Journal:Current approaches in laboratory testing for SARS-CoV-2

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Full article title Current approaches in laboratory testing for SARS-CoV-2
Journal International Journal of Infectious Diseases
Author(s) Xu, Yuzhong; Cheng, Minggang; Chen, Xinchun; Zhu, Jialou
Author affiliation(s) Shenzhen Baoan Hospital, Shenzhen University
Primary contact Email: zhujialou at szu dot edu dot cn
Year published 2020
Volume and issue 100
Page(s) 7–9
DOI 10.1016/j.ijid.2020.08.041
ISSN 1201-9712
Distribution license Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International
Website https://www.sciencedirect.com/science/article/pii/S1201971220306718
Download https://www.sciencedirect.com/science/article/pii/S1201971220306718/pdfft (PDF)

Abstract

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus, which originated in Wuhan, Hubei Province, China, has rapidly spread to produce a global pandemic. It is now clear that person-to-person transmission of SARS-CoV-2 has been occurring and that the virus has been dramatically spreading in recent months. Early, rapid, and accurate diagnosis is of great significance for curtailing the spread of SARS-CoV-2. There are currently several diagnostic techniques (e.g., viral culture and nucleic acid amplification test) being used to detect the virus. However, the sensitivity and specificity of these methods are quite different, with the sample source and detection limit varying greatly. This study reviewed all types and characteristics of the currently available laboratory diagnostic assays for detecting SARS-CoV-2 infection and summarized the selection strategies of testing and sampling sites at different disease stages to improve the diagnostic accuracy of testing for the virus' associated disease, coronavirus disease 2019 (COVID-19).

Keywords: novel coronavirus, SARS-CoV-2, COVID-19, laboratory testing, laboratory diagnosis

Discussion

An outbreak of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was discovered in Wuhan, China, in December 2019. It then rapidly developed into a global pandemic. As of May 29, 2020 a total of 5,701,337 laboratory-confirmed COVID-19 cases had been reported worldwide, with 357,688 deaths confirmed. Among the effective control measures to reduce transmission in the community, early and reliable laboratory confirmation of SARS-CoV-2 infection is of crucial importance. This review summarizes the advances made in technologies for rapid diagnosis and confirmation of respiratory infections caused by SARS-CoV-2, as well as the selection strategies of testing and sampling sites in SARS-CoV-2 detection.

Since the initial cases of pneumonia of unknown cause were first reported, viral culture and genetic sequencing of isolates obtained from these patients in January 2020 identified within 10 days a novel coronavirus as the etiology. This benefitted understanding of the disease occurrence and transmission, as well as diagnostic test development.[1] Although viral culture is relatively time-consuming and labor-intensive, it is much more useful in the initial phase of emerging epidemics before other diagnostic assays are clinically available. Besides, unbiased, high-throughput sequencing has been proven as a powerful tool for discovering pathogens (Table 1). A detection assay (BGI, Shenzhen, China), based on next-generation sequencing, was approved for emergency use authorization (EUA) by the National Medical Products Administration (NMPA) in China (see Table S1 in the Supplementary data). However, whole genome sequencing is time-consuming and requires specialized instruments with high technical thresholds, and thus is not recommended for widespread clinical use.

Table 1. Laboratory testing for detection of SARS-CoV-2. NAAT, nucleic acid amplification test; RT-PCR, reverse transcription polymerase chain reaction.
Testing type Specimen type Characteristics Testing time Limitation
Viral culture Respiratory sample Gold standard for virus diagnosis and useful in the initial phase of emerging epidemics 3–7 days Time- and labor-consuming, biosafety level 3 laboratory needed, cannot be widely used in clinical settings
NAAT, whole genome sequencing Respiratory sample and blood Detects all pathogens in a given specimen, including SARS-CoV-2, as well as viral genome mutations 20 hours Time-consuming, specialized instruments with high technical thresholds, and high cost
NAAT, real-time RT-PCR Respiratory sample, stool and blood Most widely used for laboratory confirmation of SARS-CoV-2 infection 1.5–3 hours Time-consuming procedure, requires biosafety conditions, expensive equipment, skilled personnel, and can have false negative results
NAAT, isothermal amplification Respiratory sample, stool and blood Requires only a single temperature for amplification, takes less time yet has comparable performance with real-time RT-PCR, and does not require specialized laboratory equipment 0.5–2 hours False negative results, as real-time RT-PCR
Serological testing Serum, plasma and blood Less time required, simple to operate, useful in disease surveillance and epidemiologic research 15–45 minutes Cross-reaction with other subtypes of coronaviruses
Point-of-care test Respiratory sample Provides rapid actionable information with good sensitivity and specificity for patient care outside of the clinical diagnostic laboratory 5–30 minutes Risk of quality loss and lack of cost-effectiveness

Real-time reverse transcription polymerase chain reaction (RT-PCR) is routinely used in acute respiratory infection to detect causative viruses from respiratory specimens. The World Health Organization (WHO) recommends that patients who meet the case definition for suspected SARS-CoV-2 should be screened for the virus using a nucleic acid amplification test (Table 1). Various real-time RT-PCR assays for detecting SARS-CoV-2 RNA have been developed worldwide, with different targeted viral genes or regions (Table S1, Supplementary data). To date, 13 and 52 commercial SARS-CoV-2 real-time RT-PCR diagnostic panels have been issued for EUA by China and the U.S., respectively, with the limit of detection varying from 100 to 1000 copies/mL (Table S1, Supplementary data). Although RT-PCR has relatively high sensitivity, there have been reports of multiple false negative tests for the same patients infected with SARS-CoV-2 in China[2][3], suggesting that negative results do not preclude the presence of SARS-CoV-2 in a clinical specimen. In addition, fluctuating RT-PCR results have been observed in several patients who first tested positive for SARS-CoV-2, then tested negative in the following test, and returned to being positive in a final test.[4] False negative results may be due to the selection of sampling locations, poor sample quality, low viral load of the specimen, incorrect storage and transportation, as well as laboratory testing conditions and personnel operations. If a highly suspected patient is negative for the virus, the nucleic acid amplification test should be repeated or a more suitable sample should be collected.

Isothermal amplification techniques offer a good alternative to real-time RT-PCR, with comparable performance (Table 1). They take less time and generally do not need specialized laboratory equipment. These techniques include loop-mediated isothermal amplification (LAMP), nucleic acid sequence-based amplification (NASBA), and cross priming amplification (CPA). A recent study suggested that a reverse transcription LAMP (RT-LAMP) assay could detect as low as 20 copies of SARS-CoV-2 ORF1ab RNA, with 100% agreement with the commercial real-time RT-PCR in 130 swabs and bronchoalveolar lavage fluid samples.[5] Another RT-LAMP assay, targeting the N gene of the virus, displayed a detection limit of 100 RNA copies in 30 minutes combined with colorimetric visualization.[6] These results suggest that RT-LAMP assays could be used as a sensitive and specific early detection method with which to identify SARS-CoV-2 cases. Currently, several isothermal amplification-based nucleic acid tests for SARS-CoV-2 detection have received EUAs from China's NMPA (Table S1, Supplementary data).

Serological assays that test for immunoglobin M (IgM) and immunoglobin G (IgG) antibodies provide an alternative diagnostic approach for the current rapidly growing demand for rapid diagnosis of suspected patients and asymptomatic infections. The entire test can be completed in a short time, and be independent of specific equipment or places. They are suggested to be used either in combination with molecular testing or for additional testing in suspected cases with negative nucleic acid results to improve detection accuracy of COVID-19. In a study of 397 real-time RT-PCR-confirmed COVID-19 patients and 128 virus-negative patients, IgM/IgG assays showed a sensitivity and specificity of 88.66% and 90.63% in blood samples, respectively.[7] Combined IgM–IgG tests provided better sensitivity than tests for only IgM or IgG. However, cross-reactivity of the serological assay to other coronaviruses has been observed.[8] However, serological testing remains critically useful in disease surveillance and epidemiologic research. A community seroprevalence study of 863 individuals showed that the prevalence of antibodies to SARS-CoV-2 was 4.65% in Los Angeles County[9]; 367,000 people were estimated to be infected with SARS-CoV-2, which is 43.53 times higher than the cumulative number (8,430) of confirmed cases by the time of the survey.

Point-of-care (POC) diagnostic tests provide rapid actionable information for patient care outside of centralized facilities such as airports, local emergency departments and clinics, and other locations. It has been shown to have an immediate impact on patient management and control of infectious disease epidemics.[10] At the time of writing, three detection assays have been issued EUAs for point-of-care diagnosis of SARS-CoV-2 in the U.S. (Table S1, Supplementary data), including the Xpert Xpress SARS-CoV-2 test (Cepheid, USA) (real-time RT-PCR assay), ID NOW COVID-19 test (Abbott, USA) (isothermal nucleic acid amplification), and Sofia 2 SARS Antigen FIA assay (Quidel, USA) (antigen test). These emerging POC assays would be a powerful tool for effective patient care and outbreak containment of SARS-CoV-2 infection.

Lastly, the selection of specimens for molecular assays is crucial in the laboratory diagnosis of SARS-CoV-2 (Table 2). To prevent misdiagnosis caused by insufficient viral load, bronchoalveolar lavage fluid (BALF) is the most preferred specimen, as the viral loads of respiratory tract specimens are highest in BALF, followed by sputum, nasopharyngeal swabs, and oropharyngeal swabs.[11][12] Due to the prolonged presence of SARS-CoV-2 viral RNA in fecal samples and potential fecal-oral transmission, fecal testing for SARS-CoV-2 is highly recommended when there is virus negativity in respiratory tract specimens.[13] In addition, sampling different sites in suspected people or repeatedly sampling at different infected stages may help to prevent false negative results.

Table 2. Sampling location recommended for patients with COVID-19. a All patients were confirmed by SARS-CoV-2 detection[11][12]
Specimen type Positive rate a Early stage/initial diagnosis Advanced stage Recovery/follow-up Remarks
Oropharyngeal swab 32–48% Recommended Recommended Recommended Viral loads in the upper respiratory tract peak soon within one week after symptom onset then steadily decline after that.
Nasopharyngeal swab 63% Highly recommended Highly recommended Highly recommended Nasopharyngeal swab samples generally show higher viral loads and positive rates than oropharyngeal swab samples.
Bronchoalveolar lavage fluid (BALF) 79–93% Not recommended Highly recommended Not recommended BALF could be collected from patients presenting with more severe disease or undergoing mechanical ventilation.
Sputum 72–76% Highly recommended Highly recommended Highly recommended For patients who develop a productive cough, sputum should be collected and tested for SARS-CoV-2.
Stool/anal swab 29% Not recommended Not recommended Highly recommended Fecal testing for SARS-CoV-2 is highly recommended after viral clearance in the respiratory samples.

This comprehensive review examined all available diagnostic assays of SARS-CoV-2 infection, including virus culture, whole genome sequencing, real-time RT-PCR, isothermal amplification, antibody test, and POC testing. The choice of a diagnostic assay for COVID-19 should take the characteristics and advantages of various technologies, as well as different clinical scenarios and requirements, into full consideration. Moreover, to improve the detection accuracy of infectious diseases with COVID-19, proper collection of specimens is of great importance.

Supplementary data

  • Table S1. Laboratory diagnostic assays issued EUAs for SARS-CoV-2 detection. 33kb Word document

Acknowledgements

Funding

This work was supported by Guangdong Provincial Science and Technology Program (No. 2019B030301009).

Conflict of interest

The authors declare that they have no competing financial interests.

References

  1. ↑ Zhu, N.; Zhang, D.; Wang, W. et al. (2020). "A Novel Coronavirus from Patients with Pneumonia in China, 2019". New England Journal of Medicine 382 (8): 727–33. doi:10.1056/NEJMoa2001017. PMC PMC7092803. PMID 31978945. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7092803. 
  2. ↑ Xie, X.; Zhong, Z.; Zhao, W. et al. (2020). "Chest CT for Typical Coronavirus Disease 2019 (COVID-19) Pneumonia: Relationship to Negative RT-PCR Testing". Radiology 296 (2): E41–45. doi:10.1148/radiol.2020200343. PMC PMC7233363. PMID 32049601. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7233363. 
  3. ↑ Xiao, A.T.; Tong, Y.X.; Zhang, S. et al. (2020). "False negative of RT-PCR and prolonged nucleic acid conversion in COVID-19: Rather than recurrence". Journal of Medical Virology 92 (10): 1755–56. doi:10.1002/jmv.25855. PMC PMC7262304. PMID 32270882. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7262304. 
  4. ↑ Li, Y.; Yao, L.; Li, J. et al. (2020). "Stability issues of RT-PCR testing of SARS-CoV-2 for hospitalized patients clinically diagnosed with COVID-19". Journal of Medical Virology 92 (7): 903-908. doi:10.1002/jmv.25786. PMC PMC7228231. PMID 32219885. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7228231. 
  5. ↑ Yan, C.; Cui, J.; Huang, L. et al. (2020). "Rapid and visual detection of 2019 novel coronavirus (SARS-CoV-2) by a reverse transcription loop-mediated isothermal amplification assay". Clinical Microbiology and Infection 26 (6): 773-779. doi:10.1016/j.cmi.2020.04.001. PMC PMC7144850. PMID 32276116. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7144850. 
  6. ↑ Baek, Y.H.; Um, J.; Antigua, K.J.C. et al. (2020). "Development of a reverse transcription-loop-mediated isothermal amplification as a rapid early-detection method for novel SARS-CoV-2". Emerging Microbes and Infections 9 (1): 998–1007. doi:10.1080/22221751.2020.1756698. PMC PMC7301696. PMID 32306853. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7301696. 
  7. ↑ Li, Z.; Yi, Y.; Lu, X. et al. (2020). "Development and clinical application of a rapid IgM-IgG combined antibody test for SARS-CoV-2 infection diagnosis". Journal of Medical Virology 92 (9): 1518-1524. doi:10.1002/jmv.25727. PMC PMC7228300. PMID 32104917. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7228300. 
  8. ↑ Li, G.; Ren, L.; Yang, S. et al. (2020). "Profiling Early Humoral Response to Diagnose Novel Coronavirus Disease (COVID-19)". Clinical Infectious Diseases 71 (15): 778-785. doi:10.1093/cid/ciaa310. PMC PMC7184472. PMID 32198501. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7184472. 
  9. ↑ Sood, N.; Simon, P.; Ebner, P. et al. (2020). "Seroprevalence of SARS-CoV-2-Specific Antibodies Among Adults in Los Angeles County, California, on April 10-11, 2020". JAMA 323 (23): 2425-2427. doi:10.1001/jama.2020.8279. PMC PMC7235907. PMID 32421144. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7235907. 
  10. ↑ Kozel, T.R.; Burnham-Marusich, A.R. (2017). "Point-of-Care Testing for Infectious Diseases: Past, Present, and Future". Journal of Clinical Microbiology 55 (8): 2313–20. doi:10.1128/JCM.00476-17. PMC PMC5527409. PMID 28539345. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5527409. 
  11. ↑ 11.0 11.1 Wang, W.; Xu, Y.; Gao, R. et al. (2020). "Detection of SARS-CoV-2 in Different Types of Clinical Specimens". JAMA 323 (18): 1843–44. doi:10.1001/jama.2020.3786. PMC PMC7066521. PMID 32159775. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7066521. 
  12. ↑ 12.0 12.1 Yang, Y.; Yang, M.; Shen, C. et al. (2020). "Evaluating the accuracy of different respiratory specimens in the laboratory diagnosis and monitoring the viral shedding of 2019-nCoV infections". medRxiv. doi:10.1101/2020.02.11.20021493. 
  13. ↑ Wu, Y.; Guo, C.; Lantian, T. et al. (2020). "Prolonged presence of SARS-CoV-2 viral RNA in faecal samples". The Lancet Gastroenterology and Hepatology 5 (5): 434-435. doi:10.1016/S2468-1253(20)30083-2. PMC PMC7158584. PMID 32199469. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7158584. 

Notes

This presentation is faithful to the original, with only a few minor changes to presentation. Some grammar, punctuation, and repetition was cleaned up to improve readability. In some cases important information was missing from the references, and that information was added. The "priority of specimen" column was left off Table 2 for this version, as the column in the original version is non-sensical and unexplained. Nothing else was changed in accordance with the NoDerivatives portion of the license.

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