Evaluation of Diagnostic Modalities for SARS-Cov-2: A Review Study

Background and aims: In late December 2019, a cluster of progressive pneumonia-like respiratory syndromes broke out in Wuhan, China. As the number of cases continued to rise, the 2019 coronavirus disease (COVID-19) has been declared a global public health emergency. The causative agent, i.e., SARS-CoV-2, is a highly contagious strain, which has resulted in the rapid worldwide outbreak of COVID-19. COVID-19 is an overwhelmingly transmissible disease that requires early and accurate diagnosis for proper and timely treatment of suspected cases. Materials and Methods: In order to access the scientific documentation and evidence related to the subject published during 2019 to 2021, English keywords including “COVID-19”, “SARS-CoV-2”, “Diagnosis”, “Immunoglobulin G (IgG)”, “Immunoglobulin M (IgM)”, and ‘Polymerase Chain Reaction (PCR)” were searched in Medline, PubMed, and Google Scholar databases and Persian versions of these keywords were also looked for in Jihad-e Daneshgahi’s Scientific Information Database (SID) and Iranian Journals


Introduction
A class of enveloped, positive-sense single-stranded RNA viruses, namely coronaviruses (CoV), which can be commonly isolated from different animal species, cause adverse effects in the respiratory, neurological, and enterohepatic systems of human beings. CoVs are recognized as important pathogens due mostly to their widespread global distribution and high infectivity. 1 During 2002-2003, the severe acute respiratory syndrome coronavirus (SARS-CoV) led to an outbreak in Guangdong, China, which resulted in the infection of 8000 patients and 774 cases of death in 37 countries. During 2012-2013, a novel strain of CoVs was first reported in Saudi Arabia, which was duly named Middle East respiratory syndrome coronavirus (MERS-CoV). This new strain resulted in fewer confirmed cases, albeit higher fatalities, with 2,494 confirmed cases and 858 deaths. 2,3 The current SARS-CoV-2 outbreak in 2020 initially presented with clinical signs of idiopathic pneumonia in Wuhan, China. After a while, nucleic acid sequencing and laboratory findings detected the culprit as a new strain of CoV. 4 As expected, several similarities and discrepancies have been reported in the incidence, clinical signs, and the treatment for SARS, MERS, and COVID-19. 3,5 Unlike its predecessors, SARS-CoV-2 is spreading at a much faster rate and can even result in asymptomatic infections. 6 According to R0 values, COVID-19 is more infectious than SARS and MERS. Generally, the aged and/or immunocompromised individuals are at extreme risk of developing severe disease. Despite the odds, no medication has been approved for COVID-19. 1 Some clinical features of SARS-CoV, MERS-CoV, and SARS-CoV-2 are summarized in Table 1.
From a biological viewpoint, high incidence of lymphopenia among patients with COVID-19 suggests that the virus might affect the lymphocytes, especially T-cells, in a manner similar to SARS-CoV. 7 SARS-CoV-2 particles are thought to spread through the respiratory epithelium by means of angiotensin-converting enzyme 2 (ACE2) receptors that are expressed on the ciliated bronchial epithelial cells. A massive infection can elicit generalized immune reactions like cytokine storm and induce hazardous alterations in the count and function of peripheral white blood cells. 3   is not restricted to one organ in the body but rather manifests with a broad spectrum of clinical features, ranging from septic shock and multi-organ dysfunction to no symptoms at all. Based on the severity of the disease, COVID-19 is commonly classified 8 as mild, moderate, severe, and critical types. Clinical features mostly include low-grade fever, shortness of breath, dry cough, lymphopenia, fatigue, diarrhea, and ground-glass opacifications on the chest computed tomography (CT). 9 Similar to SARS, the incubation period of COVID-19 is approximately five days (2-14 days). 10 The diagnostic methods for accurate detection of COVID-19 have changed over time with increasing awareness of the pathophysiology of this disease. Today, serum immunoglobulin assays, nucleic acid sequencing, and chest imaging are recognized as the standard diagnostic methods. 11 The definite diagnosis of COVID-19 is made once the oropharyngeal swab collected from a suspected case is confirmed to contain viral nucleic acid, 8,12 which is complemented by additional tests such as measuring the serum levels of Immunoglobulin M (IgM) and Immunoglobulin G (IgG) against the virus. 13 The serologic method has several advantages because it is simple to perform and yields a high sensitivity. 11 Quantitative realtime reverse transcriptase-polymerase chain reaction assay, or simply quantitative reverse transcription polymerase chain reaction (RT-qPCR), has been extensively used for detecting SARS-CoV-2 RNA in the respiratory secretions of suspected cases. 6 Based on available kits, this method offers a sensitivity of 45%-60%; therefore, repeated testing is recommended in the early stages of the disease. 14 CT is another diagnostic modality that can reveal the pathologic changes of lungs associated with COVID-19. CT as a widely available and cost-effective method can offer high sensitivity when performed and interpreted by experienced radiologists. 15,16 Considering the prominent role of early diagnosis in the management of COVID-19, in this paper, we will present an in-depth review of COVID-19 diagnosis through IgG/M monitoring, PCR, and CT imaging.

COVID-19 Epidemiology
As reported earlier, the majority of people infected with COVID-19 were males with a mean age of 59 years (15-89 years). Besides, the mean incubation period of SARS-CoV-2 is approximately 4.8±2.6 days, ranging from 2 to 11 days. 17 There is not much data available concerning the typical chest CT imaging patterns of children infected with SARS-CoV-2. 18 As of February 15, 2020, COVID-19 pneumonia has resulted in a mortality rate of around 2%. 19 On February 12, an abrupt rise in new SARS-CoV-2 cases was due to a modified diagnosis method based on the 5th edition of national treatment guideline, a combination of SARS-CoV-2 nucleic acid test and clinical COVID-19 features. 6 Not surprisingly, COVID-19 cases who already had underlying diseases were more susceptible to severe disease and mortality. 20

Biology
Coronaviruses belong to CoVs; order Nidovirales, family Coronaviridae, subfamily Orthocoronavirinae, which include four genera: alpha coronavirus, beta coronavirus, delta coronavirus, gamma coronavirus, as well as multiple subgenera and species. Nidovirales are enveloped, nonsegmented, positive-sense RNA viruses with a wellconserved genome that encodes numerous non-structural genes. Moreover, these viruses are characterized by multiple unusual enzymatic activities encoded within the replicase-transcriptase polyprotein, 3' nested sub-genomic mRNAs. Coronavirus virions are spherical, with diameters ranging between 100 and 160 nm. 21 These viruses have been isolated from bats and show high sequence homology with isolates from humans, suggesting that bats are natural hosts and reservoirs of this virus. 3 HCoV-19 has a spike protein (S protein), an envelope protein (E), a membrane protein (M), and a nucleocapsid protein (N protein). Among these proteins, the N-protein is the most abundant one, which is relatively conserved among different coronaviruses; hence, it is frequently used as a diagnostic antigen. 22 With regard to genetic properties, coronavirus is comprised of two strains from Alphacoronavirus (HCoV-229E and HKUNL63) and four from Betacoronavirus subfamily (HCoV-OC43, HCoV-HKU1, SARS-CoV, and MERS-CoV). 23 As long single-stranded RNA viruses, human coronaviruses appear to spread via human to human. The genome of human SARS-CoV-2 matched the typical CoVs and consisted of more than ten open reading frames (ORFs). The first ORF, ORF1a/b, processes into two large viral polyproteins. The other ORFs of SARS-CoV-2 encode the main structural proteins. 24 It is believed that SARS-CoV-2 spreads from person to person through respiratory droplets. SARS-CoV-2 can also spread via other routes of infection (i.e., close contact) if the virus persists on surfaces; however, it is not clear whether these routes are required for the distribution of the virus. SARS-CoV-2 binds to ACE2 receptors on type II pneumocystis 25 during entry into cells. Several pieces of evidence suggest that SARS-CoV-2 might change over time, or simultaneously, there may be more than one strain of the virus in circulation. 26

Clinical Manifestations
Basically, Human COVs present with either pulmonary or extrapulmonary signs. COVID-19 cases are clinically categorized into four types: mild, normal, severe, and critical, according to "Diagnosis and Treatment Plan for Novel Coronavirus Pneumonia" (Fifth Edition). 27 The majority of COVID-19 cases present with early symptoms of headache, high fever (39°C), and abnormal respiratory features, including cough and changes in breathing pattern. The virus passes through the nasal and larynx mucous membranes, and at that point, it enters the lungs via respiratory tract and subsequently spreads to peripheral blood to cause viremia. Then, SARS-CoV-2 adheres to ACE2 receptors that are highly expressed in various organs (e.g., lungs, heart, kidneys, and gastrointestinal tract). The principal pathogenesis of SARS-CoV-2 infection is viremia and severe pneumonia combined with acute cardiac injury. 17 Besides early symptoms, digestive manifestations were widely reported in COVID-19 patients with mild disease severity. 28 In more severe cases, SARS-CoV-2 could cause kidney failure and even death. 29 In addition to respiratory symptoms, fever, cough, diarrhea, and fatigue were widely seen in COVID-19 patients, whereas the chest pain and muscle ache were less commonly reported. 30 Although fever, headache, and myalgia are often reported in COVID-19 patients after 4 to 5 days, respiratory symptoms develop several days after the onset of initial symptoms. 31 In an immunocompetent individual, these COVs could clinically manifest with self-limiting upper respiratory infections and common colds, whereas in an elderly and immunocompromised host, they can severely infect the lower respiratory tract. SARS-CoV-2-positive RT-PCR results have also been reported for asymptomatic patients from Croatia and Thailand. 32 Similar to other coronavirus pneumonias like SARS that is caused by a coronavirus as well as MERS, COVID-19 can lead to acute respiratory distress syndrome (ARDS). 33 Estimated frequency of symptoms observed to date indicates that 50-80% of COVID-19 patients present with cough, 69.6% have fatigue, 20%-40% experience dyspnea, and most importantly, 85% of the cases show fever. 34 In a systematic review study, it has been shown that the most frequent symptoms of COVID-19 patients are fever, cough, and dyspnea; however, some young infected cases presented with no symptoms. 35 In another systematic review and meta-analysis including 656 patients, fever (88.7%), cough (57.6%), and dyspnea (45.6%) were the most commonly reported symptoms. 3 Table 2 summarizes some case reports on the clinical presentation of COVID-19.

General Laboratory Findings
In patients with COVID-19, elevated prothrombin time, lactate dehydrogenase (LDH), D-dimer, alanine aminotransferase (ALT), amylase, C-reactive protein (CRP), total bilirubin and BUN/creatinine, creatine kinase (CK), ferritin, and normal or low procalcitonin have been frequently reported laboratory findings. 26 Basically, the immune response of T helper (Th) cells plays a pivotal role in adaptive immunity following the recognition of viruses. After detection of the virus by antigen-presenting cells (APC) such as dendritic cells (DCs), these cells release cytokines and generate a microenvironment that is necessary for direct T-cell responses. Moreover, the production of virus-specific antibodies is mediated by CD4 + T-cells, which activate the T-dependent B-cells. 36,37 Besides, significant changes in numerous serum cytokines were found in patients with COVID-19 infection. During the initial stages of COVID-19 infection, a significant decrease in CD4 and CD8 lymphocytes can also be observed. Increased levels of interleukin IL-2, IL-7, IL-10, granulocyte colonystimulating factor) GCSF), interferon gamma-induced protein 10 (IP-10(, MCPI (monocyte chemotactic protein 1), MIPIA (macrophage inflammatory protein alpha), and TNF-α (tumor necrosis factor-a) have been reported in patients in the intensive care unit as well. 1 In addition, leukopenia, lymphopenia (+80%), and thrombocytopenia have been seen in patients with COVID-19. 26 In a systematic review and meta-analysis by Zhao et al including 19 related studies, elevated CRP, D-dimer, and LDH, as well as decreased blood platelet and lymphocytes counts were markedly correlated to severe COVID-19. Similarly, increased CRP, myoglobin, aspartate aminotransferase (AST), and ferritin, and decreased hemoglobin were noted in Wuhan patients relative to those outside Wuhan. 20 In agreement with these findings, another meta-analysis has reported that the most common abnormal laboratory changes in COVID-19 patients include lymphopenia, elevated CRP, as well as increased AST and LDH. In this case, higher levels of angiotensin II also contribute to acute lung injury. Results of the study conducted by Rodriguez-Morales et al revealed that diminished albumin, increased CRP and LDH, lymphopenia, as well as higher ESR levels were the most common laboratory findings observed in a total of 2,874 positive COVID-19 cases. 3 In the meantime, non-survivors are assumed to present with higher D-dimer, as well as longer PT and PTT associated with lower fibrinogen and antithrombin levels. 35 As shown in Table 3, there has been no consistent change in CRP levels among case reports as mildly increased CRP level has been observed in some case reports. 38 At the same time, a lower mean concentration of CRP was noted in asymptomatic patients confirmed with COVID-19. 19

IgG/IgM Titers
Acute antibody responses to SARS-CoV-2 have been reported earlier in several papers. [39][40][41] Therefore, an immediate, simple, and precise diagnosis of suspected COVID-19 is essential for monitoring, management, and treatment of the patient. Although several methods are used in clinical practice, the diagnostic value of IgM and IgG has been relatively overlooked. In a related study, within 19 days after the onset of symptoms, almost all the patients tested positive for antiviral IgG, followed by seroconversion for IgG and IgM. The titers of both immunoglobulins plateaued within few days after seroconversion. 42 Rapid detection of SARS-CoV-2-specific IgM and IgG was shown to be associated with high specificity (100% for IgM and 99.2% for IgG), demonstrating that this assay is beneficial for determining previous virus exposure. Nevertheless, negative results may be unreliable during the first weeks after SARS-CoV-2 exposure. 43 Rapid and simple point-of-care lateral flow immunoassays have been lately practiced for simultaneous detection of IgM and IgG antibodies against SARS-CoV-2 in symptomatic or asymptomatic humans infected with the virus at different stages. 44 Using a chemiluminescenceimmunoassay method, Lin et al found that IgG testing was more reliable than IgM for identifying SARS-CoV-2 infections from the confirmed patients, reaching 82.28% sensitivity and 97.5% specificity. 23 In a study by Jia et al on 57 suspected COVID-19 patients, the positive rate for single detection was 60.61% (for IgM) and 5.45% (for IgG). However, the positive diagnostic rate of the combination of IgM and IgG detection was 72.73% in 33 patients with COVID-19 negative RT_PCT results and it was 87.50% in 24 patients with COVID-19 negative RT-PCT results. 11 Nucleic Acid Detection: PCR Nowadays, precise genome detection serves a substantial function in COVID-19 diagnosis. In this regard, RT-PCR is the primary technique for detection of SARS-CoV-2 nucleic acid. However, RT-PCR is a time-and laborconsuming method for monitoring the increasing number of suspected individuals and asymptomatic patients. 45 In January 2020, the first protocols of RT-PCR assays for specific amplification of SARS-CoV-2 RNA-dependent Frequently observed symptoms at onset were fever (73%) and dry cough (59%), dizziness (2%), diarrhea (4%), vomiting (5%), headache (6%), and generalized weakness (9%).
Abbreviations: HRCT, high-resolution computed tomography; NGS, next-generation sequencing; RT-PCR, reverse transcription polymerase chain reaction Evaluation of Diagnostic Modalities for SARS-Cov-2 RNA polymerase (RdRp), envelope (E), and nucleocapsid (N) genes were released. 46 Although RT-PCR is the gold standard for COVID-19 diagnosis, this technique lacks enough sensitivity for the detection of low viral load present in test specimens. In many areas, RT-PCR kits are in short supply. 16 Although RT-PCR is not easily performed with the global shortage of testing kits, RT-PCR kits are variable and yield relatively low sensitivity; therefore, repeated testing may be necessary to ensure diagnosis, particularly early in the course of the infection, 14 reflecting the experience with MERS-CoV. In a study conducted by Alfaraj et al on 336 patients infected with MERS-CoV, approximately 89% of patients were positive for MERS-CoV after 1 swab, while 96.5% had positive results after 2 consecutive swabs and 97.6% had positive results after 3 swabs. 47 A large body of evidence has recently shown the limitations of RT-PCR. First, more than 7 SARS-CoV-2 nucleic acid PCR tests are already available. 48 Second, it has been demonstrated that the samples taken from the upper respiratory tract show their highest viral loads almost three days after the onset of clinical symptoms. It has also been shown that nasal samples have higher viral loads than throat samples. 49 Zhang et al reported low sensitivity (30%-50%) of RT-PCR in single upper respiratory specimen testing. 48 Multiple factors have been suggested to explain the false-negative rate of this method. For instance, the variation of viral RNA sequences might affect the results of RT-qPCR. Concerning sampling procedures, it has been urgently suggested that sputum and bronchoalveolar lavage fluid collected from the lower respiratory tract of patients must be used for testing SARS-CoV-2 infection. 50 Results of the study conducted by Long et al revealed that the sensitivity of initial RT-PCR was only 83.3%, and initially missed cases became positive for SARS-CoV-2 in the third round of RT-PCR tests after 8 days. This suggests that RT-PCR assay should be repeated in patients with typical chest imaging findings and initial negative RT-PCR results. 51 On the other hand, RT-PCR is a timeconsuming technique since it often requires 5 to 6 hours, while chest imaging results can be obtained over a shorter period of time. Ai et al proposed a combinative approach of RT-PCR, CRISPR-based assay, and metagenomic nextgeneration sequencing (mNGS) for COVID-19 diagnosis. According to their findings, it might be advantageous to combine such molecular diagnostic techniques to rule out other pathogens for an accurate diagnosis of SARS-CoV-2. 52 Chest Imaging: CT Besides RT-PCR, initial attempts focused on the use of chest CT in the forefront of COVID-19 diagnosis. 53 Imaging can show the number, size (patchy, large block, nodular, lumpy, etc), density (ground glass density, paving stones-like change, consolidation, fibrosis, etc.), and distribution of these lesions. 54 Recent studies have shown that approximately 96% of COVID-19 cases manifest with chest CT abnormalities, including multiple bilateral and peripheral ground-glass opacity (GGO) with or without consolidation, 55 frequently with a rounded spherical morphology and a peripheral lung distribution. 56 Previously, it was observed that lung lesions of patients infected with SARS progressed rapidly, producing "white lungs" characterized by diffuse infiltration of both lungs. On the contrary, lung lesions in patients with COVID-19 infection are comparably mild and might be wrongly diagnosed as common viral infections or bronchopneumonia. 57 There were apparent similarities between chest CT imaging patterns of COVID-19 and SARS infections. To name a few, both subpleural GGO and consolidations are prevalent and mainly observed in bilateral subpleural areas while other features such as cavities, pleural effusion, and enlarged lymph nodes are hardly seen. However, within the absorption phase of pneumonia, more severe interstitial fibrosis was discovered in patients with SARS. 27 Upon performing a systematic review of case reports and case series, Tahvildari et al reported that CT images of 80% of confirmed COVID-19 patients displayed GGO patterns, while 69% of patients presented with bilateral lung involvement. 58 Another systematic review and metaanalysis including a total of 2,874 positive COVID-19 patients showed that 68.5% of lung lesions were GGOs, 25% of the lesions were unilateral and 72.9% of them were bilateral. 3 Table 4 represents some case reports performed on COVID-19 patients focusing on their CT findings.
Multiple reports published to date have outlined the higher sensitivity of chest imaging in early detection CT scans showed ground-glass opacities (75%), increased crazypaving pattern (53%) and total CT score after 8 days; consolidation (91%) after 9-13 days and a peak in the total CT score after 14 days followed by a decrease in the total CT score without crazy-paving pattern. of COVID-19 in comparison with RT-PCR. This inconsistency resulted in suggesting that a CT scan could be more sensitive than RT-PCR. 14 Compared to RT-PCR, chest CT imaging might have some advantages. First of all, a CT scan can detect early lung lesions, and if performed by expert radiologists, it is able to yield high sensitivity. Besides, imaging techniques are widely available and economical. Jin et al developed an efficient artificial intelligence system for rapid diagnosis of COVID-19 with an accuracy as good as trained radiologists. 16 This deep convolutional neural network showed an accuracy of 94.98%, a sensitivity of 94.06%, and a specificity of 95.47% based on an independent dataset of 1255 cases. Further tests revealed that only one radiologist was marginally more accurate than the developed diagnostic system. 16 Long et al reported the CT sensitivity of 97.2%, suggesting that this technique presents markedly higher sensitivity than initial RT-PCR (83.3%). Based on their observations, results of CT imaging would be more reliable since RT-PCR may produce initial false-negative results. 51 As recommended by Wang et al, combining RT-PCR testing with clinical features should be used for accurate diagnosis of COVID-19 in order to facilitate the management of COVID-19 outbreak because some CT imaging features, including GGO and patchy shadows in bilateral lungs, have been reported in many "suspected" cases with negative RT-PCR results for the virus, making lung CT a non-specific diagnostic tool when used alone. 50

Conclusion
COVID-19, the most catastrophic epidemic in recent memory, is spreading globally at an overwhelming rate. In the absence of specific medications or an effective vaccine, early diagnosis constitutes to be the pillar upon which all other therapeutic strategies lie. Besides timely diagnosis, factors associated with the evolution of the virus and the outcomes of treatment must be carefully addressed and investigated when a new infectious disease has emerged. Serum antibody assays, PCR, and CT are invaluable assets at our disposal that can greatly help reveal the presence and severity of the disease in suspected and confirmed cases, respectively. Early diagnosis matters in that it not only contributes to the prevention of further transmission of the virus by asymptomatic carriers but also paves the way for clinicians to accurately choose the best therapeutic approach depending on the status of the patients.

Ethical Approval
Not applicable.

Conflict of Interest Disclosures
None declared.

Funding
This paper received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.