logics of closed systems, and conversely, it is hypothetically available for distribution in large quantities to any microbiological laboratory. The kit is currently distributed worldwide (called MOLgen-COVID-19; Adaltis). A new version of the kit for detecting the S gene is also available.


Subject(s)
COVID-19 Nucleic Acid Testing/methods , COVID-19/diagnosis , SARS-CoV-2/genetics , COVID-19/genetics , COVID-19 Testing/methods , Clinical Laboratory Techniques/methods , Coronavirus Envelope Proteins/genetics , Coronavirus Nucleocapsid Proteins/genetics , Coronavirus RNA-Dependent RNA Polymerase/genetics , Humans , Pandemics , Phosphoproteins/genetics , Qualitative Research , RNA, Viral/genetics , Reverse Transcriptase Polymerase Chain Reaction/methods , SARS-CoV-2/pathogenicity , Sensitivity and Specificity
19.
PLoS One ; 16(9): e0256813, 2021.
Article in English | MEDLINE | ID: covidwho-1410652

ABSTRACT

There is a worldwide need for reagents to perform SARS-CoV-2 detection. Some laboratories have implemented kit-free protocols, but many others do not have the capacity to develop these and/or perform manual processing. We provide multiple workflows for SARS-CoV-2 nucleic acid detection in clinical samples by comparing several commercially available RNA extraction methods: QIAamp Viral RNA Mini Kit (QIAgen), RNAdvance Blood/Viral (Beckman) and Mag-Bind Viral DNA/RNA 96 Kit (Omega Bio-tek). We also compared One-step RT-qPCR reagents: TaqMan Fast Virus 1-Step Master Mix (FastVirus, ThermoFisher Scientific), qPCRBIO Probe 1-Step Go Lo-ROX (PCR Biosystems) and Luna® Universal Probe One-Step RT-qPCR Kit (Luna, NEB). We used primer-probes that detect viral N (EUA CDC) and RdRP. RNA extraction methods provided similar results, with Beckman performing better with our primer-probe combinations. Luna proved most sensitive although overall the three reagents did not show significant differences. N detection was more reliable than that of RdRP, particularly in samples with low viral titres. Importantly, we demonstrated that heat treatment of nasopharyngeal swabs at 70°C for 10 or 30 min, or 90°C for 10 or 30 min (both original variant and B 1.1.7) inactivated SARS-CoV-2 employing plaque assays, and had minimal impact on the sensitivity of the qPCR in clinical samples. These findings make SARS-CoV-2 testing portable in settings that do not have CL-3 facilities. In summary, we provide several testing pipelines that can be easily implemented in other laboratories and have made all our protocols and SOPs freely available at https://osf.io/uebvj/.


Subject(s)
COVID-19 Testing/methods , COVID-19/diagnosis , Hot Temperature , RNA, Viral/genetics , SARS-CoV-2/genetics , Virus Inactivation , COVID-19/epidemiology , COVID-19/virology , Epidemics/prevention & control , Humans , Nasopharynx/virology , Reagent Kits, Diagnostic , Reproducibility of Results , Reverse Transcriptase Polymerase Chain Reaction/methods , SARS-CoV-2/physiology , Sensitivity and Specificity , Specimen Handling/methods , Workflow
20.
Genes Genomics ; 43(11): 1277-1288, 2021 11.
Article in English | MEDLINE | ID: covidwho-1409152

ABSTRACT

BACKGROUND: Coronavirus disease of 2019 (COVID-19) is well known as a fatal disease, first discovered at Wuhan in China, ranging from mild to death, such as shortness of breath and fever. Early diagnosis of COVID-19 is a crucial point in preventing global prevalence. OBJECTIVE: We aimed to evaluate the diagnostic competency and efficiency with the Allplex™ 2019-nCoV Assay kit and the Dr. PCR 20 K COVID-19 Detection kit, designed based on the qRT-PCR and dPCR technologies, respectively. METHODS: A total of 30 negative and 20 COVID-19 positive specimens were assigned to the diagnostic test by using different COVID-19 diagnosis kits. Diagnostic accuracy was measured by statistical testing with sensitivity, specificity, and co-efficiency calculations. RESULTS: Comparing both diagnostic kits, we confirmed that the diagnostic results of 30 negative and 20 positive cases were the same pre-diagnostic results. The diagnostic statistics test results were perfectly matched with value (1). Cohen's Kappa coefficient was demonstrated that the given kits in two different ways were "almost perfect" with value (1). In evaluating the detection capability, the dilutional linearity experiments substantiate that the Dr. PCR 20 K COVID-19 Detection kit could detect SARS-CoV-2 viral load at a concentration ten times lower than that of the Allplex™ 2019-nCoV Assay kit. CONCLUSIONS: In this study, we propose that the dPCR diagnosis using LOAA dPCR could be a powerful method for COVID-19 point-of-care tests requiring immediate diagnosis in a limited time and space through the advantages of relatively low sample concentration and small equipment size compared to conventional qRT-PCR.


Subject(s)
COVID-19 Nucleic Acid Testing/methods , Polymerase Chain Reaction/methods , Reverse Transcriptase Polymerase Chain Reaction/methods , SARS-CoV-2/isolation & purification , COVID-19 , Genes, Viral/genetics , Humans , Republic of Korea , SARS-CoV-2/genetics , Sensitivity and Specificity , Viral Load
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