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

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. 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. 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. 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.

Pan Y ，Long L ，Zhang D ，Yuan T ，Cui S ，Yang P ，Wang Q ，Ren S ... -  《-》

The impacts of viral inactivating methods on quantitative RT-PCR for COVID-19.

This paper aims to explore the effect of four virus inactivation methods on the rapid detection results of COVID-19 nucleic acid. Collected samples of nasopharyngeal swabs from 2 patients diagnosed with COVID-19 at the First People's Hospital of Zhaoqing City, each of sample was divided into 5 groups (groupA∼E): A:Non-inactivated raw sample; B:75 % ethanol inactivation; C:56 ℃ incubation for 30 min inactivation; D:65 ℃ incubation for 10 min inactivation; E:Pre-inactivation using RNA virus special preservation fluid added into the sampling tube to treated the nasopharyngeal swab sample separately, using real-time fluorescent RT-PCR to detect the N gene of COVID-19 and the ORF1ab gene simultaneously. All the groups are diluted in 1:2, 1:4, 1:8 ratios. The objectives are to compare the effect of the varied inactivation method on CT(Cycle Threshold)results in PCR, conduct correlation and Bland-Altman analysis. For the N gene and ORF1ab gene, the CT values of 4 inactivated and Non-inactivated treatment were correlated (P＜0.001). The results of the four treatment methods and specimens without inactivated treatment have shown good consistency. The treatment of nasopharyngeal swab specimens using mentioned four inactivated methods had no significant effect on the subsequent detection of the new COVID-19 nucleic acid test. Lab test-persons can flexibly adopt pre-inactivation methods to ensure the accuracy of virus nucleic acid test results, meanwhile guarantee the safety of lab test-persons.

Wang Y ，Song W ，Zhao Z ，Chen P ，Liu J ，Li C ... -  《-》

Extraction-free SARS-CoV-2 detection by rapid RT-qPCR universal for all primary respiratory materials.

Fast and reliable detection of SARS-CoV-2 is crucial for efficient control of the COVID-19 pandemic. Due to the high demand for SARS-CoV-2 testing there is a worldwide shortage of RNA extraction reagents. Therefore, extraction-free RT-qPCR protocols are urgently needed. To establish a rapid RT-qPCR protocol for the detection of SARS-CoV-2 without the need of RNA extraction suitable for all respiratory materials. Different SARS-CoV-2 positive respiratory materials from our routine laboratory were used as crude material after heat inactivation in direct RT-qPCR with the PrimeDirect™ Probe RT-qPCR Mix (TaKaRa). SARS-CoV-2 was detected using novel primers targeted to the E-gene. The protocol for the detection of SARS-CoV-2 in crude material used a prepared frozen-PCR mix with optimized primers and 5 μl of fresh, undiluted and pre-analytically heat inactivated respiratory material. For validation, 91 respiratory samples were analyzed in direct comparison to classical RNA-based RT-qPCR. Overall 81.3 % of the samples were detected in both assays with a strong correlation between both Ct values (r = 0.8492, p < 0.0001). The SARS-CoV-2 detection rate by direct RT-qPCR was 95.8 % for Ct values <35. All negative samples were characterized by low viral loads (Ct >35) and/or long storage times before sample processing. Direct RT-qPCR is a suitable alternative to classical RNA RT-qPCR, provided that only fresh samples (storage <1 week) are used. RNA extraction should be considered if samples have longer storage times or if PCR inhibition is observed. In summary, this protocol is fast, inexpensive and suitable for all respiratory materials.

Lübke N ，Senff T ，Scherger S ，Hauka S ，Andrée M ，Adams O ，Timm J ，Walker A ... -  《-》

Value of Viral Nucleic Acid in Sputum and Feces and Specific IgM/IgG in Serum for the Diagnosis of Coronavirus Disease 2019.

A new type of coronavirus-induced pneumonia eventually termed "coronavirus disease 2019" (COVID-19) was diagnosed in patients in Wuhan (Hubei Province, China) in December 2019, and soon spread worldwide. To improve the detection rate of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), we analyzed the results of viral nucleic acid and serum-specific antibody tests on clinical samples from 20 patients with SARS-CoV-2 infection diagnosed at the First Affiliated Hospital of Guangzhou Medical University in China. By comparing various sample types collected from COVID-19 patients, we revealed multiple pathways for SARS-CoV-2 shedding, and a prolonged detectable period for viral nucleic acid test in sputum specimens, demonstrating that the timeline of the viral shedding is of great value in determining the time of release from quarantine or discharge from hospital. We also recommend for the application of serological test to assist in confirming SARS-CoV-2 infection judged by viral nucleic acid test, especially when COVID-19-related symptoms have appeared and the viral nucleic acid test was negative. Our findings are critical for the diagnosis of SARS-CoV-2 infection and for determining deadline of restriction measures to prevent transmission caused by convalescent patients with COVID-19.

He Y ，Luo J ，Yang J ，Song J ，Wei L ，Ma W ... -  《Frontiers in Cellular and Infection Microbiology》

Distribution of SARS-CoV-2 PCR Cycle Threshold Values Provide Practical Insight Into Overall and Target-Specific Sensitivity Among Symptomatic Patients.

We examined the distribution of reverse transcription polymerase chain reaction (RT-PCR) cycle threshold (CT) values obtained from symptomatic patients being evaluated for coronavirus disease 2019 (COVID-19) to determine the proportion of specimens containing a viral load near the assay limit of detection (LoD) to gain practical insight to the risk of false-negative results. We also examined the relationship between CT value and patient age to determine any age-dependent difference in viral load or test sensitivity. We collected CT values obtained from the cobas severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) assay corresponding to 1,213 combined nasopharyngeal-oropharyngeal specimens obtained from symptomatic individuals that were reported as positive or presumptive positive for SARS-CoV-2. CT values were stratified by SARS-CoV target and patient age group. In total, 93.3% to 98.4% of specimens demonstrated CT values greater than 3× the assay LoD, at which point false-negative results would not be expected. The mean of CT values between age groups was statistically equivalent with the exception of patients in age group 80 to 89 years, which demonstrated slightly lower CTs. Based on the distribution of observed CT values, including the small proportion of specimens with values near the assay LoD, there is a low risk of false-negative RT-PCR results in combined nasopharyngeal-oropharyngeal specimens obtained from symptomatic individuals.

Buchan BW ，Hoff JS ，Gmehlin CG ，Perez A ，Faron ML ，Munoz-Price LS ，Ledeboer NA ... -  《-》