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1.
PLoS One ; 17(1): e0262258, 2022.
Article in English | MEDLINE | ID: covidwho-1606499

ABSTRACT

Although patients infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza A, influenza B and respiratory syncytial virus (RSV) show comparable or very similar manifestations, the therapeutic approaches of these respiratory viral infections are different, which requires an accurate diagnosis. Recently, the novel multiplex real-time reverse transcription-polymerase chain reaction assay AMPLIQUICK® Respiratory Triplex (BioSynex SA, Illkirch-Graffenstaden, France) allows simultaneous detection and differentiation of SARS-CoV-2, influenza A, influenza B, and RSV in respiratory tract samples. We herein evaluated the performance of the AMPLIQUICK® Respiratory Triplex for the detection of the four viruses in respiratory specimens, using Allplex™ Respiratory Panel 1 and 2019-nCoV assays (Seegene, Seoul, Korea) as reference comparator assays. A total of 359 archived predetermined respiratory samples, including 83, 145, 19 and 95 positive specimens for SARS-CoV-2, influenza A, influenza B and RSV respectively, were included. The AMPLIQUICK® Respiratory Triplex showed high concordance with the reference assays, with an overall agreement for SARS-CoV-2, influenza A, influenza B, and RSV at 97.6%, 98.8%, 98.3% and 100.0%, respectively, and high κ values ranging from 0.93 to 1.00, indicating an almost perfect agreement between assays. Furthermore, high correlations of cycle threshold (Ct) values were observed for positive samples of the four viruses between the AMPLIQUICK® Respiratory Triplex and comparator assays, with an overall high agreement between Ct values assessed by Bland-Altman analyses. In conclusion, these observations demonstrate that the multiplex AMPLIQUICK® Respiratory Triplex is a reliable assay for the qualitative detection and differentiation of SARS-CoV-2, influenza A, influenza B, and RSV in respiratory specimens, which may prove useful for streamlining diagnostics during the winter influenza-seasons.


Subject(s)
COVID-19/diagnosis , Influenza, Human/diagnosis , Multiplex Polymerase Chain Reaction/methods , Real-Time Polymerase Chain Reaction/methods , Respiratory Syncytial Virus Infections/diagnosis , COVID-19/virology , Humans , Influenza, Human/virology , Molecular Diagnostic Techniques , Nasopharynx/virology , Respiratory Syncytial Virus Infections/virology , Retrospective Studies , Sensitivity and Specificity
2.
PLoS One ; 16(12): e0260884, 2021.
Article in English | MEDLINE | ID: covidwho-1581769

ABSTRACT

OBJECTIVES: To exploit the features of digital PCR for implementing SARS-CoV-2 observational studies by reliably including the viral load factor expressed as copies/µL. METHODS: A small cohort of 51 Covid-19 positive samples was assessed by both RT-qPCR and digital PCR assays. A linear regression model was built using a training subset, and its accuracy was assessed in the remaining evaluation subset. The model was then used to convert the stored cycle threshold values of a large dataset of 6208 diagnostic samples into copies/µL of SARS-CoV-2. The calculated viral load was used for a single cohort retrospective study. Finally, the cohort was randomly divided into a training set (n = 3095) and an evaluation set (n = 3113) to establish a logistic regression model for predicting case-fatality and to assess its accuracy. RESULTS: The model for converting the Ct values into copies/µL was suitably accurate. The calculated viral load over time in the cohort of Covid-19 positive samples showed very low viral loads during the summer inter-epidemic waves in Italy. The calculated viral load along with gender and age allowed building a predictive model of case-fatality probability which showed high specificity (99.0%) and low sensitivity (21.7%) at the optimal threshold which varied by modifying the threshold (i.e. 75% sensitivity and 83.7% specificity). Alternative models including categorised cVL or raw cycle thresholds obtained by the same diagnostic method also gave the same performance. CONCLUSION: The modelling of the cycle threshold values using digital PCR had the potential of fostering studies addressing issues regarding Sars-CoV-2; furthermore, it may allow setting up predictive tools capable of early identifying those patients at high risk of case-fatality already at diagnosis, irrespective of the diagnostic RT-qPCR platform in use. Depending upon the epidemiological situation, public health authority policies/aims, the resources available and the thresholds used, adequate sensitivity could be achieved with acceptable low specificity.


Subject(s)
COVID-19/virology , Real-Time Polymerase Chain Reaction/methods , SARS-CoV-2/genetics , Viral Load/methods , Adolescent , Adult , Aged , COVID-19/mortality , COVID-19 Nucleic Acid Testing/methods , Child , Child, Preschool , Female , Genome, Viral , Humans , Logistic Models , Male , Middle Aged , Retrospective Studies , Sensitivity and Specificity , Young Adult
3.
Clin Infect Dis ; 73(Suppl_5): S454-S464, 2021 12 15.
Article in English | MEDLINE | ID: covidwho-1577471

ABSTRACT

BACKGROUND: Minimally invasive tissue sampling (MITS), a postmortem procedure that uses core needle biopsy samples and does not require opening the body, may be a valid alternative to complete autopsy (CA) in highly infectious diseases such as coronavirus disease-19 (COVID-19). This study aimed to (1) compare the performance of MITS and CA in a series of COVID-19 deaths and (2) evaluate the safety of the procedure. METHODS: From October 2020 to February 2021, MITS was conducted in 12 adults who tested positive before death for COVID-19, in a standard, well-ventilated autopsy room, where personnel used reinforced personal protective equipment. In 9 cases, a CA was performed after MITS. A thorough histological evaluation was conducted, and the presence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was evaluated by real-time reverse-transcription polymerase chain reaction (RT-PCR) and immunohistochemistry. RESULTS: The diagnoses provided by MITS and CA matched almost perfectly. In 9 patients, COVID-19 was in the chain of events leading to death, being responsible for diffuse alveolar damage and mononuclear T-cell inflammatory response in the lungs. No specific COVID-19 features were identified. Three deaths were not related to COVID-19. All personnel involved in MITS repeatedly tested negative for COVID-19. SARS-CoV-2 was identified by RT-PCR and immunohistochemistry in the MITS samples, particularly in the lungs. CONCLUSIONS: MITS is useful for evaluating COVID-19-related deaths in settings where a CA is not feasible. The results of this simplified and safer technique are comparable to those of CA.


Subject(s)
COVID-19 , Autopsy , Humans , Personal Protective Equipment , Real-Time Polymerase Chain Reaction , SARS-CoV-2
4.
J Virol Methods ; 300: 114421, 2022 Feb.
Article in English | MEDLINE | ID: covidwho-1568894

ABSTRACT

BACKGROUND: COVID-19 is a worldwide pandemic representing the most challenging global health crisis currently. Screening tests availability are a problematic task due to resource-limited abilities of some countries using RT-qPCR technique for SARS-COV-2 detection. OBJECTIVE: To cope with these health emergencies, in particular with this COVID-19 pandemic, states with low molecular diagnostic resources must optimize their capacity in molecular tests. We aimed to design a simple and effective strategy to improve inputs in the RT-qPCR tests as we attempted to check the financial advisability of using such an approach by calculating reduction rate of the test unit cost. METHODS: The used RNA was taken from suspected Covid-19 positive people. Nasopharyngeal swabs were collected at Pasteur Institute Diagnostic Center, Constantine, Algeria, 2020. We have optimized a screening strategy by grouping 16 individuals per pool, without reducing the sensitivity of RT-qPCR. RESULTS: A 1/16 dilution of a positive sample was a practical limit that does not require the use of robotic systems or mathematical modeling to construct the pools. The financial analysis of our strategy has shown that the costs can be reduced to 90 %. The pooled testing strategy that was proven in this study could be recommended to help COVID-19 containment in countries with low potential screening infrastructures using RT-qPCR technique by reducing the number of tests required to identify all positive subjects.


Subject(s)
COVID-19 , SARS-CoV-2 , COVID-19 Testing , Humans , Pandemics , Real-Time Polymerase Chain Reaction , Sensitivity and Specificity
5.
Clin Chem ; 67(11): 1545-1553, 2021 11 01.
Article in English | MEDLINE | ID: covidwho-1561050

ABSTRACT

BACKGROUND: We evaluated the analytical sensitivity and specificity of 4 rapid antigen diagnostic tests (Ag RDTs) for severe acute respiratory syndrome coronavirus 2, using reverse transcription quantitative PCR (RT-qPCR) as the reference method and further characterizing samples using droplet digital quantitative PCR (ddPCR) and a mass spectrometric antigen test. METHODS: Three hundred fifty (150 negative and 200 RT-qPCR positive) residual PBS samples were tested for antigen using the BD Veritor lateral flow (LF), ACON LF, ACON fluorescence immunoassay (FIA), and LumiraDx FIA. ddPCR was performed on RT-qPCR-positive samples to quantitate the viral load in copies/mL applied to each Ag RDT. Mass spectrometric antigen testing was performed on PBS samples to obtain a set of RT-qPCR-positive, antigen-positive samples for further analysis. RESULTS: All Ag RDTs had nearly 100% specificity compared to RT-qPCR. Overall analytical sensitivity varied from 66.5% to 88.3%. All methods detected antigen in samples with viral load >1 500 000 copies/mL RNA, and detected ≥75% of samples with viral load of 500 000 to 1 500 000 copies/mL. The BD Veritor LF detected only 25% of samples with viral load between 50 000 to 500 000 copies/mL, compared to 75% for the ACON LF device and >80% for LumiraDx and ACON FIA. The ACON FIA detected significantly more samples with viral load <50 000 copies/mL compared to the BD Veritor. Among samples with detectable antigen and viral load <50 000 copies/mL, sensitivity of the Ag RDT varied between 13.0% (BD Veritor) and 78.3% (ACON FIA). CONCLUSIONS: Ag RDTs differ significantly in analytical sensitivity, particularly at viral load <500 000 copies/mL.


Subject(s)
Antigens, Viral/analysis , COVID-19 Testing/methods , Point-of-Care Testing , Humans , Mass Spectrometry , Real-Time Polymerase Chain Reaction/methods , SARS-CoV-2/immunology , Sensitivity and Specificity , Viral Load
6.
J Virol Methods ; 300: 114420, 2022 Feb.
Article in English | MEDLINE | ID: covidwho-1560015

ABSTRACT

The emergence and spread of SARS-CoV-2 has led to a compelling request for accurate diagnostic tests. The aim of this study was assessing the performance of a real-time RT-qPCR (rt RT-qPCR) assay and of a droplet digital RT-PCR (dd RT-PCR) targeting the nsp14 genome region for the detection of SARS-CoV-2 in nasopharyngeal swabs. A total of 258 nasopharyngeal swabs were analyzed with the nsp14 assays and, for comparison, with a reference assay targeting the RdRp and E genes. Conflicting results were further investigated by two additional protocols, the Centers for Disease Control and Prevention (CDC) real-time targeting N1/N2, and a nested RT-PCR for the spike region. Agreement of results was achieved on 226 samples (156 positive and 70 negative), 8 samples were positive in the reference assay and in the nsp14 rt RT-qPCR but negative with the dd RT-PCR, and 24 samples provided different combinations of results with the three assays. Sensitivity, specificity and accuracy (95 %C.I.) of the nsp14 assays were: 100.0 % (97.4-100.0), 98.7 % (92.1-100.0), and 99.6 % (97.5-100.0) for the rt RT-qPCR; 92.4 % (87.4-95.6), 100.0 % (94.2-100.0), and 94.7 % (91.1-97.0) for the dd RT-PCR. The results of the study support the use of the nsp14 real-time RT-qPCR and ddPCR for the detection of SARS-CoV-2 in nasopharyngeal swabs.


Subject(s)
COVID-19 , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , SARS-CoV-2 , COVID-19/diagnosis , Exonucleases , Humans , Nasopharynx/virology , RNA, Viral/genetics , SARS-CoV-2/isolation & purification , Sensitivity and Specificity
7.
J Virol Methods ; 300: 114428, 2022 02.
Article in English | MEDLINE | ID: covidwho-1559401

ABSTRACT

BACKGROUND: The World Health Organization (WHO) recommended RT-qPCR tests as the reference technique for SARS-CoV-2 molecular detection, however with the rapid spread of the infection, mutations in specific RT-qPCR target regions have been widely described could allow the presumptive identification. OBJECTIVE: In this study, we evaluated the analytical performance of the Allplex™SARS-CoV-2/FluA/FluB/RSV assay for the additional presumptive identification of SARS-CoV-2 variants in a real-life setting. RESULTS: We observed gene-specific changes in the cycle threshold (Ct) of the N and RdRp genes compared with the Ct yielded for the S gene when the SARS-CoV-2 testing was performed Allplex™SARS-CoV-2/FluA/FluB/RSV assay. Seventeen samples showed Ct variations in the N and/or RdRp. In 10 cases, the N gene was affected, delayed or negative and in 14 cases, the RdRp gene showed a delay or negative concerning the S gene. A delay in the Ct of both genes (RdRp and N) was observed in six cases. Sequencing determined that all samples identified as B.1.1.7 showed changes in the PCR curves of the N and RdRp. However, samples identified as B.1.177 only showed variations for the RdRp gene. CONCLUSIONS: Allplex™SARS-CoV-2/FluA/FluB/RSV assay, the diagnosis could presumably allow the rapid assignment of lineages B.1.1.7 and B.1.177, and emphasizes the importance of exhaustive surveillance for circulating variants of the SARS-CoV-2 virus to reduce community transmission.


Subject(s)
COVID-19 , SARS-CoV-2 , COVID-19 Testing , Humans , Real-Time Polymerase Chain Reaction , Sensitivity and Specificity
8.
Clin Chim Acta ; 525: 46-53, 2022 Jan 15.
Article in English | MEDLINE | ID: covidwho-1559179

ABSTRACT

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which has caused a global pandemic beginning in 2020, can be detected by reverse-transcription polymerase chain reaction (RT-PCR). However, owing to the urgent need for a large number of detection kits, the time spent researching and developing these kits has been shortened during the pandemic, and the kits that are being used commercially have not undergone full and independent evaluation. To ensure the accuracy of SARS-CoV-2 test results, performance verification of commercial Real-Time quantitative PCR (RT-qPCR) kits is required. METHODS: The performance of five commercial RT-qPCR diagnostic kits for SARS-CoV-2 used in China was evaluated using a coronavirus disease 2019 (COVID-19) RNA liquid performance verification reference product-manufactured by Guangzhou Bondson (BDS) Biotechnology Co., Ltd.,Guangzhou, China-that uses droplet digital RT-PCR technology combined with fluorescence quantitative PCR. The five kits of Novel Coronavirus 2019-nCoV nucleic acid detection kit (RT-qPCR method) evaluated were Da An (Da An Gene Co., Ltd. of Sun Yat-sen University), Liferiver (Shanghai ZJ Bio-Tech Co., Ltd.), Kinghawk (Beijing Kinghawk Pharmaceutical Co., Ltd.), eDiagnosis (Wuhan Easy Diagnosis Biomedicine Co., Ltd.), and Maccura (Maccura Biotechnology Co., Ltd.). Performance verification criteria included the coincidence rate, limit of detection (LoD), cross-reactivity, precision, and anti-interference. Finally, through the BDS performance verification reference product kit, clinical samples are used to verify its clinical diagnostic efficacy. RESULTS: The coincidence rate was 100% for all kits except for Kinghawk, which was 95%. The LoD for Da An, eDiagnosis and Maccura was 250copies/mL, and it was 1000 copies/ml for Liferiver. Kinghawk was not able to detect its advertised LoD of 500 copies/ml. The cross-reactivity test results were all negative. Moreover, all kits had a coefficient of variation less than 5%; however, Liferiver showed the best precision. Da An, Liferiver, and eDiagnosis showed higher sensitivity to the nucleocapsid (N) gene than they did to the open reading frame (ORF) 1ab genes. Anti-interference results for all five kits were positive. The results of clinical diagnostic efficacy were that the specificity of the four kits was 1.000 (0.877-1.000), the sensitivity of Da An was 1.000 (0.850-1.000), Liferiver was 0.964 (0.798-0.998), Maccura was 0.893 (0.706-0.972), and eDiagnosis was 0.857 (0.664-0.953). CONCLUSIONS: All commercial RT-qPCR diagnostic kits for SARS-CoV-2 passed the BDS performance verification, except for Kinghawk (batch No:20200608113) which failed to detect the LoD of 500 copies/mL. Da An and Liferiver have excellent clinical diagnostic specificity and sensitivity. This study can provide guidance for the selection or optimization of RT-qPCR diagnostic test kits for SARS-CoV-2.


Subject(s)
COVID-19 , SARS-CoV-2 , China , Humans , Pandemics , RNA, Viral/genetics , Reagent Kits, Diagnostic , Real-Time Polymerase Chain Reaction , Sensitivity and Specificity
9.
Clin Lab ; 67(12)2021 Dec 01.
Article in English | MEDLINE | ID: covidwho-1551835

ABSTRACT

BACKGROUND: The implementation of an automated nucleic acid extraction system has many advantages over the manual methods. The purpose of this study was to evaluate the validity of two different methods for nucleic acid extraction in virus transport medium. METHODS: We collected 20 nasopharyngeal swabs in viral transport medium from the emergency department of the Asia University Hospital for the detection of SARS-CoV-2. The performance of the MaelstromTM 8 (Taiwan Advanced Nanotech) and the QIAamp Viral RNA Mini Kit (Qiagen) were compared for the extraction of nucleic acid from viral transport medium. The extracts were used for the validation of the RNA extraction procedures. The RNase P target was amplified in a one-step reverse transcription-quantitative PCR (RT-qPCR) reaction, as internal control for the extraction method. RESULTS: In this study, the agreement between the two methods was good and Pearson's correlation coefficient (r) was 0.919 (p < 0.001). The mean cycle threshold value of the two methods was 29.1. CONCLUSIONS: Overall, the performance values of the MaelstromTM 8 and the QIAamp Viral RNA Mini Kit were comparable to each other. In summary, the MaelstromTM 8 provides a standardized procedure, avoidance of sample-to-sample cross contaminations, is easy to use, improves turnaround time and requires less hands-on time as compared to the manual extraction method. The MaelstromTM 8 is more suitable for clinical laboratories that carry small or medium-sized samples for nucleic acid extraction.


Subject(s)
COVID-19 , Humans , RNA, Viral/genetics , Real-Time Polymerase Chain Reaction , SARS-CoV-2 , Sensitivity and Specificity
10.
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
11.
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
12.
J Med Virol ; 93(12): 6778-6781, 2021 Dec.
Article in English | MEDLINE | ID: covidwho-1544295

ABSTRACT

A high-throughput, fully automated antigen detection test for SARS-CoV-2 is a viable alternative to reverse-transcription polymerase chain reaction (RT-qPCR) for mass screening during outbreaks. In this study, we compared RT-qPCR for viral load and the VITROS® SARS-CoV-2 Antigen Test with reference to the results of the LUMIPULSE® SARS-CoV-2 Ag Test. Of 128 nasopharyngeal swab specimens taken from patients suspected of being infected with SARS-CoV-2, 49 were positive and 79 were negative according to RT-qPCR. Consistent dose-dependent detection with VITROS® assay was successfully achieved when using nasopharyngeal swab specimens with Ct values of 32.0 or lesser, whereas the CLEIA-based LUMIPULSE® assay was able to detect lower viral loads compared with the VITROS® assay. Our results show that the performance of the VITROS® assay was satisfactory for the diagnosis of contagious COVID-19 patients in the clinical setting. Highlights The performance of the VITROS® SARS-CoV-2 Antigen Test was sufficient for the diagnosis of contagious COVID-19. This test showed high sensitivity and specificity in the detection of SARS-CoV-2 in samples with a Ct value of 32 or less.


Subject(s)
COVID-19 Serological Testing/methods , COVID-19 Testing/methods , COVID-19/diagnosis , COVID-19/immunology , Immunoenzyme Techniques/methods , Immunologic Tests/methods , SARS-CoV-2/immunology , Antigens, Viral/genetics , Antigens, Viral/immunology , COVID-19/virology , Humans , Mass Screening/methods , Nasopharynx/immunology , Nasopharynx/virology , RNA, Viral/genetics , RNA, Viral/immunology , Real-Time Polymerase Chain Reaction/methods , SARS-CoV-2/genetics , Sensitivity and Specificity , Viral Load/genetics , Viral Load/immunology
13.
J Med Virol ; 93(12): 6575-6581, 2021 12.
Article in English | MEDLINE | ID: covidwho-1530180

ABSTRACT

Reliable and rapid detection of severe acute respiratory syndrome coronavirus 2 in laboratory setting is critical to control the pandemic. We aimed to an evaluated polymerase chain reaction (PCR) efficiency of nasopharyngeal swabs stored in viral transport medium (VTM) in different temperatures. Ninety swabs taken into VTM were analyzed at the first hour, then divided into two groups with similar numbers of positive and negative samples. Positive samples of each group were also subgrouped according to Fam CT values as low CT (<25), medium CT (25-32), and high CT (32-38) groups. One group was stored at 4°C, while the other was stored at room temperature, PCR analyses were repeated every 24 h for 5 days and on Day 12. There was a total of 30 positive samples (12 low CT, 11 medium CT, and 7 high CT). The CT values of both groups remained unchanged in first 3 days while the CT values of the room temperature group increased after the third day. All of the positive samples remained positive in both groups for the first 5 days. On the 12th day, the total number of positives decreased to 8 in the room temperature group and 11 in the 4°C groups. All the low CT samples remained positive in both groups. In conclusion, it is safe to store positive samples in room temperature for up to 5 days. Only samples with high viral loads remain positive for 12 days, regardless of whether stored at room temperature or 4°C. Negative samples don't turn to invalid if stored in VTM.


Subject(s)
COVID-19 Testing/methods , Real-Time Polymerase Chain Reaction/methods , Specimen Handling/methods , COVID-19/diagnosis , Humans , Real-Time Polymerase Chain Reaction/standards , SARS-CoV-2 , Specimen Handling/standards , Time Factors
14.
Front Cell Infect Microbiol ; 11: 717068, 2021.
Article in English | MEDLINE | ID: covidwho-1528814

ABSTRACT

This study aimed to detect the SARS-COV2 viral component directly from inoculated VTM without RNA extraction. Inoculated VTMs of already tested 50 positive and 50 negative samples were divided into three groups. Group I was treated with Proteinase K (PK) followed by 3-step-heat treatment at different temperatures (25°C, 60°C, and 98°C) and stored at 4°C. Group II was directly subjected to 3-step-heat treatment without PK exposure and stored at 4°C. And group III was set-up as standard group; it was processed using Qiagen's column based QIAamp Nucleic Acid kit and the obtained nucleic acids were stored at 4°C. These stored samples were used as a template to execute real-time polymerase chain reaction, and results were noted. Group I demonstrated 96% and 88% sensitivity for N and ORF1ab genes respectively, whereas group II demonstrated 78% and 60% when compared to the results of standard group III. Overall group I showed better results than group II when compared to group III. Thus, in situations where gold-standard reagents are not available, PK exposure and heat treatment can be employed to carry out molecular detection of SARS-CoV2 viral component.


Subject(s)
COVID-19 , RNA, Viral , Endopeptidase K , Humans , RNA, Viral/genetics , Real-Time Polymerase Chain Reaction , SARS-CoV-2
15.
J Korean Med Sci ; 36(44): e301, 2021 Nov 15.
Article in English | MEDLINE | ID: covidwho-1526760

ABSTRACT

We used serial rectal swabs to investigate the amount and duration of virus secretion through the gastrointestinal tract and assessed the association between fecal shedding and gastrointestinal symptoms and to clarify the clinical usefulness testing rectal swabs. We enrolled ten adult patients hospitalized with symptomatic coronavirus disease 2019 (COVID-19). Respiratory and stool specimens were collected by physicians. The presence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was confirmed using real-time reverse-transcription polymerase chain reaction. All ten patients had respiratory symptoms, six had diarrhea, and seven were positive for SARS-CoV-2 on rectal swabs. The viral loads in the respiratory specimens was higher than those in the rectal specimens, and no rectal specimens were positive after the respiratory specimens became negative. There was no association between gastrointestinal symptoms, pneumonia, severity, and rectal viral load. Rectal swabs may play a role in detecting SARS-CoV-2 in individuals with suspected COVID-19, regardless of gastrointestinal symptoms.


Subject(s)
COVID-19 Nucleic Acid Testing/methods , COVID-19/virology , Rectum/virology , SARS-CoV-2/isolation & purification , Virus Shedding , Adolescent , Adult , Aged , Aged, 80 and over , COVID-19/complications , COVID-19/transmission , Diarrhea/etiology , Diarrhea/virology , Feces/virology , Female , Humans , Male , Middle Aged , Nasopharynx/virology , Prospective Studies , Real-Time Polymerase Chain Reaction , Severity of Illness Index , Time Factors , Viral Load
16.
J Clin Microbiol ; 59(12): e0144621, 2021 11 18.
Article in English | MEDLINE | ID: covidwho-1522905

ABSTRACT

To provide an accessible and inexpensive method to surveil for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mutations, we developed a multiplex real-time reverse transcription-PCR (rRT-PCR) assay, the Spike single-nucleotide polymorphism (SNP) assay, to detect specific mutations in the spike receptor binding domain. A single primer pair was designed to amplify a 348-bp region of spike, and probes were initially designed to detect K417, E484K, and N501Y. The assay was evaluated using characterized variant sample pools and residual nasopharyngeal samples. Variant calls were confirmed by SARS-CoV-2 genome sequencing in a subset of samples. Subsequently, a fourth probe was designed to detect L452R. The lower limit of 95% detection was 2.46 to 2.48 log10 genome equivalents (GE)/ml for the three initial targets (∼1 to 2 GE/reaction). Among 253 residual nasopharyngeal swabs with detectable SARS-CoV-2 RNA, the Spike SNP assay was positive in 238 (94.1%) samples. All 220 samples with threshold cycle (CT) values of <30 for the SARS-CoV-2 N2 target were detected, whereas 18/33 samples with N2 CT values of ≥30 were detected. Spike SNP results were confirmed by sequencing in 50/50 samples (100%). Addition of the 452R probe did not affect performance for the original targets. The Spike SNP assay accurately identifies SARS-CoV-2 mutations in the receptor binding domain, and it can be quickly modified to detect new mutations that emerge.


Subject(s)
COVID-19 , SARS-CoV-2 , Humans , Mutation , RNA, Viral/genetics , Real-Time Polymerase Chain Reaction , Reverse Transcription
17.
J Virol Methods ; 299: 114333, 2022 01.
Article in English | MEDLINE | ID: covidwho-1525873

ABSTRACT

The increasing prevalence of N501Y variants of SARS-CoV-2 has kindled global concern due to their enhanced transmissibility. Genome sequencing is the gold standard method to identify the emerging variants of concern. But it is time-consuming and expensive, limiting the widespread deployment of genome surveillance in some countries. Health authorities surge the development of alternative assay to expand screening capacity with reduced time and cost. In this study, we developed an in-house TaqMan minor groove binder (MGB) probe-based one-step RT-qPCR assay to detect the presence of N501Y mutation in SARS-CoV-2. A total of 168 SARS-CoV-2 positive respiratory specimens were collected to determine diagnostic accuracy of the RT-qPCR assay. As a reference standard, PANGO lineages and the mutation patterns of all samples were characterised by whole-genome sequencing. The analytical sensitivity and the ability of the assay to detect low frequency of N501Y variants were also evaluated. A total of 31 PANGO lineages were identified from 168 SARS-CoV-2 positive cases, in which 34 samples belonged to N501Y variants, including B.1.1.7 (n = 20), B.1.351 (n = 12) and P.3 (n = 2). The N501Y RT-qPCR correctly identified all 34 samples as N501Y-positive and the other 134 samples as wildtype. The limit-of-detection of the assay consistently achieved 1.5 copies/µL on four different qPCR platforms. N501Y mutation was successfully detected at an allele frequency as low as 10 % in a sample with mixed SARS-CoV-2 lineage. The N501Y RT-qPCR is simple and inexpensive (US$1.6 per sample). It enables robust high-throughput screening for surveillance of SARS-CoV-2 variants of concern harbouring N501Y mutation.


Subject(s)
COVID-19 , SARS-CoV-2 , Humans , Real-Time Polymerase Chain Reaction , Whole Genome Sequencing
18.
J Infect Dev Ctries ; 15(10): 1408-1414, 2021 10 31.
Article in English | MEDLINE | ID: covidwho-1518651

ABSTRACT

INTRODUCTION: In this study, we aimed investigate the relationship of SARS-CoV-2 viral load cycle threshold (Ct) values with pneumonia. METHODOLOGY: A total of 158 patients in whom SARS-CoV-2 was confirmed in upper respiratory tract (URT) samples with molecular method and who had computed tomography (CT) of the chest, between April 2020 and June 2020 were included in this retrospective cross-sectional study. RESULTS: Mean age of 158 PCR positive patients was 45.22 ± 17.89 and 60.8% of them were male. Pneumonia was detected in 40.5% of the patients on their chest CT. A weak but significant correlation was found between SARS-CoV-2 Ct value detected with PCR in analysis of oropharyngeal/ nasopharyngeal (OP/NP) samples and chest CT score (Pearson's r: 0.197, p = 0.01). No correlation was found between the first detected viral load Ct value and age, gender and mortality. There was no significant correlation between chest CT score and mortality. While the areas remaining under ROC curve for Ct value in analysis of OP/NP samples in prediction of chest CT score ≥ 1, ≥ 5 and ≥ 10 were 0.564, 0.640 and 0.703 respectively. CONCLUSIONS: We found that the amount of SARS-CoV-2 viral load (inverse relationship with Ct) detected in OP/NP samples of patients with COVID-19 pneumonia did not reflect the increasing severity of pulmonary lesions on chest CT. Although primary target of SARS-CoV-2 is all epithelial cells of the respiratory tract we believe studies comparing viral loads in lower respiratory tract samples are needed to determine the severity of pulmonary disease.


Subject(s)
COVID-19/virology , Real-Time Polymerase Chain Reaction/methods , SARS-CoV-2/genetics , Viral Load/methods , Adolescent , Adult , Aged , Aged, 80 and over , COVID-19/diagnostic imaging , Child , Child, Preschool , Cross-Sectional Studies , Female , Humans , Lung/diagnostic imaging , Lung/pathology , Lung/virology , Male , Middle Aged , Nasopharynx/virology , Oropharynx/diagnostic imaging , Retrospective Studies , Tomography, X-Ray Computed , Young Adult
19.
Methods Mol Biol ; 2392: 185-197, 2022.
Article in English | MEDLINE | ID: covidwho-1513948

ABSTRACT

Real-time quantitative PCR is currently the most widely used method for the human pathogen severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) identification. Due to the rapid evolution of the SARS-CoV-2 genome, novel mutations on the primer binding sites will cause the failure of PCR. Therefore, in addition to a well-designed primer set, these primers need to be updated and evaluated regularly to ensure that the rapidly evolving genome primers can be amplified. In this protocol, (1) we firstly use assembled genome sequences in the SARS-CoV-2 database to identify and characterize indels and point mutations; (2) design primers skipping the sites of mutations; (3) check the coverage of the primers with the daily update SARS-CoV-2 database; (4) redesign them if novel mutations found in the primer binding sites. Although this protocol takes SARS-CoV-2 as an example, it is suitable for other species that have genomes accumulating mutations over time.


Subject(s)
COVID-19 , Genome, Viral , Real-Time Polymerase Chain Reaction , SARS-CoV-2 , COVID-19/diagnosis , COVID-19 Testing , Humans , SARS-CoV-2/genetics
20.
Front Cell Infect Microbiol ; 11: 741147, 2021.
Article in English | MEDLINE | ID: covidwho-1512020

ABSTRACT

The coronavirus disease 2019 (COVID-19) has caused and is still causing tremendous damage to the global economy and human health. Qualitative reverse transcription-PCR (RT-qPCR) is the golden standard for COVID-19 test. However, the SARS-CoV-2 variants may not only make vaccine less effective but also evade RT-qPCR test. Here we suggest an innovative primer design strategy for the RT-qPCR test of SARS-CoV-2. The principle is that the primers should be designed based on both the nucleic acid sequence and the structure of the protein encoded. The three nucleotides closest to the 3' end of the primer should be the codon which encodes the tryptophan in the structure core. Based on this principle, we designed a pair of primers targeting the nucleocapsid (N) gene. Since tryptophan is encoded by only one codon, any mutation that occurs at this position would change the amino acid residue, resulting in an unstable N protein. This means that this kind of SARS-CoV-2 variant could not survive. In addition, both our data and previous reports all indicate that the mutations occurring at other places in the primers do not significantly affect the RT-qPCR result. Consequently, no SARS-CoV-2 variant can escape detection by the RT-qPCR kit containing the primers designed based on our strategy.


Subject(s)
COVID-19 , SARS-CoV-2 , Humans , Mutation , RNA, Viral/genetics , Real-Time Polymerase Chain Reaction , Sensitivity and Specificity
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