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Diagnostic assays for COVID-19: a narrative review

Zhang, Lidinga,b; Liang, Xiaohana,b; Li, Yanqingb; Zheng, Huac; Qu, Wenshengd; Wang, Baofenge; Luo, Haiminga,b,∗

Author Information
doi: 10.1097/JBR.0000000000000077
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Abstract

Introduction

Currently, there is no definitive evidence that SARS-CoV-2 originated from the seafood market and the exact origin of the virus is still unknown.[1–5] Millions of diagnosed cases have shown that the main route of transmission is person-to-person and that coronavirus disease 2019 (COVID-19) has higher levels of transmissibility and pandemic risk than SARS-CoV and other coronaviruses.[6–9] Owing to the continuous and rapid increase in number of infected people, the World Health Organization (WHO) has declared the COVID-19 outbreak a pandemic and public health emergency.

SARS-CoV-2 belongs to the β genus of the coronaviridae family and is similar to SARS-CoV (identity of approximately 79%) but distinct from Middle East respiratory syndrome coronavirus (identity of approximately 50%).[1,10] SARS-CoV-2 is the largest known single-stranded positive-sense RNA virus (around 30 kb in length) with a diameter of 60 to 140 nm that encodes structural and non-structural proteins.[1,11–13] Four main structural proteins, spike (S), membrane (M), envelope (E), and nucleocapsid (N) (Fig. 1A), have similar structures in all known CoVs. These proteins promote the viral infection of host cells and subsequent replication. S protein is responsible for recognizing host cell surface receptors and for mediating fusion of the viral envelope and cell membrane, which directly determines host tropism and transmission capabilities. S protein is more variable than the other three structural proteins.[12,14–16] M protein has three transmembrane domains and is involved in the formation and budding of the viral envelope, which helps shape the virion particles and binding to nucleocapsid.[17,18] E protein plays a role in the assembly and release of particles, and in viral pathogenesis,[19] while N protein contains two domains and aids binding of the genome to a replication transcription complex, which is required for the replication of genomic material.[16,20] Many clinical cases indicate that people with poor immune function (eg, children, infants) and patients with pre-existing diseases (eg, heart disease, high blood pressure, diabetes, chronic respiratory disease, and cancer) have a higher risk of succumbing to SARS-CoV-2 infection.[21]

Figure 1
Figure 1:
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and clinical manifestation of coronavirus disease 2019 (COVID-19). (A) The structure of SARS-CoV-2. (B) Symptoms reported in COVID-19 cases. (C) Percentage of COVID-19 patients with different symptoms collected from different studies. Data were collected from Guan et al,[33] Chen et al,[34] Shi et al,[35] Huang et al,[2] Yang et al,[36] Gane et al,[27] and Wang et al.[37].

The COVID-19 outbreak poses a huge threat to human health and is of tremendous public interest around the world. Owing to the continuous persistence of pathogenic COVID-19 infections, effective therapeutics and vaccines are urgently needed, but none are currently approved by the US Federal Drug Administration for the treatment of COVID-19 patients.[22] However, two different studies confirmed that the key to SARS-CoV-2 infection of human cells is the combination of the coronavirus S protein and the human angiotensin-converting enzyme 2 (ACE2) protein. ACE2, which is involved in controlling blood pressure, is recognized by the S protein and mediates infection of human cells.[23] Amino acid changes at certain sites of S protein can significantly affect the ability of the S protein to bind to the ACE2 receptor, and the degree of binding between the S protein and ACE2 is directly related to infectivity and pathogenicity of the virus.[6] This binding is, therefore, very important for drug development and vaccine design to combat COVID-19.

Rapid and point-of-care (POC) diagnostic methods are the first line of defence against COVID-19. These methods enable the rapid implementation of control measures to limit its spread through case identification, isolation, and contact tracing. However, the accuracy of the currently reported diagnosis tools is variable and highly accurate assays are crucial for identifying and detecting COVID-19. This review describes currently available diagnostic tools and analyzes their performance characteristics. We also introduce the concept of a smartphone platform combined with POC tests based on multiple SARS-CoV-2 biomarkers that would enable early diagnosis of COVID-19 and underpin contact tracing strategies.

Database search strategy

The authors used the following inclusion criteria for literature search: studies that discussed the co-occurrence of COVID-19, SARS-CoV-2, infection symptoms, diagnosis tools. Full-text articles in English published between January 1990 and September 2020 were included in this review. The authors searched the PubMed database to identify relevant publications. The literature search strategy was as follows: each of two synonymous phrases, that is, (1) COVID-19, (2) SARS-CoV-2, were combined with each of: (a) nucleic acid tests, (b) serological tests, (C) imaging tests; (D) point-of-care diagnostics eg, “diagnosis tools”, viz. (1) + (a); “nucleic acid detection”, viz. (1) + (b), etc. Four queries were obtained. The authors screened the reference list of included studies to identify other potentially useful studies.

Clinical manifestation and diagnosis of COVID-19

Many studies have reported that the common clinical symptoms of COVID-19 include fever, cough, myalgia or fatigue, pneumonia, complicated dyspnea, and anosmia, whereas the symptoms of diarrhea, hemoptysis, headache, runny nose, and phlegm-producing cough are less common (Fig. 1B).[2,24–32] However, the complete clinical manifestation is not yet clear, because reported symptoms range from mild to severe. Several detailed studies conducted by Guan et al,[33] Chen et al,[34] Shi et al,[35] Huang et al,[2] Yang et al,[36] Clemency et al,[27] and Wang et al[37] describe the clinical symptoms of COVID-19. These symptoms are highly varied, posing a challenge for clinical diagnosis (Fig. 1C). Moreover, over 20% of patients infected with COVID-19 show no symptoms, and the WHO has confirmed that asymptomatic patients can spread the virus, indicating the importance of early detection and subsequent patient support and isolation.[38,39] Considering that the symptoms of COVID-19 vary among individuals, a symptom-based test, such as temperature measurement, cannot be used as a robust diagnostic test for COVID-19. The development of accurate and sensitive diagnosis methods is crucial for detecting SARS-CoV-2 and identifying COVID-19.

Nucleic acid testing

Owing to the poor performance of symptom-based tests, molecular diagnosis technologies, such as nucleic acid-based assays, have received much attention. Molecular diagnosis plays a key role in the detection of various pathogenic microorganisms and numerous diseases.[40–42] Molecular diagnosis tools based on nucleic acids include sequencing, reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR), droplet digital PCR (ddPCR), multiplex PCR (mPCR), loop-mediated isothermal amplification (LAMP), real-time LAMP (RT-LAMP), and DNA microarray hybridization. Sequencing,[43–47] RT-PCR,[48–50] mPCR,[51] LAMP,[52,53] and RT-LAMP[54–56] have been applied to screen for COVID-19, and promising results have been reported by several studies. CRISPR-based assays are also potentially rapid nucleic acid tests.

Sequencing

Sequencing is one of the most valuable technologies for measuring the outbreak of COVID-19.[43–47] It can rapidly obtain genome sequence information of unknown viruses and has irreplaceable significance and value in detecting SARS-CoV-2.[1,10,12] The rapid sequencing of SARS-CoV-2 is very useful for preventing and controlling sudden outbreaks and for subsequent research, such as designing PCR primers and probes for rapid nucleic acid tests, constructing evolutionary trees to reveal pathogen-related characteristics, tracing the outbreak origin and identifying drivers and transmission chains and mapping the spread, as well as monitoring the evolution of etiological agents.[43–47] At present, three high-throughput sequencing methods, multiplex PCR amplicon sequencing, hybrid capture (capture)-based sequencing, and ultra-high-throughput metatranscriptomic (meta) sequencing, are used in SARS-CoV-2 detection and research (Fig. 2). The general characteristics of the three approaches are summarized in Table 1. In clinical practice, metagenomics next-generation sequencing (mNGS) is currently the most commonly used method for gene sequencing of pathogens.[43] mNGS does not require pathogen cultivation, provides non-preferred pathogen detection, and can detect various pathogens, such as bacteria, fungi, viruses, and parasites, at the same time. mNGS can also simultaneously identify mixed infections and can achieve a whole network of all pathogens. The first genomic sequence of SARS-CoV-2 was determined using metagenomic RNA sequencing, which provides the possibility to collect base-pair level information of the strain to track viral mutations in different tissues.[1,10,12] Although mNGS has several obvious advantages, its complicated protocols, expensive platform, and relatively long detection time (requires 24–72 hours) greatly limit its application. An advance would be for mNGS to be used in combination with RT-PCR to obtain complementary advantages. Unfortunately, whole-genome sequencing of SARS-COV-2 based on multiplex PCR or hybridization capture-based sequencing has not been reported.[47] We look forward to more advanced sequencing technologies that can be used for COVID-19 detection.

Figure 2
Figure 2:
The sequencing workflow for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) with reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR), recombinase polymerase amplification (RPA), loop-mediated isothermal amplification (LAMP) combined with magnetic immunocapture (IC-LAMP), and CRISPR-based amplification. (A) Different kinds of sample were pretreated, followed to nucleic acid extraction (B), and ampliciated by RT-PCR, RPA, IC-LAMP, or CRISPR (C), then products were used for sequencing (D).
Table 1 - General characteristics of the three approaches described in this study
Metatranscriptomic sequencing Hybrid capture- based sequencing Multiplex PCR amplicon-based sequencing
Sequencing objective Microbiome+human Target genome Target genome
2nd strand synthesis Y Y N
Fragmentation Y Y N
Library preparation Y Y N
PCR 18 cycles 18+18 cycles 15+25 cycles
Estimated time for presequencing sample processing 10.5h 20.5h 7.5h
Oligo synthesis 120nt × 506 40–60nt × 2×(113+14+10)
Cost estimated for pre-sequencing samples processing Moderate High Low
Estimated minimum data for downstream analyses (basel level) >10Gb Mb Mb
Evenness High Moderate Low
Sensitivity + ++ +++
Accuracy (SNV) +++ ++ +++
Accuracy (iSNV) +++ ++ +

PCR-based assay

As the primary method for diagnosing COVID-19, RT-qPCR is regarded as “gold standard” and is widely used worldwide.[48] The process includes the extraction of RNA from patient samples followed by reverse transcription of the RNA into cDNA and then PCR amplification using a premix and primers that specifically bind to the SARS-COV-2 viral genome.[57] To achieve visual and quantitative detection of SARS-COV-2, qPCR is performed using intercalating dyes such as SYBR Green or Taqman probes. SYBR Green, a dye with a green excitation wavelength, binds to the dsDNA double helix minor groove. The dye is non-specific for DNA products, and any amplification (specific or non-specific) will result in increased fluorescence readout.[58] TaqMan probes are a short oligonucleotides that contain a 5′ fluorophore and a 3′ quencher. They anneal to sequences in specific DNA amplicons.[58] Their fluorescence is proportional to the number of amplified molecules and is specifically related to the sequence of the correct amplified product, which can be measured in real-time on a qPCR instrument. The RT-qPCR assays for the diagnosis of COVID-19 target conserved sequences, such as the N gene,[48–50] ORF1 ab, E gene,[49] RdRp,[49] and ORF1b-nsp14. Figure 3A summarizes the 55 studies, which include ∼14,878 patients exhibiting some degree of clinical symptoms. The average accuracy is 87.6%, which is significantly higher than that of symptom-based tests. However, the sensitivity of RT-qPCR has been questioned by some researchers, who claim that the sensitivity of RT-qPCR is less than 58%,[59] even as low as 38%.[60] Moreover, other groups have reported that the sensitivity of RT-qPCR ranges from 20% to 100%.[61] Therefore, the use of RT-qPCR to “rule out” infection faces a challenge, that is, should RT-qPCR be regarded as the gold standard for the diagnosis of COVID-19? The sensitivity of RT-qPCR depends on the predictive value, which varies with the time from exposure and symptom onset. The dynamic changes in RT-PCR detection sensitivity can explain the significant discrepancies among studies. As shown in Figure 3B, the positive rate of RT-qPCR tests gradually declined with the time after the onset of symptoms. Therefore, the optimal window for COVID-19 detection based on RT-qPCR is within 12 days after the onset of symptoms. Beyond this, the RT-qPCR assay is not recommended because the result is unreliable; two or more tests are needed for reliability (Fig. 3C).[62] Moreover, the requirement for complex equipment and the relatively time-consuming assay (1.5–2 hours) greatly limit its ability to meet the rapidly growing need to detect COVID-19 infection, and to screen individuals who have come into close contact with confirmed cases. mPCR first reported by Visseaux et al[51] for the detection of COVID-19 is another molecular diagnostic tool. mPCR provides a highly sensitive, robust and accurate assay for the rapid detection of COVID-19, which allows rapid application by non-PCR trained staff or for POC testing. Compared with high-throughput platforms, mPCR analysis brings higher reagent cost and can process fewer samples, but the same kit can detect multiple relevant respiratory viruses, which is important for patient diagnosis workflows, especially when multiple viruses in addition to SARS-CoV-2 are prevalent. QIAstat-SARS can be used in all laboratories without PCR training, and has a quick turnaround time (approximately 67 minutes) for immediate testing when needed. POC testing for respiratory viruses using mPCR has been suggested to improve global patient management by enabling faster and more appropriate clinical decisions.[63,64] This results in benefits for patient classification, isolation management and overall patient assessment, which are crucial for COVID-19 management. Unfortunately, mPCR has not been confirmed by other independent studies, and its exact performance is not known.

Figure 3
Figure 3:
Schematic diagrams of representative nucleic acid testing methods. (A) The positive rates of reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR), loop-mediated isothermal amplification (LAMP), or computed tomography (CT) diagnoses collected from different studies. (B) The dynamics of RT-PCR testing monitored from the onset of symptoms. (C) The cumulative positive RT-PCR assay ratio from the second to the fifth test. Adapted from references [62]. (D–G) Magnetic immunocapture assay (G) and three rapid nucleic acid detection methods, including recombinase polymerase amplification (RPA) (D), LAMP (E), and CRISPR-based assay (F) that can be combined as RPA combined with loop-mediated isothermal amplification (RAMP), CRISPR-based detection, and immunocapture LAMP (IC-LAMP) to achieve a more efficient procedure for SARS-CoV-2 detection. Adapted from references [58,123,126].

Isothermal amplification

Isothermal amplification is a promising detection technique that does not require expensive thermocycling or professional skills. Loop-mediated isothermal amplification (LAMP) and recombinase polymerase amplification (RPA) combined with LAMP (RAMP) have been used to detect SARS-CoV-2 (Fig. 3D–E). LAMP is a simple and sensitive isothermal amplification technique that has been widely used for the detection of various pathogens because it is rapid (about 50 minutes) and is not dependent on precision equipment.[40,65,66] A strand displacement DNA polymerase, together with four (FIP, BIP, F3, B3) or six (FIP, BIP, F3, B3, LB, LF) primers that recognize distinct target sites of the DNA sequence are the core of LAMP assays. The amplification is divided into two stages, called the initial stage and the amplification cycle stage. At the initial stage, the F2 sequence of FIP first binds to the F2c region of the template and extends forward under the action of DNA polymerase strand displacement to initiate strand displacement synthesis. Then, the external primer (F3) binds to and extends the F2c region of the template, thereby replacing the complete FIP-linked complementary single strand. Because F1c and F1 are complementary structures, self-based pairs form a circular structure. Using this strand as a template, synthesis begins from BIP and B3 to form a single-strand with a dumbbell-shaped structure, which is the starting structure of the LAMP amplification cycle. During the amplification cycle, many single-strands with a dumbbell-shaped structure are formed as described in the initial stage. Furthermore, real-time detection of positive products can be judged by white precipitate or green fluorescence with the naked eye; therefore, LAMP is applicable for POC testing. LAMP has an average accuracy of 93.6% from 29 studies including about 2369 patients, which is significantly higher than RT-qPCR tests and symptom-based tests. Although LAMP has many advantages including high sensitivity and rapidity, any aerosol produced can lead to false positives. LAMP-based assays require enrichment of the virus and genomic extraction, which is time-consuming, uses dangerous reagents, and is technically demanding. However, LAMP can be combined with magnetic immunocapture (IC-LAMP), which efficiently enriches pathogen cells without the need for plasmid or genome extraction and has been widely used for the detection of various pathogenic microorganisms (Fig. 3G).[40,67,68] IC-LAMP uses the specificity of a monoclonal antibody and is more convenient, rapid, and sensitive than other detection methods. It should be able to meet the challenge of large-scale diagnosis of COVID-19, although this has not been confirmed. Excitingly, several other different combinations have been used for COVID-19 detection, including RPA combined with LAMP (RAMP) (Fig. 3D–E), and CRISPR-Cas12a,[69–71] 12b,[71] or 13[72,73] (Figure 3E-F). Furthermore, combined RT-LAMP-assisted nucleic acid detection shows good performance with high accuracy and low detection limit.

CRISPR-based assay

CRISPR is an effective gene-editing technique but, recently, its ability to detect nucleic acids has attracted attention because it has been applied to SARS-COV-2 detection. It has potential for efficient, simple, and reliable on-site diagnosis because CRISPR-based nucleic acid assays do not require precision equipment and takes less than 1hour. CRISPR detection systems consist of CRISPR nuclease, a guide RNA (gRNA), and a single-strand oligo reporter modified with fluorescein and a quencher at each terminus.[69] For specific detection of SAR-CoV-2, several specific domains of the SAR-CoV-2 RNA genome, including sites in N, E, S, RdRp, ORF1a, and ORF1b genes are chosen for amplification from extracted nucleic acid samples and are targeted by gRNA.[69,71,72,74,75] When the gRNA targets a SARS-CoV-2 sequence in the resulting amplicons, CRISPR nuclease specifically cleaves the target amplicons and simultaneously cleaves the oligo reporters nearby to free the quencher from the fluorescent molecule. Thus, this assay can be analyzed with a fluorescent reader or lateral flow strips.[69,71,72,74,75] For detection systems with lateral flow readout, the processed sample flows onto the strip and the uncleaved reporter oligos are captured on the control line, whereas the cleaved reporter molecules flow to the second detection line (test line), reflecting the existence of SAR-CoV-2 specific sequence.[69] To date, four CRISPR nucleases, including CRISPR-Cas12a, CRISPR-Cas12b, CRISPR-Cas13, and CRISPR-Cas3 have been reported for COVID-19 diagnosis.[69,71,72,74–76] The nucleic acid amplification technologies are available here mainly including RFA, recombinase-aided amplification (RAA), LAMP, PCR or RT-RFA, RT-RAA, RT-LAMP, RT-PCR, and the introduction of reverse transcription depends on the type of CRISPR nuclease.[69,71,72,74,75] Several studies have demonstrated a good overall agreement of CRISPR-based assays with the “gold standard” RT-PCR test; however, because these assessments were based on small sample sizes, the reliability of CRISPR-based assay requires further testing.[69,74] Furthermore, the reaction mixture may be contaminated by aerosols if it is exposed to the environment midway between amplification and CRISPR detection. Thus, Guo et al[71] proposed a tube with a special structure to keep both reactions in a single tube, which increases the possibility of deploying a CRISPR-based assay in areas at great risk of SAR-CoV-2 infection or with poor-resources.

Serological testing

RT-PCR or LAMP-based nucleic acid tests and other nucleic acid-based assays have provided promising results. However, the sensitivity of these methods depends directly on the viral load, which varies from patient to patient, resulting in decreased performance, especially 12 days after the onset of symptoms (Fig. 3B–C). Given the increasing pressure for large-scale screening in hospitals and communities, reliable and rapid serological diagnostic methods are urgently needed to screen COVID-19 patients and subjects without obvious symptoms. Much attention is now focused on serological tests (Fig. 4A), either in the format of enzyme-linked immunosorbent assays (ELISA) (Fig. 4B) or immunochromatographic lateral flow assays (ILFA) (Fig. 4C).[77,78] Viral protein antigens and antibodies are secreted into the serum in response to SARS-CoV-2 infection, which can be targeted and can provide a larger time window for indirect detection of SARS-CoV-2.

Figure 4
Figure 4:
Representative results of serological testing. (A) The dynamic changes of viral and antibody load in blood after SARS-CoV-2 infection. Reprinted with permission from reference [80]. Enzyme-linked immunosorbent assays (ELISA). (B) and immunochromatographic lateral flow assays (ILFA). (C) were commonly used for serological testing. (D) Comparison of the positive rate of antibodies detected by rN-based ELISA and rS-based ELISA. (E) The positive rates of immunoglobulin M (IgM), immunoglobulin G (IgG), IgM/IgG, and immunoglobulin A (IgA) diagnoses were collected from different studies. (F–I) Kinetics of IgM, IgG, IgM/IgG, and IgA of patients monitored from the onset of symptoms.

IgM/G-based testing

Immunoglobulin M (IgM) is the largest immunoglobulin and the first antibody to be produced in response to an infection. It is usually produced 5–7 days after antigen stimulation and accounts for about 1/10 of total serum antibody (Fig. 4A).[79,80] Therefore, detection of its concentration can be used for early diagnosis of infectious diseases. In contrast, immunoglobulin G (IgG) is the smallest immunoglobulin but accounts for the largest proportion in serum (approximately 3/4 of all antibodies), and is produced at 10 to 15 days after infection (Fig. 4A).[79] Notably, IgG is the only antibody that can pass through the placenta. Currently, ILFA and ELISA serological tests that target N protein, S protein or both have been used to diagnose COVID-19 (Fig. 4D).[81–86] Of note, the positive rate for S protein-based assays was better than that for N protein-based assays and the positive rate for combined IgM and IgG was better than that for single N protein or S protein-based assays (Fig. 4D). We have summarized the published IgM-based studies, which include approximately 1731 patients exhibiting some degree of clinical symptoms, and they have an average sensitivity of 63.6% (Fig. 4E). Compared with IgM-based assays, the sensitivity of IgG-based assays is higher with an average sensitivity of 67.9%, as summarized from 10 different studies that include 2852 patients (Fig. 4E). However, the average sensitivity of the combined IgM and IgG assay is significantly improved, up to 81.5%, which is better than the IgM or IgG assay alone (Fig. 4D–E). Therefore, we recommend using reliable IgG-IgM combined antibody detection kits for screening COVID-19 patients rather than individual IgG or IgM antibody kits. At the same time, the sensitivity of IgM or IgG, and the determination based on IgM and IgG was observed in the group of ≤7 days (average sensitivity is 21.7%) from the perspective of the kinetics of anti-SARS-CoV-2 IgM and anti-SARS-CoV-2 IgM in COVID-19 patients, confirming that some acutely sick patients presenting shortly after symptom onset should receive nucleic acid testing (Fig. 4F–H). In contrast, when the test sample was collected 8 days after a certain threshold of clinical symptoms, high sensitivity was observed with IgM or IgG, as well as IgM and IgG-based assays (average sensitivity of 84.3%) (Fig. 4F–H). Interestingly, the sensitivity of IgM-based assays was lower in the group having symptoms for ≥15-day than that in the group having symptoms for between 8 and 14 days, which may reflect that IgM antibodies have been converted to IgG antibodies (Fig. 4H). In summary, the rapid detection of antibodies may be useful and can be applied in remote areas where qPCR analysis cannot be performed.

IgA-based testing

Recently, IgA has been found in the serum of COVID-19 patients, and its level is significantly higher in severely ill patients than in mild or moderate cases.[81,87–89] IgA mainly exists in mucosal membrane tissues such as the digestive tract, respiratory tract, and urogenital system, accounting for 10–20% of total serum antibodies, second only to IgG.[90] SARS-CoV-2 has been described to enter the human body through the respiratory tract, oral mucosa, and conjunctival epithelium.[91–93] Mucosal surfaces are undoubtedly the first line of defense and IgA plays a key role in protecting against virus infection through the mucosa-associated lymphoid tissues.[91,92,94] However, only a few papers have reported the detection of SARS-CoV-2-specific IgA in serum.[87,89] As shown in Figure 4I, only one study by Padoan et al[89] has shown detailed dynamic test results from 67 COVID-19 patients with an average accuracy of 87.3%. Ma et al,[79] Guo et al,[87] and Paces et al[88] confirmed that specific IgA responses are stronger and more durable than IgM responses. In conclusion, IgA-based assays can be used for the rapid screening of SARS-CoV-2 carriers with or without symptoms in hospitals, clinics, and testing laboratories.

Imaging-based assays

Molecular imaging, defined as “non-invasive visualization, dynamic acquisition, and comprehensive reflection”, is widely used in the diagnosis of various diseases. In the early stages of the disease outbreak, owing to a lack of COVID-19-related diagnostic kits, computed tomography (CT) scans were used as the main clinical diagnosis tool. CT scans are non-invasive and can provide cross-sectional images. Chest CT plays an important role in the detection of COVID-19 and is a key supplement to nucleic acid testing, especially in the face of asymptomatic patients or patients with low viral load.[59,95] Many studies have reported CT manifestations in COVID-19 patients, but they are diverse because CT findings directly depend on the stage of infection after the onset of symptoms.[59,80,95] All studies have shown that in COVID-19 patients, typical imaging manifestation is opacity of the glass lens, especially in the surrounding and lower lobes, bilateral multi-leaflet lobules, and nodular consolidation areas.[96,97] Linear consolidations and other signs are observed very frequently, indicating that organizing pneumonia occurs a few days after the onset of disease. Several non-typical findings, including pleural effusion, masses, cavitations, and lymphadenopathies are rarely observed and be used for alternative diagnosis.[98,99] Previous studies[33,59,95,100,101] described dynamic changes in chest CT scans of COVID-19 patients at different times and revealed that the optimal period for CT scans is >13 days after the onset of clinical symptoms with the average sensitivity of 86–100%. However, because most COVID-19 imaging features overlap with other viral pneumonias, the specificity of CT scans is compromised, even as low as 25%. Another imaging method, positron-emission tomography (PET), plays an important role in assessing infections, monitoring treatment response, and helping to predict recovery time.[43] The combined use of PET and CT (PET/CT) has been used in multiple studies to diagnose patients with COVID-19. It can not only obtain anatomical structure information of a lesion but can also accurately reflect the physiological and metabolic state of the disease. PET/CT scans have higher sensitivity, but are not specific, and are challenging for the differential diagnosis of lung infections. More importantly, CT or PET are both device-dependent and can not be used for diagnosis in rare cases. Therefore, PET/CT imaging is unlikely to be routinely used in the diagnosis of COVID-19, but it is an important auxiliary tool, especially when the clinical symptoms are unclear and the differential diagnosis is challenging. However, patients with positive imaging results but negative RT-PCR results should be isolated and repeated RT-PCR or serological tests performed to prevent misdiagnosis. Additionally, owing to the fast-spread of COVID-19 pneumonia, radiologists are facing a tremendous workload. Thus, a computer-aided diagnostic tool is necessary to rapidly screen and assess COVID-19 pneumonia. Many tools based on deep learning or machine learning have been developed for chest CT images that can recognize typical lesion characteristics of COVID-19. Because the inputs to these tools are different, they could be used for lesion segmentation, COVID-19 diagnosis, and severity prediction, providing potential to increase the efficiency of COVID-19 diagnosis and to improve management.[102]

Other assay methods

Nanoparticle-based assay

Nanotechnology can also play an important role in controlling the spread of COVID-19 because it provides great potential for optimizing immunological and nucleic acid testing for POC diagnostics of SARS-CoV-2. In recent years, colloidal gold nanoparticles (AuNPs) have attracted much attention as qualitative labeling probes because of their exclusive properties, which enable the detection of proteins and nucleic acids via color change.[103,104] AuNP-based lateral flow immunochromatography provides a simple, timely, and on-site method to detect anti-SARS-CoV-2 IgG and IgM from human serum.[105,106] For specific detection, the SARS-CoV-2 nucleocapsid proteins are coated on the test line of a lateral flow strip for target capture, and the anti-human IgG/M is conjugated with AuNPs as the reporter, or these roles can be reversed.[104–106] However, because colloidal gold is relatively insensitive, Wang et al[104] introduced selenium nanoparticles as an alternative, which are more sensitive, economical and insensitive to electrolytes. Furthermore, Chen et al[107] proposed a semi-quantitative LFA method based on lanthanide-doped polystyrene nanoparticles (LNPs) to detect anti-SARS-CoV-2 IgG in human serum. LNPs can emit fluorescence at the excitation wavelength of 365 nm; thus, the ratio of the fluorescence peak area of the test line to the control line reflects the concentration of lgG. This allows identification of positive cases and monitoring of the progression of COVID-19. As for the application of nanoparticles in nucleic acid testing, Moitra et al[103] proposed a colorimetric assay based on AuNPs capped with thiol modified antisense oligonucleotides, simultaneously targeting two sites of the SARS-CoV-2 N gene. When there is a specific RNA sequence in the nucleic acid extraction, the AuNPs will agglomerate. RNaseH is then added, which causes visible precipitation so that positive cases can be identified within 10 minutes. Similarly, nanoparticle-based LFA is suitable for nucleic acid testing. For example, Zhu et al[108] have developed an AuNPs-based lateral flow biosensor coupled with multiplex RT-LAMP that amplified the ORFlab and N gene of SARS-CoV-2 using primers labeled with fluorescein/digoxin and biotin. Thus, mRT-LAMP produced numerous fluorescein-/digoxin- and biotin-amplicons, which could be captured with the test lines and AuNPs modified with matching anti-molecules. The total diagnostic test based on this approach can be completed within 1hour with high sensitivity and specificity, providing a promising tool for reliable, user-friendly POC diagnostics. In addition to virus detection, silver[109] iron oxide,[110] and lipid nanoparticles[111] are also promising tools to treat and control COVID-19 infection because of their antiviral properties or their ability to enhance drug intranasal or intravenous delivery. Cellular nanosponges, made of plasma membranes from human lung epithelial type II cells or human macrophages, have been reported as a broad-spectrum therapeutic method to neutralize SARS-CoV-2 or other viruses via the binding of specific surface protein receptors.[112]

Aptamer-based assay

Aptamers are single-stranded folded RNAs or DNAs that can detect a great variety of molecules with high affinity and specificity. Compared with antibody-based assays, aptamer-based detection is more flexible, less costly, more stable, and easier to produce.[113] Although aptamers have been widely used to detect and treat various infectious diseases, including viral infections, their application in SARS-CoV-2 is rarely reported. Owing to the sequence homology between the N proteins of SARS-CoV and SARS-CoV-2, the DNA aptamer for the N protein of SARS-CoV-2 is different from the aptamer that can specifically bind to the N protein of SARS-CoV, which permits a novel aptamer-based method for SARS-CoV-2 detection.[114] Song et al[115] used an ACE2 competition selection strategy and a machine learning screening algorithm to identify aptamers targeting the receptor-binding domain of the SARS-CoV-2 spike glycoprotein, which can prevent the binding of SARS-CoV-2 RBD to ACE2. This has potential for early detection of SARS-CoV-2.[116] However, SARS-CoV-2 detection by all the above-mentioned aptamers needs further investigation.

Conclusions

Nucleic acid-based tests (eg, RT-qPCR and RT-LAMP), imaging tests (eg, PET/CT), and serological testing (eg, IgM/G and IgA) have high diagnostic value in the different stages of COVID-19. The sensitivity of nucleic acid-based tests mainly depends on two aspects: one is the detection technology and the other is the viral load, which depends on the time from onset of the disease. The first 12 days after onset is the most reliable time for the detection of SARS-CoV-2 and from 12 days after the onset of symptoms, RT-PCR becomes unreliable.[117,118] The dynamic characteristics of LAMP-based detection are poorly defined; therefore, we do not know the exact sensitivity of this technique at different stages of the disease. However, we can infer LAMP results through the dynamic results of RT-PCR detection. Considering the differences in viral load among patients, negative test results from respiratory samples cannot exclude the disease. Moreover, incorrect sampling techniques and mutations in the viral genome are the main factors leading to negative results. Currently, imaging testing is particularly important because it can visualize and dynamically evaluate FDG-uptake patterns in other parts of the patient.[119] Moreover, PET/CT scans can evaluate the damage to other organs, monitor treatment response, and help in predicting recovery time, which is important for monitoring the progress of COVID-19. PET/CT scans are not suitable for routine COVID-19 management but are important for emergencies. However, for large-scale screening or screening in remote areas, nucleic acid-based detection or imaging tests seem impractical because of time constraints and the specialist equipment required. In contrast, serological tests, especially in the form of LFA, can be performed within 15 minutes. However, the cross-reaction of serum samples from the acute phase of different viral infections (particularly in the IgM portion) and the antibody response from different SARS-CoV-2 strains or genotypes should be considered to avoid false positives. Together, we comprehensively introduce the current developed methods for SARS-CoV-2 detection, analyze advantages and disadvantages, and provide the optium period of each diagnosis tool. However, there are still some limitations are should be raised, such as several novel diagnosis tools will developed in the futher that not be discussed in this review should be summarized. Although, some novel detection tools have been mentioned as soon as our possible, detail and comprehensively to disscuss the futher developed assays, which has been beyond the scope of our capabilities. This still needs more research to continue to report and update some newly developed detection methods, which is extremely important for human health.

Future perspectives

In the face of this pandemic, there is an urgent need for highly accurate detection methods that enable the quick implementation of control measures to limit the spread of SARS-CoV-2. A variety of detection methods have been developed and applied to diagnose COVID-19, which can be mainly divided into nucleic acid-based detection, imaging, and serological testing. All currently available methods have limitations owing to poor performance or dependence on specialized equipment. Therefore, it is strongly recommended to perform multiple tests on patients because it is difficult to accurately detect COVID-19 with single detection methods. Dynamic changes in the ability of RT-PCR to detect SARS-CoV-2 and the sensitivity of IgG/M detection mean that RT-PCR can provide higher accuracy in the early stage of the disease, while the accuracy of IgG/M tests increases from 15 days after the onset of symptoms and reaches peak accuracy from 21–25 days, with the peak level being maintained for days 31–41. Therefore, IgG is a powerful diagnostic marker at a later stage of the disease. Taken together, the combined use of RT-PCR and IgG/M assays will be more reliable than a single method. However, for remote areas without PCR equipment, the combination of IC-RT-LAMP and IgG/M assays can achieve POC diagnosis with higher sensitivity compared with a combination of RT-PCR or RT-LAMP and IgG/M. IgA, as a recently reported biomarker, provides encouraging results, which are comparable to IgG/M assays given the dynamic process of sensitivity. Thus, the combination of IgM, IgG, and IgA in one LFA is preferable than any one or two of these three, especially in emergencies that require large-scale population screening. The specificity and sensitivity of two or more biomarkers combined are higher than that of single assays, but few studies have explored this approach.

In addition to the above methods, several novel sensors should be developed, such as electrochemical biosensors,[120] optical biosensors,[121–123] plasmonic photothermal biosensors,[124] and nanoparticle-based biosensors.[67,123] These sensors are powerful technology, enabling real-time in situ detection of biomarkers with high spatial and temporal resolution, as indicated by low detection limits.[125] In other diagnostic fields, different kinds of biosensors should not be used for isolation tests, so there is an urgent need to rationally combine these biosensors to maximize their benefits. There have been encouraging results from recently developed biosensors that combined nanoparticles with optical biosensors (Fig. 5A), electrochemical biosensors (Fig. 5B), nanoparticles biosensors (Fig. 5C), and paper biosensors combined with a smartphone (Fig. 5D) to realize rapid detection of different pathogenic microorganisms.[120–127].

Figure 5
Figure 5:
Schematic diagrams of several novel sensors. (A) Ultrasensitive detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) using surface-enhanced Raman scattering-based lateral flow immunoassay strips. Adapted from references [123,126]. (B) Electrical pulse-induced electrochemical biosensor for SARS-CoV-2 detection. Adapted from reference [127]. (C) Visual and point-of-care (POC) detection of SARS-CoV-2 using the combination of nanoparticle-based assay and optical biosensors. Adapted from references [123,126]. (D) Dual-functional plasmonic photothermal biosensors for detection of SARS-CoV-2. Adapted from reference [124]. (E) Integrating chemiluminescence detection on smartphones. Adapted from reference [131]. (F) Aptamers-based sensors for detection or treatment of SARS-CoV-2. Adapted from reference [116].

Many diseases, including COVID-19, are usually caused by complex interactions that commonly present more than one biomarker. Thus, the development of probes simultaneously targeting two or more biomarkers would be extremely valuable for improving sensitivity and specificity. Bispecific probes or multi-targeted probes are expected to recognize different epitopes with high binding affinity and specificity. Several bispecific probes used for cancer diagnosis have yielded promising results over single-target probes, but currently bispecific probes or multi-targeted probes have not been developed or implemented for COVID-19.

The use of sensitive probes and accurate diagnostic tools will not be sufficient to prevent COVID-19 from continuing to spread. How the pandemic can be controlled has received tremendous attention from all over the world, and two factors have been highlighted. One is to monitor patients, and another is to share epidemiological data.[128,129] We can achieve the former by the above methods, but in the latter case, we need smartphones or computers to perform COVID-19 surveillance via the Internet. Sensitive detection methods together with accurately confirmed cases can speed up the management of the disease (Fig. 5E and F), while insufficient communication and reporting will enhance the global spread of COVID-19.[128,129] The SARS outbreak in Canada was a case where the disease was not well managed because there was little communication between regional medical institutions.[130] More importantly, the use of smartphones in conjunction with POC tests can instantly record the number of diagnosed patients in different regions and can accurately reflect the trajectory of diagnosed patients. This can help determine a propagation model and prevent the cross-infection of healthy people. This technique has been used to treat patients with mental health problems, but it has not been developed to diagnose COVID-19.[131]

Acknowledgments

None.

Author contributions

HL coordinated the writing of the review, provided writing guidance and manuscript revision. LZ contributed to writing the first draft. XL and YL contributed to table preparation. HZ, WQ, and BW provided valuable suggestions to manuscript revisions. All authors reviewed and approved the final manuscript.

Financial support

This study was supported by the Science Fund for Creative Research Groups of the National Natural Science Foundation of China (No. 61721092), grants from the National Natural Science Foundation of China (No. 81971025), and the Startup Fund of Huazhong University of Science and Technology of China.

Conflicts of interest

The authors have no conflicts of interest to disclose..

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Keywords:

COVID-19; imaging tests; nucleic acid tests; point-of-care diagnostics; serological tests

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