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

Abstract

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 guanidiniumbased 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.

Recurrence of SARS-CoV-2 viral RNA in recovered COVID-19 patients: a narrative review

“…In a retrospective analysis involving 161 COVID-19 patients, the authors showed that false-negative test results of SARS-CoV-2 viral RNA were mainly caused by poor-quality sampling and that swabs did not contain a sufficient quantity of cellular materials [ 72 ]. Furthermore, thermal inactivation also decreases the sensitivity of RT-PCR tests for SARS-CoV-2 [ 73 ].…”

The production and clinical implications of SARS-CoV-2 antibodies

“…20 This study demonstrated a rate of re-detectable positive nucleic acid as high as 52.7% (39/74) despite strict adherence to domestic guidelines on discharge criteria, which may be attributed to the fact that SARS-CoV-2 mainly resides in the lower respiratory system. 21,22 In addition, possible deviation in specimen collection may lead to false negativity of nucleic testing and consequently inappropriate discharge decisions, 23,24 resulting in recurrence and even deterioration of the disease. The 20 patients with re-detectable positive nucleic acid displayed decline in lymphocyte subsets on the second admission, which was similar to the changes of initial infection.…”

Diagnosis of COVID-19: Considerations, Controversies and Challenges in South Africa

“…(10,13) In mild disease, especially in the early stages, the RT-PCR false negative rate is ~30-40%. (8,14,15) A meta-analysis reported that a single test ~10 days post symptom development had a ~33% false negative rate using a nasopharyngeal swab (52.89% for a throat swab). (16) Ai et al reported a false negative rate of 41% in a cohort of 1014 hospitalised patients; the estimated median interval between the initial negative test and subsequent positive RT-PCR result was 5.1 ± 1.5 days.…”

“…(19) This false negativity phenomenon may be due to several factors, including a low viral load below the detection limit of the assay, low sample volume or cellular mass during acquisition, sampling location (upper versus lower respiratory tract), sample degradation during transport or storage, sample processing methodology and the timing of sampling in relation to the stage of the disease (RT-PCR positivity may progressively increase during the course of the disease). (14) Test accuracy will depend on the quality of the specimen collected. (20) It has since been shown that specimens from the lower respiratory tract have a higher viral load and hence more likely to test positive than specimens from the upper respiratory tract.…”