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

Yang Pan; Luyao Long; Daitao Zhang; Tingting Yuan; Shujuan Cui; Peng Yang; Quanyi Wang; Simei Ren

doi:10.1093/clinchem/hvaa091

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

Clin Chem. 2020 Jun 1;66(6):794-801. doi: 10.1093/clinchem/hvaa091.

Authors

Yang Pan^{1

2

3}, Luyao Long^{4

5

6}, Daitao Zhang^{1

2

3}, Tingting Yuan⁷, Shujuan Cui^{1

2

3}, Peng Yang^{1

2

3}, Quanyi Wang^{1

2

3}, Simei Ren^{4

5

6}

Affiliations

¹ Institute for Infectious Disease and Endemic Disease Control, Beijing Center for Disease Prevention and Control, Beijing, P. R. China.
² Research Centre for Preventive Medicine of Beijing, Beijing, China.
³ School of Public Health, Capital Medical University, Beijing, China.
⁴ National Center for Clinical Laboratories, Beijing Hospital, National Center of Gerontology, Institute of Geriatric Medicine, Chinese Academy of Medical Sciences, Beijing, China.
⁵ Graduate School, Chinese Academy of Medical Sciences, Peking Union Medical College, Beijing, China.
⁶ Beijing Engineering Research Center of Laboratory Medicine, Beijing Hospital, Beijing, China.
⁷ Department of Clinical Laboratory, Beijing Hospital, National Center of Gerontology, Beijing, China.

Abstract

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.

Keywords: COVID-19; RT-PCR; SARS-CoV-2; false negative; nucleic acid test; thermal inactivation.

MeSH terms

Betacoronavirus / chemistry*
COVID-19
COVID-19 Testing
COVID-19 Vaccines
Clinical Laboratory Techniques / methods
Coronavirus Infections / diagnosis*
False Negative Reactions
Feces / virology
Guanidine / chemistry
Hot Temperature*
Humans
Pandemics
Pneumonia, Viral / diagnosis*
RNA, Viral / analysis*
Real-Time Polymerase Chain Reaction / methods
Real-Time Polymerase Chain Reaction / statistics & numerical data
SARS-CoV-2
Specimen Handling / methods
Sputum / virology
Time Factors
Viral Load*
Virus Inactivation / drug effects

Substances

COVID-19 Vaccines
Covid-19 aAPC vaccine
RNA, Viral
Guanidine