One-step and sequential SARSCOV-2 polymerase chain reaction tests would not work every time.

INTRODUCTION: RT-PCR is widely used as a diagnostic test for the detection of SARS-CoV-2. In this study, we aim to describe the clinical utility of serial PCR testing in the final detection of COVID-19. METHOD: We collected multiple nasopharyngeal swab samples from patients who had negative RT-PCR test on the first day after hospitalization. RT-PCR tests were performed on the second day for all patients with initial negative result. For the patients with secondary negative results on day 2, tertiary RT-PCR tests were performed on day 3 after hospitalization. RESULT: Among 68 patients with initial negative test results, at the end of follow-up, the mortality number was 20 (29.4%). About 33.8% of patients had subsequent positive PCR test results for the second time and 17.4% of the patients who performed third PCR test had positive result. CONCLUSION: Based on this study, serial RT-PCR testing is unlikely to yield additional information.

Severe Acute Respiratory Syndrome Coronavirus 2 Total and Subgenomic RNA Viral Load in Hospitalized Patients.

BACKGROUND: Previous studies demonstrated that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA can be detected for weeks after infection. The significance of this finding is unclear and, in most patients, does not represent active infection. Detection of subgenomic RNA has been proposed to represent productive infection and may be a useful marker for monitoring infectivity. METHODS: We used quantitative reverse-transcription polymerase chain reaction (RT-qPCR) to quantify total and subgenomic nucleocapsid (sgN) and envelope (sgE) transcripts in 185 SARS-CoV-2-positive nasopharyngeal swab samples collected on hospital admission and to relate to symptom duration. RESULTS: We find that all transcripts decline at the same rate; however, sgE becomes undetectable before other transcripts. The median duration of symptoms to a negative test is 14 days for sgE and 25 days for sgN. There is a linear decline in subgenomic compared to total RNA, suggesting that subgenomic transcript copy number is dependent on copy number of total transcripts. The mean difference between total and sgN is 16-fold and the mean difference between total and sgE is 137-fold. This relationship is constant over duration of symptoms, allowing prediction of subgenomic copy number from total copy number. CONCLUSIONS: Subgenomic RNA may be no more useful in determining infectivity than a copy number threshold determined for total RNA.

Diagnostic Accuracy Estimates for COVID-19 Real-Time Polymerase Chain Reaction and Lateral Flow Immunoassay Tests With Bayesian Latent-Class Models.

Our objective was to estimate the diagnostic accuracy of real-time polymerase chain reaction (RT-PCR) and lateral flow immunoassay (LFIA) tests for coronavirus disease 2019 (COVID-19), depending on the time after symptom onset. Based on the cross-classified results of RT-PCR and LFIA, we used Bayesian latent-class models, which do not require a gold standard for the evaluation of diagnostics. Data were extracted from studies that evaluated LFIA (immunoglobulin G (IgG) and/or immunoglobulin M (IgM)) assays using RT-PCR as the reference method. The sensitivity of RT-PCR was 0.68 (95% probability interval (PrI): 0.63, 0.73). IgG/M sensitivity was 0.32 (95% PrI :0.23; 0.41) for the first week and increased steadily. It was 0.75 (95% PrI: 0.67; 0.83) and 0.93 (95% PrI: 0.88; 0.97) for the second and third weeks after symptom onset, respectively. Both tests had a high to absolute specificity, with higher point median estimates for RT-PCR specificity and narrower probability intervals. The specificity of RT-PCR was 0.99 (95% PrI: 0.98; 1.00). and the specificity of IgG/IgM was 0.97 (95% PrI: 0.92, 1.00), 0.98 (95% PrI: 0.95, 1.00) and 0.98 (95% PrI: 0.94, 1.00) for the first, second, and third weeks after symptom onset. The diagnostic accuracy of LFIA varies with time after symptom onset. Bayesian latent-class models provide a valid and efficient alternative for evaluating the rapidly evolving diagnostics for COVID-19, under various clinical settings and different risk profiles.

A retrospective analysis of 12,400 SARS-CoV-2 RNA tests in patients with COVID-19 in Wuhan.

ABSTRACT: The outbreak and widely spread of coronavirus disease 2019 (COVID-19) has become a global public health concern. COVID-19 has caused an unprecedented and profound impact on the whole world, and the prevention and control of COVID-19 is a global public health challenge remains to be solved. The retrospective analysis of the large scale tests of SARS-CoV-2 RNA may indicate some important information of this pandemic. We selected 12400 SARS-CoV-2 tests detected in Wuhan in the first semester of 2020 and made a systematic analysis of them, in order to find some beneficial clue for the consistent prevention and control of COVID-19.SARS-CoV-2 RNA was detected in suspected COVID-19 patients with real-time fluorescence quantitative PCR (RT-qPCR). The patients' features including gender, age, type of specimen, source of patients, and the dynamic changes of the clinical symptoms were recorded and statistically analyzed. Quantitative and qualitive statistical analysis were carried out after laboratory detection.The positive rate of SARS-CoV-2 was 33.02% in 12,400 suspected patients' specimens in Wuhan at the first months of COVID-19 epidemics. SARS-CoV-2 RT-qPCR test of nasopharyngeal swabs might produce 4.79% (594/12400) presumptive results. The positive rate of SARS-CoV-2 RNA was significantly different between gender, age, type of specimen, source of patients, respectively (P < .05). The median window period from the occurrence of clinical symptom or close contact with COVID-19 patient to the first detection of positive PCR was 2 days (interquartile range, 1-4 days). The median interval time from the first SARS-CoV-2 positive to the turning negative was 14 days (interquartile range, 8-19.25 days).This study reveals the comprehensive characteristics of the SARS-CoV-2 RNA detection from multiple perspectives, and it provides important clues and may also supply useful suggestions for future work of the prevention and treatment of COVID-19.

Detection profile of SARS-CoV-2 using RT-PCR in different types of clinical specimens: A systematic review and meta-analysis.

Testing is one of the commendable measures for curbing the spread of coronavirus disease (COVID-19). But, it should be done using the most appropriate specimen and an accurate diagnostic test such as real-time reverse transcription-polymerase chain reaction (qRT-PCR). Therefore, a systematic review was conducted to determine the positive detection rate of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in different clinical specimens using qRT-PCR. A total of 8136 pooled clinical specimens were analyzed to detect SARS-CoV-2, the majority were nasopharyngeal swabs (69.6%). A lower respiratory tract (LRT) specimens had a positive rate (PR) of 71.3% (95% confidence interval [CI]: 60.3%-82.3%) while no virus was detected in the urinogenital specimens. Bronchoalveolar lavage fluid (BLF) specimen had the PR of 91.8% (95% CI: 79.9%-103.7%), followed by rectal swabs; 87.8% (95% CI: 78.6%-96.9%) then sputum; 68.1% (95% CI: 56.9%-79.4%). A low PR was observed in oropharyngeal swabs; 7.6% (95% CI: 5.7%-9.6%) and blood samples; 1.0% (95% CI: -0.1%-2.1%) whereas no SARS-CoV-2 was detected in urine samples. Feces had a PR of 32.8% (95% CI:1 5.8%-49.8%). Nasopharyngeal swab, a widely used specimen had a PR of 45.5% (95% CI: 31.2%-59.7%). In this study, SARS-CoV-2 was highly detected in LRT specimens while no virus was detected in urinogenital specimens. BLF had the highest PR followed by rectal swab then sputum. Nasopharyngeal swab which is widely used had moderate PR. Low PR was recorded in oropharyngeal swab and blood samples while no virus was found in urine samples. Last, the virus was detected in feces, suggesting SARS-CoV-2 transmission by the fecal route.

A simple lung ultrasound protocol for the screening of COVID-19 pneumonia in the emergency department.

The most relevant manifestation of coronavirus disease 2019 (COVID-19) is interstitial pneumonia. Several lung ultrasound (US) protocols for pneumonia diagnosis are used in clinical practice, but none has been proposed for COVID-19 patients' screening in the emergency department. We adopted a simplified 6-scan lung US protocol for COVID-19 pneumonia diagnosis (LUSCOP) and compared its sensitivity with high resolution computed tomography (HRCT) in patients suspected for COVID-19, presenting to one Emergency Department from February 21st to March 15th, 2020, during the outbreak burst in northern Italy. Patients were retrospectively enrolled if both LUSCOP protocol and HRCT were performed in the Emergency Department. The sensitivity of LUSCOP protocol and HRCT were compared. COVID-19 pneumonia's final diagnosis was based on real-time reverse-transcription polymerase chain reaction from nasal-pharyngeal swab and on clinical data. Out of 150 suspected COVID-19 patients, 131 were included in the study, and 130 had a final diagnosis of COVID-19 pneumonia. The most frequent lung ultrasonographic features were: bilateral B-pattern in 101 patients (77%), B-pattern with subpleural consolidations in 26 (19.8%) and lung consolidations in 2 (1.5%). LUSCOP Protocol was consistent with HRCT in correctly screening 130 out of the 131 COVID-19 pneumonia cases (99.2%). In one case COVID-19 pneumonia was excluded by both HRCT and lung US. LUSCOP protocol showed optimal sensitivity and can be proposed as a simple screening tool for COVID-19 pneumonia diagnosis in the context of outbreak burst areas where prompt isolation of suspected patients is crucial for patients' and operators' safety.

Comparison of Primer-Probe Sets among Different Master Mixes for Laboratory Screening of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2).

BACKGROUND: There is a shortage of chemical reagents for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) diagnosis and a surge of SARS-CoV-2 cases, especially in limited-resource settings. Therefore, the combination of an optimal assay kit is necessary. METHODS: We compared the ability to screen SARS-CoV-2 among three primer-probe sets in two different master mixes, Invitrogen™ SuperScript™ III One-Step RT-PCR and LightCycler Multiplex RNA Virus Master. RESULTS: The assay with TIB-Molbiol, IDT, and Phu Sa sets for LightCycler Multiplex RNA Virus Master or Invitrogen™ SuperScript™ III One-Step RT-PCR showed positive results from a single reaction of triplicate in the three days of 4.8 copies per reaction. R squared and amplification efficiency were 0.97 and ranged from 107 to 108%, respectively. CONCLUSIONS: Our findings indicated that TIB-Molbiol, IDT, and Phu Sa primer-probe sets could be beneficial for the laboratory screening of SARS-CoV-2 by RT-qPCR assay of E gene. There is a need to consider the combination of these reagent sets as a new strategy to increase the testing capacity of screening programs for COVID-19.

A reverse-transcription recombinase-aided amplification assay for the rapid detection of N gene of severe acute respiratory syndrome coronavirus 2(SARS-CoV-2).

The current outbreak of coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was reported in China firstly. A rapid, highly sensitive, specific, and simple operational method was needed for the detection of SARS-CoV-2. Here, we established a real-time reverse-transcription recombinase-aided amplification assay (RT-RAA) to detect SARS-CoV-2 rapidly. The primers and probe were designed based on the nucleocapsid protein gene (N gene) sequence of SARS-CoV-2. The detection limit was 10 copies per reaction in this assay, which could be conducted within 15 min at a constant temperature (39 °C), without any cross-reactions with other respiratory tract pathogens, such as other coronaviruses. Furthermore, compared with commercial real-time RT-PCR assay, it showed a kappa value of 0.959 (p < 0.001) from 150 clinical specimens. These results indicated that this real-time RT-RAA assay may be a valuable tool for detecting SARS-CoV-2.

Primer design for quantitative real-time PCR for the emerging Coronavirus SARS-CoV-2.

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

Potential False-Negative Nucleic Acid Testing Results for Severe Acute Respiratory Syndrome Coronavirus 2 from Thermal Inactivation of Samples with Low Viral Loads.

BACKGROUND: Coronavirus disease-2019 (COVID-19) has spread widely throughout the world since the end of 2019. Nucleic acid testing (NAT) has played an important role in patient diagnosis and management of COVID-19. In some circumstances, thermal inactivation at 56°C has been recommended to inactivate severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) before NAT. However, this procedure could theoretically disrupt nucleic acid integrity of this single-stranded RNA virus and cause false negatives in real-time polymerase chain reaction (RT-PCR) tests. METHODS: We investigated whether thermal inactivation could affect the results of viral NAT. We examined the effects of thermal inactivation on the quantitative RT-PCR results of SARS-CoV-2, particularly with regard to the rates of false-negative results for specimens carrying low viral loads. We additionally investigated the effects of different specimen types, sample preservation times, and a chemical inactivation approach on NAT. RESULTS: Our study showed increased Ct values in specimens from diagnosed COVID-19 patients in RT-PCR tests after thermal incubation. Moreover, about half of the weak-positive samples (7 of 15 samples, 46.7%) were RT-PCR negative after heat inactivation in at least one parallel testing. The use of guanidinium-based lysis for preservation of these specimens had a smaller impact on RT-PCR results with fewer false negatives (2 of 15 samples, 13.3%) and significantly less increase in Ct values than heat inactivation. CONCLUSION: Thermal inactivation adversely affected the efficiency of RT-PCR for SARS-CoV-2 detection. Given the limited applicability associated with chemical inactivators, other approaches to ensure the overall protection of laboratory personnel need consideration.