Udugama (ASC nano 2020, see below) presented an overview on the techniques used at the start of the epidemic, as well as on emerging diagnostic methods in March 2020. More recently, a review by Ravi (Biosens Bioelectron 2020, see below) focused on molecular and serological assays with FDA Emergency Use Authorization. A list of assays commercially available for diagnosis of COVID-19 is updated by FIND (https://www.finddx.org/covid-19/pipeline/).
Assays that are still in development stage are also presented. As indicated by Premraj (Diagnostics Basel 2020, see below), there are now serious debates globally and regionally on the sensitivity and specificity of these tests and about the overall accuracy and reliability of the tests for decision making on COVID-19 control strategies.
Molecular methods
In April 2020, a review by Shen (J Pharm Anal 2020, see below) summarized available detection methods for SARS-CoV-2 nucleic acid. The paper is short, but provides very clear explanations about the different methods that have been developed. A list of currently available molecular assays can be found at FIND (https://www.finddx.org/covid-19/pipeline/?section=molecular-assays#diag_tab).
RT-PCR
Initially developed RT-PCR assays
In acute respiratory infection, RT-PCR is routinely used to detect causative viruses from respiratory secretions. Early reports presented various assays for COVID-19 diagnosis. A real-time reverse-transcription PCR (rtRT-PCR) was used to identify SARS-CoV-2 through preliminary and validation detection of its E gene, RNA-dependent RNA polymerase (RdRp) gene, and N gene (Yu Micr Inf 2020, see below). Chu (Clin Chem 2020, see below) reported the development of two 1-step quantitative rtRT-PCR assays detecting the ORF1b and N regions of the viral genome. The primer and
probe sets were designed to react with SARS-CoV-2 and its closely related viruses, such as SARS coronavirus. These assays were evaluated using a panel of positive and negative controls and shown to have a dynamic range of at least seven orders of magnitude (2x10−4-2000 TCID50/reaction).
Fluorescence-based quantitative PCR kit were rapidly distributed by the Chinese CDC for laboratory confirmation of disease in China. And a whole array of commercial tests became available. Wang (on medRxiv:
https://www.medrxiv.org/content/10.1101/2020.02.12.20022327v2) reported for instance the use of a detection kit (Bioperfectus, Taizhou, China) to detect the ORF1ab gene and the N gene using real-time RT-PCR. Positive results on both the ORF1ab gene and the N gene are required for laboratory confirmation of the disease.
In Europe, the envelope (E)-gene screening test as published by Corman (Euro Surv 2020, see below) has been widely implemented.
Sharfstein (JAMA 2020, see below) and Babiker (Am J Clin Pathol 2020, see below) provided a detailed explanation of the issues faced in the USA at the beginning of the epidemic, which delayed COVID-19 PCR testing in the country. Lu (Em Inf Dis 2020, see below) described the design and validation of the US CDC rRT-PCR panel and presented comprehensive data on its performance with multiple specimen types and clinical specimens. The panel consisted of 3 RT-PCR assays targeting the N gene. The US FDA issued an Emergency Use Authorization on Feb 4 2020 to enable emergency use of this panel.
Improving PCR assays
Chan (J Clin Microb 2020, see below) developed a novel real-time RT-PCR assay targeting the RNA-dependent RNA polymerase (RdRp)/helicase (Hel) (COVID-19-RdRp/Hel assay). The assay has a low limit of detection (1.8 TCID50/ml with genomic RNA and 11.2 RNA copies/reaction with in vitro RNA transcripts). It was compared to the RdRp-P2 assay currently used in European laboratories. Among 273 specimens from 15 patients with laboratory-confirmed COVID-19 in Hong Kong, 77 (28.2%) were positive by both the COVID-RdRp/Hel and RdRp-P2 assays. The COVID- COVID-19-RdRp/Hel assay was positive for an additional 42 RdRd-P2-negative specimens [119/273 (43.6%) vs 77/273 (28.2%), P<0.001], including 29/120 (24.2%) respiratory tract specimens and 13/153 (8.5%) non-respiratory tract specimens.
The mean viral load of these specimens was 3.21×104 RNA copies/ml (range, 2.21×102 to 4.71×105 RNA copies/ml).
The COVID-19-RdRp/Hel assay did not cross-react with other human-pathogenic coronaviruses and respiratory pathogens in cell culture and clinical specimens, whereas the RdRp-P2 assay cross-reacted with SARS-CoV in cell culture.
Won (ExpNeurobiol 2020, see below) presented a low cost, rapid alternative RT PCR protocol for COVID-19 diagnosis, composed of specimen self-collection by the patient via pharyngeal swab, Trizol-based RNA purification, and SYBR Green-based RT PCR.
Automated platforms
Pfefferle (Euro Surveill 2020, see below) evaluated the performance of a molecular assay for detection of SARS-CoV-2 on a high-throughput platform, the cobas 6800, using the 'open channel' for integration of a laboratory-developed assay. The authors observed good analytical performance in clinical specimens. The fully automated workflow enabled high-throughput testing with minimal hands-on time, while offering fast and reliable results.
Cepheid announced that it has received Emergency Use Authorization (EUA) from the U.S. FDA for Xpert® Xpress SARS-CoV-2, a rapid molecular diagnostic test for qualitative detection of SARS-CoV-2 (http://cepheid.mediaroom.com/2020-03-21-Cepheid-Receives-Emergency-Use-Authorization-from-FDA-for-Rapid-SARS-CoV-2-Test). The test has been designed to operate on any of Cepheid's automated GeneXpert® Systems, with a detection time of approximately 45 minutes.
Validation data & assay limitations
Xie (Int J Inf Dis 2020, see below) compared nucleic acid amplification testing performed with 3 different fluorescent RT-PCR kits on different samples, including oropharyngeal swab, blood, urine and stool. Nine out of the 19 patients tested were found positive for SARS-CoV-2 using oropharyngeal swab samples, and the virus nucleic acid was also detected in eight of these nine patients using stool samples. None of positive results was identified in the blood and urine samples. Similar data were obtained with the 3 kits.
Of note, a lack of assay sensitivity was reported by Xie (Radiol 2020, see below), who described five patients with SARS-CoV-2 infection who had initial negative RT-PCR results in mouth swabs but typical imaging findings, including ground-glass opacity (5 patients) and/or mixed ground-glass opacity and mixed consolidation (2 patients). All patients were eventually confirmed with SARS-CoV-2 infection by repeated swab tests. Similar cases were reported by various authors:
Huang (Radiol 2020, see below).
Winichakoon (J Clin Microb 2020, see below) reported a case of COVID-19 pneumonia diagnosed from bronchoalveolar lavage fluid in Thailand, who initially had negative tests from nasopharyngeal/oropharyngeal swabs.
A publication by Wang, Kang et al. (J Med Vir 2020, see below) further illustrates the sensitivity limitation of current RT-PCR based diagnosis, previously reported by others. Although the paper does not provide details, the authors described a COVID-19 case not confirmed by SARS-CoV-2 RT-qPCR testing at the first three evaluations within three weeks, before bronchoalveolar lavage fluid was acquired and results from both RT-qPCR and next-generation sequencing (NGS) testing became positive for SARS-CoV-2.
Ruan (Chin Med J 2020, see below) presented a case with negative RT PCR result until day 11 of disease onset.
Interestingly, a study by Li, Yao et al. (J Med Vir 2020, see below) evaluated the sensitivity of RT-PCR testing on sequential samples. The study illustrated well on one hand the importance of retest for improving detection of positive cases, and on the other hand the instability of results over time in a same patient.
Wang (Clin Chem 2020, see below) showed that the limit of detection (LOD) of the six commercial kits approved in China differ substantially, with the poorest LODs likely leading to false-negative results when RT–PCR is used to detect SARS-CoV-2 infection.
Rhoads (J Clin Microbiol 2020, see below) compared the Abbott ID Now, Diasorin Simplexa, and CDC FDA EUA methods for the detection of SARS-CoV-2 from nasopharyngeal and nasal swabs. The 95% CIs for the positive percent agreement was overlapping for the ID Now and Simplexa assays when using the modified CDC method as the reference standard.
The sample size of this study was not large enough to conclude one of these assays had clearly superior or inferior performance for the detection of SARS-CoV-2 from upper respiratory specimens in liquid transport media.
Another report by Moran (J Clin Microbiol 2020, see below) compared results from specimens tested with Cepheid Xpert Xpress SARS-CoV-2 and Roche cobas SARS-CoV-2 assays. Of these 103 specimens, 42 tested positive and 60 tested negative with both systems for agreement of 99%.
A possible technical limitation of current RT-PCR was raised by Fan, Zhang et al. (Chin Med J 2020, see below). The authors evaluated the potential impact of SARS-CoV-2 genome evolution on RT-PCR performance by analysing published primer sets and their match with 77 publicly available whole genome sequences. They found five RT-qPCR primer sets (targeting Orf1ab or N) that may potentially cause false negative results. Targeting the more conserved nsp12 (RdRp) gene was thus recommended.
Tahamtan (Expert Rev Mol Diagn. 2020, see below) provided an overview of the performance issues with RT-PCR.
Conclusions were that in case of negative RT-PCR result with clinical features suspicion for COVID-19, especially when
only upper respiratory tract samples were tested, multiple sample types in different time points, including from the lower respiratory tract if possible, should be tested. Combination of real time RT-PCR and clinical features especially CT image could facilitate disease management. Proper sampling procedures, good laboratory practice standard, and using high quality extraction and real-time RT-PCR kit could improve the approach and reduce inaccurate results.
RT-LAMP
Other molecular-based detection techniques include reverse transcription loop-mediated isothermal amplification (RT-LAMP), which feasibility for detection of SARS-CoV-2 has been established by various groups. Isothermal amplification, unlike PCR, enables amplification at a constant temperature using two or three sets of primers and a polymerase with high strand displacement activity, avoiding the need for thermal cycling (Ravi Biosens Bioelectron 2020, see below). To achieve comparable specificity, four different primers are used to amplify six distinct regions on the target gene. As a result, isothermal amplification can achieve higher amounts of nucleic acid copies in a shorter amount of time compared to standard PCR.
Lamb (manuscript on medRxiv: https://www.medrxiv.org/content/10.1101/2020.02.19.20025155v1) demonstrated the feasibility of rapid screening diagnosis completed in under 30 minutes, using Reverse Transcription Loop-Mediated Isothermal Amplification (RT-LAMP) . No validation data have been presented yet. Only simulated patient samples were used, which were created by spiking serum, urine, saliva, oropharyngeal swabs, and nasopharyngeal swabs with a portion of the COVID-19 nucleic sequence.
Yu (Clin Chem 2020, see below) developed an isothermal LAMP based method for COVID-19, amplifying a fragment of the ORF1ab gene. The assay detected synthesized RNA equivalent to 10 copies of virus. Reaction time varied from 15-40 minutes, depending on the loading of virus in the collected samples. 42/43 patient samples initially diagnosed with RTqPCR showed consistent signal after 40 min incubation with the new assay (97.6% sensitivity).
Another LAMP method was described by Baek (Em Micr Inf 2020, see below).
Among the commercially available assays, the ID NOW COVID-19 Test relies on isothermal nucleic acid amplification, targeting a unique region of the RNA-dependent RNA polymerase (RdRP) gene of SARS-CoV-2 (Ravi Biosens Bioelectron 2020, see below). ID NOW COVID-19 Test provides results in 13 minutes or less from throat, nasal or nasopharyngeal swab samples, with reported analytical sensitivity of 125 copies/mL. The ID NOW COVID-19 Test is performed on the ID NOW Instrument, which gives a simple method for mixing sample with test reagents and transferring to the test base through a cartridge.
Another test is Cue Health's Cue COVID-19 Test, a rapid, portable assay that delivers results to a mobile phone in less than 25 minutes. Similar to ID NOW, Cue's test also uses isothermal amplification on nasal swabs, but it detects the SARS-CoV-2 N gene. Additionally, Cue's disposable POC test cartridge forms a connected diagnostic platform with a mobile phone that enables a patient to have convenient access to their health information.
CRISPR-based methods
Several CRISPR-based diagnostic methods have also been described:
Hou (manuscript on medRxiv: https://www.medrxiv.org/content/10.1101/2020.02.22.20025460v1) reported the development of an isothermal, CRISPR-based diagnostic. The assay demonstrated a near single-copy sensitivity. It was evaluated on 61 specimens with suspected infection (52 positives) and showed great clinical sensitivity with a shorter turn-around time (40 min) than RT-PCR.
Proof-of-principle of another CRISPR-based detection method was also described by Curti (manuscript on bioRxiv: https://www.biorxiv.org/content/10.1101/2020.02.29.971127v1).
Broughton (Nat Biotech 2020, see below) reported development of a rapid (<40 min), easy-to-implement and accurate CRISPR-Cas12-based lateral flow assay for detection of SARS-CoV-2 from respiratory swab RNA
extracts. The method was validated using clinical samples from patients in the United States, including 36 patients with COVID-19 infection and 42 patients with other viral respiratory infections. The CRISPR-based DETECTR assay provided a 95% positive predictive agreement and 100% negative predictive agreement compared to the US CDC SARS-CoV-2 real-time RT-PCR assay.
Javalkote (Methods 2020, see below) presented a compilation of state-of-the-art detection techniques for COVID-19 using CRISPR technology, stating that this approach has tremendous potential to transform diagnostics and epidemiology.
Other molecular methods
Guan (Chin Med J 2020, see below) reported a case with inconsistent fluorescence quantitative-PCR results, for which high-throughput sequencing was used to make a further diagnosis of SARS-CoV-2 infection. Although high-throughput sequencing appears too costly and labour-intensive for routine diagnosis, the authors believe that it can be used for further diagnosis of COVID-19 patients with unclear PCR results under the condition of strict operation and quality control.
Serological methods
A review paper by Infantino (Isr Med Assoc J 2020, see below) provided an overview on serological diagnostic assays.
Bryant (Sci Imm 2020, see below) provided important considerations with regards to use cases of serological assays and implications in terms of assay validation. The review by Pecora (Clin Lab Med 2020, see below) is the most recent summary pertaining to SARS-CoV-2 serology testing. As of today, an impressive number of antibody assays is described
(https://www.finddx.org/covid- 19/pipeline/?avance=Commercialized&type=all&test_target=Antibody&status=all§ion=show-all&action=default).
ELISA
Various ELISA protocols for detection of antibodies to SARS-CoV-2 emerged rapidly, in the first few weeks of the epidemic. For instance, a SARS-CoV-2 IgM and IgG ELISA has been reported by Zhang (Em Micr Inf 2020, see below).
The assay is based on recombinant N. A preliminary evaluation was conducted in 16 patients (incl. 3 patients with severe disease). As shown by Figure 20, an increase of specific antibodies was seen in part of the patients as early as by day 5.
Figure 20 Serological detection of SARS-CoV-2. Dashed line indicates cut-off, which was determined based on data from healthy controls (from Zhang Em Micr Inf 2020).
Xiang (manuscript on medRxiv https://www.medrxiv.org/content/10.1101/2020.02.27.20028787v1) reported the evaluation of 2 serological assays: an IgG and IgM ELISA and a colloidal gold-immunochromatographic assay kit for detection of COVID-19. Using 63 samples for the ELISA and 91 plasma samples for the colloidal
gold-immunochromatographic assay, they found a sensitivity of the combined ELISA IgM and ELISA IgG of 55/63 (87.3%), and that of the colloidal gold-immunochromatographic IgM and IgG assay of 75/91 (82.4%). Both methods displayed a specificity of 100%.
Liu (J Clin Micr 2020, see below) reported the evaluation of N and S protein-based IgM and IgG ELISAs in 214 confirmed COVID-19 patients (Table 11).
Table 11 IgM and IgG detection in the 214 serum samples from patients with COVID-19 (from Liu J Clin Micr 2020)
The sensitivity of the S-based ELISA for IgM detection was found significantly higher than that of the N-based ELISA.
An increase in the sensitivity of IgM and IgG detection was observed with an increasing number of days post-disease onset. The positive rate of N-based and S-based IgM and IgG ELISAs was less than 60% during the early stage of the illness (day 0-10), and increased after 10 days.
A study by Zhao (Clin Inf Dis 2020, see below) investigated the dynamics of total Ab, IgM and IgG antibody against SARS-CoV-2 in serial blood samples collected from 173 confirmed COVID-19 patients. Testing was performed ELISA kits supplied by Beijing Wantai Biological Pharmacy Enterprise. Antibodies were found in <40% of patients within 1-week since onset, and rapidly increased to 100.0% (Ab), 94.3% (IgM) and 79.8% (IgG) by day-15 after onset. In contrast, RNA detectability decreased from 66.7% (58/87) in samples collected before day-7 to 45.5% (25/55) between day 15 and 39. Combining RNA and antibody detections significantly improved the sensitivity of pathogenic diagnosis for COVID-19.
Okba (Em Inf Dis 2020, see below) presented validation data for in house S and N based ELISAs as well as for a β version of the Euroimmun commercial S1 IgG or IgA ELISAs. In the 3 in-house ELISAs tested, the RBD and N protein ELISAs were more sensitive than S1 ELISA in detecting antibodies in mildly infected patients and showed stronger correlations with PRNT50 titers. Therefore, the authors indicated that detecting antibodies against 2 different antigens might be needed to avoid false-negative results in surveillance studies.
Wang (J Clin Microb 2020, see below) reported that middle-high level of rheumatoid factor-IgM in sera could lead to false-positive reactivity in SARS-CoV-2 IgM GICA (colloidal gold immunochromatography assay) and ELISA assays. The authors suggested that urea dissociation tests would be helpful in reducing such false-positive SARS-CoV-2 IgM results.
Hicks (manuscript on medRxiv : https://doi.org/10.1101/2020.06.22.20137695) evaluated the serologic reactivity of pre-pandemic archival blood serum samples (pre2019) and samples collected in April 2020 in the US (New York and New Jersey) from a community highly affected by SARS-CoV-2. Utilizing twelve previously reported ELISAs, the authors tested IgG, IgM and IgA reactivity against S proteins from SARS-CoV-2, MERS-CoV, SARS-CoV, OC43, and HCoV-HKU1. They detected the potential cross-reactivity of antibodies against SARS-CoV-2 towards the four other coronaviruses, with the strongest cross-recognition between SARS-CoV-2 and SARS /MERS-CoV antibodies. They advised that the interpretation of current serological results should take into account archival patient sera (pre2019), including sera from known SARS-CoV and MERS convalescent patients, to properly analyse the resulting data and adjust any estimates of seropositivity as needed.
Of note, Stadlbauer (Curr Protoc Microbiol 2020, see below) described a detailed protocol for expression of antigens derived from the S protein of SARS-CoV-2 that can serve as a substrate for immunological assays, as well as the protocol of a two-stage ELISA.
Chemiluminescence (CLIA)
Jin (Int J Infect Dis 2020, see below) investigated the diagnostic value of serological test and evolution of test results over time in 43 COVID-19 patients. SARS-CoV-2 IgM and IgG chemiluminescence immunoassay (CLIA) kits from Shenzhen YHLO Biotech Co., Ltd (China) were used, with two antigens of SARS-CoV-2 coated on the magnetic beads of these CLIA assays (N and S proteins). Compared to molecular test, the sensitivity of serum IgM and IgG antibodies to diagnose COVID-19 was 48.1% and 88.9%, and the specificity was 100% and 90.9%. However, IgG positive rate increased till 100% over time.
Padoan (Clin Chem Lab Med 2020, see below) and Lippi (Clin Chem Lab Med 2020, see below) reported on the validation of MAGLUMI 2000 Plus CLIA assay for the measurement of specific IgM and IgG in sera. Results of MAGLUMI IgM and IgG were well aligned with those of Euroimmun Anti-SARS-CoV-2 IgA and IgG, especially concerning the IgG and the cumulative immunoglobulin profile.
Pseudotype neutralization assay
Nie (Emerg Microbes Infect 2020, see below) reported on a pseudovirus neutralization assay for SARS-CoV-2 and its validation. The assay is based on a VSV pseudovirus system. The key parameters for this assay were optimized, including cell types, cell numbers, virus inoculum. With this test, SARS-CoV-2 convalescent patient sera showed high neutralizing potency. The assay showed relatively low coefficient of variations with 15.9% and 16.2% for the intra- and inter-assay analyses respectively.
Rapid test for antibody detection
Li, Yi et al. (J Med Vir 2020, see below) reported the development of a rapid and simple point-of-care lateral flow SARS-CoV-2 immunoassay which can detect IgM and IgG antibodies simultaneously in human blood within 15 minutes. The clinical detection sensitivity and specificity of this test were measured using blood samples collected from 397 PCR-confirmed COVID-19 patients and 128 negative patients at 8 different clinical sites. The overall testing sensitivity reached 88.66% and specificity 90.63%. The assay was evaluated on fingerstick blood samples, as well as serum and plasma from venous blood.
In March 2020, Cassaniti (J Med Vir 2020, see below) reported a study aimed at validating the VivaDiagTM COVID-19 IgM/IgG Rapid Test lateral flow immunoassay (LFIA) for the rapid diagnosis of COVID-19, in real-life conditions. The performance of VivaDiagTM COVID-19 test was assessed in 50 patients at their first access at emergency room department with fever and respiratory syndrome in comparison with results of nasal swab molecular screening.
Sensitivity of the VivaDiagTM COVID-19 IgM/IgG Rapid Test was only 18.4%, specificity was 91.7%, while NPV was 26.2%
Sensitivity of the VivaDiagTM COVID-19 IgM/IgG Rapid Test was only 18.4%, specificity was 91.7%, while NPV was 26.2%