Influence of Different Inactivation Methods on Severe Acute Respiratory Syndrome Coronavirus 2 RNA Copy Number.

The outbreak of coronavirus disease 2019 (COVID-19) has spread across the world and was characterized as a pandemic. To protect medical laboratory personnel from infection, most laboratories inactivate the virus causing COVID-19, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), in clinical samples before testing. However, the effect of inactivation on the detection results remains unknown. Here, we used a digital PCR assay to determine the absolute SARS-CoV-2 RNA copy number in 63 nasopharyngeal swab samples and assess the effect of inactivation methods on viral RNA copy number. Viral inactivation was performed by three different methods: (i) incubation with the TRIzol LS reagent for 10 min at room temperature, (ii) heating in a water bath at 56°C for 30 min, and (iii) high-temperature treatment, including autoclaving at 121°C for 20 min, boiling at 100°C for 20 min, and heating at 80°C for 20 min. Compared to the amount of RNA in the original sample, TRIzol treatment destroyed 47.54% of the nucleocapsid protein (N) gene and 39.85% of open reading frame (ORF) 1ab. For samples treated at 56°C for 30 min, the copy number of the N gene and ORF 1ab was reduced by 48.55% and 56.40%, respectively. The viral RNA copy number dropped by 50 to 66% after heating at 80°C for 20 min. Nearly no viral RNA was detected after autoclaving at 121°C or boiling at 100°C for 20 min. These results indicate that inactivation reduced the quantity of detectable viral RNA and may cause false-negative results, especially in weakly positive cases. Thus, use of the TRIzol reagent rather than heat inactivation is recommended for sample inactivation, as the TRIzol reagent had the least effect on the RNA copy number among the tested methods.

Evaluation of the clinical performance of single-, dual-, and triple-target SARS-CoV-2 RT-qPCR methods.

BACKGROUND: The coronavirus disease 2019 (COVID-19) has become a pandemic. Reverse transcription quantitative PCR (RT-qPCR) has played a vital role in the diagnosis of COVID-19, but the rates of false negatives is not ideal in dealing with this highly infectious virus. It is thus necessary to systematically evaluate the clinical performance of the single-, dual-, triple-target detection kits to guide the clinical diagnosis of this disease. METHODS: A series of reference materials calibrated by droplet digital PCR (ddPCR) and 57 clinical samples were used to evaluate the clinical performance of six single-, dual-, triple-target SARS-CoV-2 nucleic acid detection kits based on RT-qPCR. RESULTS: The dual-target kits, kit B and kit C had the highest and the lowest detection sensitivity, which was 125 copies/mL and 4000 copies/mL, respectively. Among the 57 clinical samples from patients with COVID-19, 47 were tested positive by the kit B, while 35, 29, 28, 30, and 29 were found positive by the kits A, C, D, E, and F, respectively. The number of targets in a detection kit is not a key factor affecting sensitivity, while the amount of sample loading may influence the performance of a detection kit. CONCLUSIONS: This study provides a guide when choosing or developing a nucleic acid detection kit for the diagnosis of COVID-19. Also, the absolute-quantification feature and high-sensitivity performance of ddPCR, suggesting that it can be used to review clinically suspected samples.

Effect of Convalescent Plasma Therapy on Time to Clinical Improvement in Patients With Severe and Life-threatening COVID-19: A Randomized Clinical Trial.

Importance: Convalescent plasma is a potential therapeutic option for patients with coronavirus disease 2019 (COVID-19), but further data from randomized clinical trials are needed. Objective: To evaluate the efficacy and adverse effects of convalescent plasma therapy for patients with COVID-19. Design, Setting, and Participants: Open-label, multicenter, randomized clinical trial performed in 7 medical centers in Wuhan, China, from February 14, 2020, to April 1, 2020, with final follow-up April 28, 2020. The trial included 103 participants with laboratory-confirmed COVID-19 that was severe (respiratory distress and/or hypoxemia) or life-threatening (shock, organ failure, or requiring mechanical ventilation). The trial was terminated early after 103 of a planned 200 patients were enrolled. Intervention: Convalescent plasma in addition to standard treatment (n = 52) vs standard treatment alone (control) (n = 51), stratified by disease severity. Main Outcomes and Measures: Primary outcome was time to clinical improvement within 28 days, defined as patient discharged alive or reduction of 2 points on a 6-point disease severity scale (ranging from 1 [discharge] to 6 [death]). Secondary outcomes included 28-day mortality, time to discharge, and the rate of viral polymerase chain reaction (PCR) results turned from positive at baseline to negative at up to 72 hours. Results: Of 103 patients who were randomized (median age, 70 years; 60 [58.3%] male), 101 (98.1%) completed the trial. Clinical improvement occurred within 28 days in 51.9% (27/52) of the convalescent plasma group vs 43.1% (22/51) in the control group (difference, 8.8% [95% CI, -10.4% to 28.0%]; hazard ratio [HR], 1.40 [95% CI, 0.79-2.49]; P = .26). Among those with severe disease, the primary outcome occurred in 91.3% (21/23) of the convalescent plasma group vs 68.2% (15/22) of the control group (HR, 2.15 [95% CI, 1.07-4.32]; P = .03); among those with life-threatening disease the primary outcome occurred in 20.7% (6/29) of the convalescent plasma group vs 24.1% (7/29) of the control group (HR, 0.88 [95% CI, 0.30-2.63]; P = .83) (P for interaction = .17). There was no significant difference in 28-day mortality (15.7% vs 24.0%; OR, 0.59 [95% CI, 0.22-1.59]; P = .30) or time from randomization to discharge (51.0% vs 36.0% discharged by day 28; HR, 1.61 [95% CI, 0.88-2.95]; P = .12). Convalescent plasma treatment was associated with a negative conversion rate of viral PCR at 72 hours in 87.2% of the convalescent plasma group vs 37.5% of the control group (OR, 11.39 [95% CI, 3.91-33.18]; P < .001). Two patients in the convalescent plasma group experienced adverse events within hours after transfusion that improved with supportive care. Conclusion and Relevance: Among patients with severe or life-threatening COVID-19, convalescent plasma therapy added to standard treatment, compared with standard treatment alone, did not result in a statistically significant improvement in time to clinical improvement within 28 days. Interpretation is limited by early termination of the trial, which may have been underpowered to detect a clinically important difference. Trial Registration: Chinese Clinical Trial Registry: ChiCTR2000029757.