key: cord-0759369-zpq0g7kp
authors: Jing, Ran; Kudinha, Timothy; Zhou, Meng-Lan; Xiao, Meng; Wang, He; Yang, Wen-Hang; Xu, Ying-Chun; Hsueh, Po-Ren
title: Laboratory Diagnosis of COVID-19 in China: A review of challenging cases and analysis
date: 2020-10-17
journal: J Microbiol Immunol Infect
DOI: 10.1016/j.jmii.2020.10.004
sha: ef6771b4b3e6eb0dc16538807bf2c666a1fc81a5
doc_id: 759369
cord_uid: zpq0g7kp

Since the initial emergence of coronavirus disease 2019 (COVID-19) in Wuhan, Hubei province, China, a rapid spread of the disease occurred around the world, rising to become an international global health concern at pandemic level. In the face of this medical challenge threatening humans, the development of rapid and accurate methods for early screening and diagnosis of COVID-19 became crucial to containing the emerging public health threat, and prevent further spread within the population. Despite the large number of COVID-19 confirmed cases in China, some problematic cases with inconsistent laboratory testing results, were reported. Specifically, a high false-negative rate of 41% on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection by real-time reverse transcription-polymerase chain reaction (qRT-PCR) assays was observed in China. Although serological testing has been applied worldwide as a complementary method to help identify SARS-CoV-2, several limitations on its use have been reported in China. Therefore, the use of both qRT-PCR and serological testing in the diagnosis of COVID-19 in China and elsewhere, presented considerable challenges, but when used in combination, can be valuable tools in the fight against COVID-19. In this review, we give an overview of the advantages and disadvantages of different molecular techniques for SARS-CoV-2 detection that are currently used in several labs, including qRT-PCR, gene sequencing, loop-mediated isothermal amplification (LAMP), nucleic acid mass spectrometry (MS), and gene editing technique based on clustered regularly interspaced short palindromic repeats (CRISPR/Cas13) system. Then we mainly review and analyze some causes of false-negative qRT-PCR results, and how to resolve some of the diagnostic dilemma.

acid mass spectrometry (MS), and gene editing technique based on 24 clustered regularly interspaced short palindromic repeats (CRISPR/Cas13) 25 system. Then we mainly review and analyze some causes of false-negative 26 qRT-PCR results, and how to resolve some of the diagnostic dilemma. China and elsewhere, involving inconsistent laboratory testing results, mainly 58 caused by false-negative real-time reverse transcription-polymerase chain 59 reaction (qRT-PCR) detection. In this review, we summarize and discuss 60 some possible causes of false-negative results, including how to resolve the 61 diagnostic dilemma. We also review and discuss the advantages and 62 SARS-CoV-2 etiological characteristics and genome 69 organization with known SARS-CoV-2 strain. This is especially valuable in cases when 115 only one SARS-CoV-2 gene target is detected for the known βCoVs by qRT-116 PCR. For example, Wang et al. have developed a nanopore target 117 sequencing (NTS) method targeting 11 viral regions that is able to detect as 118 few as 10 viral copies/mL within 1 hour of sequencing. 8 In addition, next 119 generation sequencing (NGS) also played an important role in studying the 120 origin of SARS-CoV-2 and was very valuable in the early stages of COVID-121 19 outbreak in China. Based on phylogenetic analysis, SARS-CoV-2 is 122 closely related (with 88% sequence identity) to bat-SL-CoVZC45 and bat-SL-123

CoVZXC21, 9 and most closely related (with 96.3% of sequence similarity) to 124 bat-CoV RaTG13, all detected in bats. 10 However, it is not very closely 125 related to SARS-CoV and MERS-CoV, with about 79% and 50% sequence 126 similarity, respectively. 9 127 Molecular sequencing is also used to study the evolution of SARS-CoV-2 128 and monitoring the virus variability. For example, in Guangdong province 129 (China), 53 genomes from COVID-19 confirmed cases were generated by 130 assay, and a negative or positive result can be visually differentiated by 161 using a colorimetric change without requiring a machine to read the results. 162

In addition, LAMP results are available in 1 hour, and there is no requirement 163 for expensive reagents or specialized equipment, making it useful for POC 164 diagnosis in remote clinical facilities without sufficient laboratory capacity. 165

Moreover, some studies have demonstrated that the LAMP assay has higher 166 sensitivity and specificity compared to qRT-PCR assays as it utilizes six 167 primers to identify multiple regions on the target in a single reaction. 15 

Despite the significant increase in the number of laboratory-confirmed cases, URT before onset of symptoms (Figure 2) . 30 Then the viral load (in throat 275 swabs) peaks during the first week of illness and gradually decreases in the 276 second week (Figure 2) , and the researchers supposed that infectiousness 277 peaked on or before symptom onset, as per data obtained from 23 patients. 30 278

However, a research study in Germany indicated that viral shedding in 279 pharyngeal swabs reached a peak in the first week of symptomatic 280 presentation. 28 Feng et al. reported on a case from China with fever and 281 patchy ground-glass opacity on chest CT on admission, but with four 282 negative sequential qRT-PCR results on the pharyngeal swabs. 31 It was not 283 until the fifth day of admission, that the fifth qRT-PCR test was positive. This 284 case indicates that the four negative serial qRT-PCR testing results were 285 possibly false-negatives. One possible reason is that although the virus had 286 already started shedding in his pharyngeal site before or after admission, it 287

was not detected until day 5 due to the low viral load below the detectable 288 limit of the qRT-PCR assay. In Korea, a similar case was reported in a 289 patient with a fever who had SARS-CoV-2 detected from a mixed specimen 290 of nasopharyngeal and oropharyngeal swabs on day 2 of symptom onset. 32 291 However, the viral load started to decline from day 7, and viral RNA was 292 undetectable by qRT-PCR for two successive days from day 15 in spite of 293 the ongoing infection, suggesting that viral load kinetics, sampling time and 294 duration of the illness, can have an influence on qRT-PCR results. 32 295

Multiple COVID-19 cases which were SARS-CoV-2 positive by qRT-PCR 296 assays in the respiratory tract swabs after patients had been discharged from 297 hospital, have become highly controversial in China. 33 Zhou et al. reported a 298 case who met the criteria for hospital discharge but was tested positive for 299 SARS-CoV2 again 10 days after discharge. 34 Thus a longer observation 300 period should be considered for discharged patients. It was unclear why these patients' URT specimens tested consecutively 333 negative for SARS-CoV-2 RNA. Some possible causes include improper 334 collection or handling of specimens, and low viral load due to diminished viral 335 shedding in URT specimens. Another possible explanation is the relatively 336 lower sensitivity of nasopharyngeal and oral swab qRT-PCR assays for 337 SARS-CoV-2 RNA, ranging from 56% to 83%, in comparison to lower 338 respiratory tract (LRT) specimens. 36 Although BALF specimens increase the 339 detection rates of COVID-19, their collection requires a suction device and a 340 skilled operator, and is also painful for the patients, so they are not Table 2 . 448

Specific IgM and IgG antibodies can be used in determining whether the 449 patient has a recent or previous viral infection, 48 J o u r n a l P r e -p r o o f Table 1 Summary of available SARS-CoV-2 qRT-PCR assays.

Gene target

China. CDC 

Loop mediated isothermal amplification (LAMP) 570 assays as a rapid diagnostic for COVID-19

Rapid 573 detection of SARS-CoV-2 using reverse transcription RT-LAMP method

Rapid 576 molecular detection of SARS-CoV-2 (COVID-19) virus RNA using colorimetric 577 LAMP

Laboratory testing 580 techniques for SARS-CoV-2

Development 583 and evaluation of a CRISPR-based diagnostic for 2019-novel coronavirus

Rapid 592 identification of emerging infectious agents using PCR and electrospray 593 ionization mass spectrometry

A systematic review of 596 asymptomatic infections with COVID-19

Asymptomatic carrier state, acute respiratory disease, and pneumonia due to 600 severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2): Facts and 601 myths

Early transmission 604 dynamics in Wuhan, China, of novel coronavirus-Infected pneumonia

Molecular and 607 serological investigation of 2019-nCoV infected patients: implication of 608 multiple shedding routes

A case of COVID-19 with long duration of viral 614 shedding

Virological assessment of hospitalized patients with COVID-2019

Connecting clusters of COVID-19: an epidemiological and serological 621 investigation

Temporal dynamics in viral 624 shedding and transmissibility of COVID-19

A case report of COVID-19 with false 627 negative RT-PCR test: necessity of chest CT

Viral load kinetics 630 of SARS-CoV-2 infection in first two patients in Korea

Pathological 633 evidence for residual SARS-CoV-2 in pulmonary tissues of a ready-for-634 discharge patient

CoV2 in a discharged COVID-19 case

Evaluating the 640 accuracy of different respiratory specimens in the laboratory diagnosis and 641 monitoring the viral shedding of 2019-nCoV infections

False-negative nasopharyngeal 644 swab RT-PCR assays in typical COVID-19: role of ultra-low-dose chest CT 645 and bronchoscopy in diagnosis

Negative nasopharyngeal and oropharyngeal swabs do not rule out 649 COVID-19

Consistent detection of 2019 novel coronavirus in saliva

First case of 655 severe childhood novel coronavirus pneumonia in China

Early virus clearance 662 and delayed antibody response in a case of COVID-19 with a history of co-663 infection with HIV-1 and HCV

Potential false-negative 666 nucleic acid testing results for severe acute respiratory syndrome coronavirus 667 2 from thermal inactivation of samples with low viral loads

Laboratory testing for coronavirus disease 2019

National Health Commission of the People's Republic of China

Real-time RT-PCR in COVID-19 detection: issues 678 affecting the results

mitigate the risk of loss of sensitivity by new genomic variants during the 683 COVID-19 outbreak

National Health Commission of the People's Republic of China. Diagnosis & 686 treatment scheme for coronavirus disease 2019

Antibody responses 690 to SARS-CoV-2 in patients of novel coronavirus disease 2019

Challenges in laboratory diagnosis of the novel coronavirus SARS-CoV-2

Early detection of

SARS-CoV-2 antibodies in COVID-19 patients as a serologic marker of 697 infection

Reasons and solutions for 'false positive results' of 2019 699 novel coronavirus•specific antibodies detection

Testing 702 for SARS-CoV-2 (COVID-19): a systematic review and clinical guide to 703 molecular and serological in-vitro diagnostic assays

Cellex Emergency Use Authorization

The 708 incubation period of coronavirus disease 2019 (COVID-19) from publicly 709 reported confirmed cases: estimation and application

Diagnostic 712 performance of COVID-19 serology assays

The clinical 714 application of 2019-nCoV IgM and IgG tests by colloidal gold method and the 715 analysis of its interfering factors

Diagnosing COVID-19: the disease and tools for detection

Cross-reactive 721 antibody response between SARS-CoV-2 and SARS-CoV infections

Molecular assays to diagnose COVID-19: Summary table of available 728 protocols

The United States Centers for Disease Control and Prevention. 2019-novel 731 coronavirus (2019-nCoV) real-time rRT-PCR panel primers and probes

The University of Hong Kong (HKU), School of Public Health. Detection of 735 2019 novel coronavirus (2019-nCoV) in suspected human cases by RT-PCR

SARS-CoV-2, severe acute respiratory syndrome coronavirus

real-time reverse transcription polymerase chain reaction

ORF, open reading frames

RNA-dependent RNA polymerase gene