Diagnostic approaches in COVID-19: clinical updatesPurva Asrania Division of Biochemistry, Indian Agricultural Research Institute, New Delhi, IndiaView further author information, Mathew Suji Eapenb Respiratory Translational Research Group, Department of Laboratory Medicine, School of Health Sciences, College of Health and Medicine, University of Tasmania, Launceston, AustraliaView further author information, Collin Chiab Respiratory Translational Research Group, Department of Laboratory Medicine, School of Health Sciences, College of Health and Medicine, University of Tasmania, Launceston, Australia;c Department of Respiratory Medicine, Launceston General Hospital, Launceston, AustraliaView further author information, Greg Haugb Respiratory Translational Research Group, Department of Laboratory Medicine, School of Health Sciences, College of Health and Medicine, University of Tasmania, Launceston, Australia;c Department of Respiratory Medicine, Launceston General Hospital, Launceston, AustraliaView further author information, Heinrich C. Weberb Respiratory Translational Research Group, Department of Laboratory Medicine, School of Health Sciences, College of Health and Medicine, University of Tasmania, Launceston, Australia;d Department of Respiratory Medicine, Tasmanian Health Services (THS), North West Hospital, Burnie, Australiahttps://orcid.org/0000-0003-0290-0355View further author information, Md. Imtaiyaz Hassane Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia, New Delhi, Indiahttps://orcid.org/0000-0002-3663-4940View further author information Sukhwinder Singh Sohalb Respiratory Translational Research Group, Department of Laboratory Medicine, School of Health Sciences, College of Health and Medicine, University of Tasmania, Launceston, AustraliaCorrespondenceSukhwinder.Sohal@utas.edu.auView further author information show allReceived 21 Jul 2020Accepted 11 Sep 2020Accepted author version posted online: 12 Sep 2020Published online: 30 Sep 20201. IntroductionMicroorganisms have great potential to infect and destruct human life [1]. They are far more lethal to any other weapon when it comes to mass destruction [2]. From ancient times, various bacterial and viral infections have been emerged and associated with pandemics owning to their higher transmission rate, difficulty in diagnosis, and lack of advanced healthcare systems [3,4]. In recent times, the world is facing a similar economic, social, and health crisis by a virus that has emerged in Wuhan city of China in late 2019 [5].1.1. SARS-CoV-2 and its featuresSevere acute respiratory syndrome (SARS-CoV-2) is an enveloped, positive-sense, and single-stranded RNA (ssRNA) virus, belonging to the β-CoV family [6]. The molecular basis of the pathogenesis of SARS-CoV-2 has been established [7]. Bat CoV sharing 96% sequence similarity to the SARS-CoV-2, suggested as most likely reservoirs [8]. However, the receptor-binding domain (RBD) in spike region of virus shares 97.4% genomic similarity to pangolins, indicating human transmission might have happened through coming in contact with them rather than bats [9,10]. SARS-CoV-2 causes respiratory syndrome and severe pneumonia [5]. These physiological conditions are a result of inflammatory cytokines produced through early virus replication [11]. Virus entry and replication trigger apoptosis/pyroptosis of macrophages causing the release of proinflammatory cytokines such as IL-1β and TNF-α [12,13]. These cytokines further mediate two types of responses: ACE2 downregulation and shedding resulting in loss of Renin-angiotensin system (RAS) [14–16] and other being the activation of TH17 cells that are responsible for triggering other proinflammatory cytokines (IL-17, IL-21, IL-22, and GM-CSF) causing vascular permeability and leakage [17–19]. This cytokine storm is similar to that as observed during SARS-CoV infection and hence causes inflammation and lung injuries [20].1.2. Lockdown effects on physical and physiological well-beingCOVID-19 led to the entire population of the world to face a complete lockdown and in following the social distancing and quarantine rules imposed by the governments of the respective countries [21]. The changing daily habits and forced period of inactivity had a huge impact on physiological well-being of the individuals [22]. Mental health issues such as depression, fear, panic, frustration, anger, and post-traumatic stress were on the rise and similarly the risk to physical health associated with the sedentary life style increased considerably [23]. Various studies emphasized on importance of physical exercise in preventing the infections and boosting the levels of immunity [24]. It is being emphasized in various studies that the period of emotional and mental stress would reduce by engaging in regular work-out and eventually would low down the risk of infections associated with the moments of physical inactivity [22]. Similar such effects of lockdown may be observed in the lifestyle and career opportunities of the sportspersons [25]. Following a long period of rest and an unexpected stop to the training sessions of the athelets, quarantine period may cause decline in their endurance capacity, muscle strength, and maximum oxygen consumption [26]. This may also predispose them to different structural alterations in the organizations of tissues and their associated mechanical properties [27]. This generates a health concern to their careers and for the entire sports sector around the world [25]. An interesting study was reported by Giustino and his team where they identified the influence of quarantine period on decreasing physical activity among the persons with different demographic and anthropometric indices such as body mass index (BMI) in Sicilia valley [28]. They found that the quarantine had a negative influence on the practising of physical activity and males, young adults and overweight individuals were in the vulnerable categories [28]. Similar kind of studies were performed across different parts of the world. For example Canadians showed practice of physical activity had a direct correlation to the well-being of the individuals. Those who were engaged more in outdoor physical activity had lower anxiety than the others who were spending time indoors [29]. A study conducted on Italians supported the same fact indicating the quarantine period had left a negative impact in terms of reduced physical activity and thus, affecting the physiological and physical well-being of the individuals [30]. People of all age groups were affected by this pandemic and the only way to restore the normal life-balance relies in praticising the physical activities as a non-pharmacological and preventive strategy to physical and mental health in the periods of forced rest [31].1.3. Diagnostic approaches of SARS-COV-2Various diagnostic approaches such as molecular diagnosis that rely upon the identification and amplification of viral RNA sequence through real-time reverse transcriptase-polymerase chain reaction (rRT-PCR) [32], a serological diagnosis that uses antibody testing for identifying the infection in patients, point of care (POC) devices, radiology-based detection to look for clinical changes in the suspected patients, and viral cell culture techniques constitutes an important part of this review [33,34]. The aim of this study is to provide an overview of current status of diagnostic strategies, their molecular basis, mechanisms of action, and the latest innovations in the field of diagnosis for controlling the pandemic. Secondly, this narrative and critical review is prepared in a way that it updates the clinicals and researchers with the latest immunodiagnostic and molecular diagnostic POC kits and devices for emergency use authorization by US Food and Drug Administration (FDA). (https://www.fda.gov/medical-devices/emergency-situations-medical-devices/emergency-use-authorizations). Thirdly, to inculcate an innovatory thinking in researchers about designing of new and efficient diagnostic strategies for future is also one of the prime objective of this study. The majority of the literature cited in this paper is collected from guidelines on protocols and other considerations on diagnostic strategies of COVID-19 issued by World Health Organization (WHO), Center for disease control and prevention (CDC) and FDA.2. rRT-PCR based molecular detectionThe basis of molecular diagnostics depends upon the identification and amplification of viral genetic material from the specimen of suspected individuals. This identification is done by rRT-PCR [35]. A set of the protocol has been issued by the CDC and WHO, which involves the designing of primers that binds with the specific areas of the viral genome and then enables their amplification [36]. When the viral RNA and nucleocapsid gene of SARS-CoV-2 were identified they were used as a template for designing of oligonucleotide primers and dual hydrolysis probe for in vitro qualitative detection of RNA using the rRT-PCR technique (Figure 1).Diagnostic approaches in COVID-19: clinical updatesAll authorsPurva Asrani, Mathew Suji Eapen, Collin Chia, Greg Haug, Heinrich C. Weber , Md. Imtaiyaz HassanSukhwinder Singh Sohalhttps://doi.org/10.1080/17476348.2021.1823833Published online:30 September 2020Figure 1. rRT-PCR-based molecular detection of SARS-CoV-2 in respiratory specimens. The test is based upon the separation of reporter and quencher dye by 5` nuclease activity of Taq polymerase. As the amplification progresses in the PCR machine, cleaving of reporter dyes generates fluorescence whose intensity is measured via a PCR software. Various POC kits and devices works on this principle of molecular detectionDisplay full sizeFigure 1. rRT-PCR-based molecular detection of SARS-CoV-2 in respiratory specimens. The test is based upon the separation of reporter and quencher dye by 5` nuclease activity of Taq polymerase. As the amplification progresses in the PCR machine, cleaving of reporter dyes generates fluorescence whose intensity is measured via a PCR software. Various POC kits and devices works on this principle of molecular detectionThe mechanism of action involves isolation and purification of RNA extracted from upper and lower respiratory regions, which are then reverse transcribed to cDNA and then subjected to Applied Biosystems 7500 Fast Dx Real-Time PCR Instrument with SDS version 1.4 software for its subsequent amplification. This process provides appropriate conditions for the binding of probes between forward and reverse primers; however, during the extension phase of the PCR cycle, these probes are degraded by the 5՛ nuclease activity of Taq polymerase separating reporter dye and a quencher dye. This separation generates a fluorescent signal and with each cycle, more and more cleaving of reporter dye occurs thereby increasing the fluorescence intensity. The intensity of each cycle is measured via a PCR detection software system. Along with the test samples, the human RNase P (RP) gene is also targeted by a set of primers and probe as a control during the PCR run. Result interpretation of rRT-PCR must always be done by professional experts (https://www.fda.gov/media/134922/download) [36] (Table 1). Diagnostic approaches in COVID-19: clinical updatesAll authorsPurva Asrani, Mathew Suji Eapen, Collin Chia, Greg Haug, Heinrich C. Weber , Md. Imtaiyaz HassanSukhwinder Singh Sohalhttps://doi.org/10.1080/17476348.2021.1823833Published online:30 September 2020Table 1. Interpretation of rRT-PCR results of SARS-CoV-2 mediated COVID-19. SARS-CoV N1 and N2 markers – two regions with nucleocapsid (N) gene; RP, human RNase P. Abberevations: CDC, Centre of Disease Control and Prevention; rRT-PCR, Real-Time Reverse Transcriptase-Polymerase Chain Reaction. References as cited within the textCSVDisplay Table Different countries have adopted different viral targets for the molecular-based detection of this pathogen. For example, China had mainly relied upon the identification of ORF1ab and N gene; CDC USA has identified three targets in the N gene of the virus; Germany focused upon RdRP, E and N gene for detection; Pasteur Institute of Paris targets 2 regions within RdRP gene; National Institute of Health, Thailand has mainly used N gene; Hong Kong had developed a diagnostic test based upon the identification of ORF1b-nsp14 and N gene; whereas spike protein was targeted by National Institute of Infectious Disease, Japan (https://www.fda.gov/media/134922/download) [36].CDC USA has developed a diagnostic panel where 3 RT-PCR reactions target the N gene. One set detects all β-CoVs while two are specific for SARS-CoV-2 infection. For confirmation, all three tests should be positive [37]. The faculty of Medicine at the University of Hong Kong uses two tests to detect the presence of viral subgenus Sarbecovirus [38]. These tests undergo screening assay of N gene followed by confirmation using ORF1b assay. Since no reports of SARS-CoV infection in humans is present at this moment, therefore, all the positive cases are thought to cause by pathogenicity of SARS-CoV-2. This test was found to be negative for other coronaviruses [39]. Berlin, Germany uses a Charite algorithm that uses two tests; one specifically targeted against E gene and other against RdRP gene of subgenus Sarbecovirus for detecting the virus-mediated infection in the patients [39]. Both the tests must be positive for carrying out the subsequent steps where RT-PCR is used for specifically targeting the RdRP gene [40]. Alphacoronaviruses and beta coronaviruses cannot be detected using this approach. FDA has approved emergency use authorization for several in vitro molecular POC diagnostic kits until now (Table 2). Diagnostic approaches in COVID-19: clinical updatesAll authorsPurva Asrani, Mathew Suji Eapen, Collin Chia, Greg Haug, Heinrich C. Weber , Md. Imtaiyaz HassanSukhwinder Singh Sohalhttps://doi.org/10.1080/17476348.2021.1823833Published online:30 September 2020Table 2. FDA approved molecular-diagnostic tests and POC kits for emergency use against COVID-19. Abbervations: ND, not disclosed; N, Nucleocapsid; E, Envelope; S, Spike; RdRP, RNA-dependent RNA Polymerase; M, Matrix; rRT-PCR, Real-Time Reverse Transcriptase-Polymerase Chain Reaction; CRISPER, Clustered Regularly Interspaced Short Palindromic Repeats; ORF, Open Reading Frame; IL, Interleukin; KL, Krebs von den Lungen; pp1ab, nonstructural polyprotein 1ab; NGS, Next generation Sequencing. References as cited within the text. https://www.fda.gov/emergency-preparedness-and-response/mcm-legal-regulatory-and-policy-framework/emergency-use-authorizationCSVDisplay Table 3. Serological testsSerological tests are used when molecular techniques have performed unsatisfactorily [41]. Antibody detection is the main basis for the serological tests conducted in the laboratories [42]. When the SARS-CoV-2 was initially identified, a lack of available molecular tools and rapid antigen test kits allowed serology to be used as a supplementary diagnostic tool [43].An antibody is a specific protein that is produced inside the host cell in response to an infection. Therefore, an antibody test identifies the presence of antibodies in the specimen that might have developed as a part of the immune response if a person had prior exposure to the viral pathogen. Four different serological assays are commonly used in laboratories for the detection of SARS-CoV-2 infection are discussed in Table 3. Enzyme-linked immunosorbent assay (ELISA) is performed for both qualitative and quantitative analysis and generally performed in a lab setting illustrated in Figure 2(a). Diagnostic approaches in COVID-19: clinical updatesAll authorsPurva Asrani, Mathew Suji Eapen, Collin Chia, Greg Haug, Heinrich C. Weber , Md. Imtaiyaz HassanSukhwinder Singh Sohalhttps://doi.org/10.1080/17476348.2021.1823833Published online:30 September 2020Table 3. Serological-based testing of COVID-19. (https://www.cdc.gov/coronavirus/2019-ncov/lab/serology-testing.html)CSVDisplay Table Diagnostic approaches in COVID-19: clinical updatesAll authorsPurva Asrani, Mathew Suji Eapen, Collin Chia, Greg Haug, Heinrich C. Weber , Md. Imtaiyaz HassanSukhwinder Singh Sohalhttps://doi.org/10.1080/17476348.2021.1823833Published online:30 September 2020Figure 2. Serological testing of COVID-19 using ELISA. In this process, a 96 well plate is coated with viral specific antigens and serum samples of patients are loaded. If the sample had a previous exposure to the virus, IgM- and IgG-specific antibodies will recognize and bind to antigen coated on the wells. Substrate bound secondary antibodies are loaded into the wells later, which are specific to the primary antibodies. (a) binding of 2° Ab induces a reaction producing a color change. More is the amount of color product; more is the presence of antibodies in the sample. (b) Binding induces a chemical reaction that emits light. More is the luminescence; more is the presence of antibodies and hence quantitative detection of virus is possible by both the approachesDisplay full sizeFigure 2. Serological testing of COVID-19 using ELISA. In this process, a 96 well plate is coated with viral specific antigens and serum samples of patients are loaded. If the sample had a previous exposure to the virus, IgM- and IgG-specific antibodies will recognize and bind to antigen coated on the wells. Substrate bound secondary antibodies are loaded into the wells later, which are specific to the primary antibodies. (a) binding of 2° Ab induces a reaction producing a color change. More is the amount of color product; more is the presence of antibodies in the sample. (b) Binding induces a chemical reaction that emits light. More is the luminescence; more is the presence of antibodies and hence quantitative detection of virus is possible by both the approachesIn this process of viral detection, the plate is usually coated with viral specific protein such as spike and then loaded with the blood serum sample. If antibodies are present, it binds with the viral antigen forming an antigen-antibody complex. Later, secondary antibodies labeled with fluorochrome or substrates are added to this plate which recognizes the previously formed antigen-antibody complex and produces a detectable color change or shows the property of fluorescence through a chemical reaction.Chemiluminescent immunoassay (CLIA) is a modified version of ELISA where luminescence is measured for the detection of pathogens (Figure 2(b)). It is a quantitative test that can measure the number of IgG, IgM, and IgA antibodies [44]. This test allows the mixing of patient samples with viral specific proteins. The formation of the antigen-antibody complex is then detected by the binding of another secondary antibody that undergoes a chemical reaction for producing light. The amount of emitted light is then calculated for measuring the number of antibodies present in the sample.Rapid diagnostic tests (RDT) are small, portable, and are considered as a point of care immunodiagnostics [45]. They work on the principle of lateral flow assay where samples in the form of nasal swabs, saliva, or blood indicate colored lines for determining the positive or negative results (Figure 3(a)). In a lateral flow assay, a membrane containing gold nanoparticle labeled antibodies (Au-Ab) and capture antibodies are present in two different lines. When the patient’s sample is loaded onto the membrane, it moves by capillary action across the membrane. It first encounters Au-Ab and the viral antigens bind to form a complex. This complex then travel furthers and gets captured by the capture antibodies in the second line and their immobilization on that surface results in the production of colored lines confirming the tests (https://www.centerforhealthsecurity.org/resources/COVID-19/serology/Serology-based-tests-for-COVID-19.html). Neutralization assay identifies the antibodies in the patient’s serum that blocks the virus replication in the in vitro culture (Figure 3(b)). The procedure starts with the collection of serum samples from the suspected patients and the growth of virus culture on Vero E6 cells. When the virus starts its process of replication, the patient’s serum sample containing antibodies is added in the culture respectively. If there is a presence of detectable titer of IgM and IgG antibodies, it binds with the virus particles and blocks the replication process confirming the exposure of SARS-CoV-2 in the patient’s serum specimen.Diagnostic approaches in COVID-19: clinical updatesAll authorsPurva Asrani, Mathew Suji Eapen, Collin Chia, Greg Haug, Heinrich C. Weber , Md. Imtaiyaz HassanSukhwinder Singh Sohalhttps://doi.org/10.1080/17476348.2021.1823833Published online:30 September 2020Figure 3. Serological testing of COVID-19 via (a) rapid diagnostic test based on lateral flow assay and (b) neutralization assay. Serum sample of patient is loaded at sample pad on the membrane. Conjugate pad contains colloidal-gold labeled control antibodies and virus specific antigens. If antibodies are present in the sample it travels on the membrane via capillary action. Upon reaching conjugate pad, it binds to viral specific proteins forming antigen-antibody complex. It further travels and gets captured by test antibodies present on test line. This generates colored line on the membrane which can be easily detected. The control antibodies also travel from conjugate pad via capillary action but gets captured by control capturing antibodies on the control line. Any waste antibodies or antigens gets later absorbed on the absorption pad. The figure (b) shows the neutralization assay where samples after serial dilution along with virus is poured on monolayer of Vero E6 cell lines. The viral replication forms plaque on the surface of agar however; the presence of antibodies blocks the replication process thus causing an inhibition of plaque formationDisplay full sizeFigure 3. Serological testing of COVID-19 via (a) rapid diagnostic test based on lateral flow assay and (b) neutralization assay. Serum sample of patient is loaded at sample pad on the membrane. Conjugate pad contains colloidal-gold labeled control antibodies and virus specific antigens. If antibodies are present in the sample it travels on the membrane via capillary action. Upon reaching conjugate pad, it binds to viral specific proteins forming antigen-antibody complex. It further travels and gets captured by test antibodies present on test line. This generates colored line on the membrane which can be easily detected. The control antibodies also travel from conjugate pad via capillary action but gets captured by control capturing antibodies on the control line. Any waste antibodies or antigens gets later absorbed on the absorption pad. The figure (b) shows the neutralization assay where samples after serial dilution along with virus is poured on monolayer of Vero E6 cell lines. The viral replication forms plaque on the surface of agar however; the presence of antibodies blocks the replication process thus causing an inhibition of plaque formationVarious immunodiagnostic POC kits have been commercialized for the emergency authorization are listed in Table 4. Two other types of kits available recognize the host antibody [46] and others detect the viral antigens [47]. Identification of virus-specific antigens is more prominent because the early infection can be detected through this approach. Hereby, antibodies are fixed to a plastic cover and the sample of patients is poured from the above. If the virus is actively replicating it binds to the antibodies and generates a visually detectable color within just 30 minutes (Figure 4). ELISA-based approach is mostly used for the designing of immunodiagnostic kits of both natures. Lateral flow assay is another important principle that is generally used for the production of POC kits [46]. Diagnostic approaches in COVID-19: clinical updatesAll authorsPurva Asrani, Mathew Suji Eapen, Collin Chia, Greg Haug, Heinrich C. Weber , Md. Imtaiyaz HassanSukhwinder Singh Sohalhttps://doi.org/10.1080/17476348.2021.1823833Published online:30 September 2020Table 4. FDA approved immunodiagnostic tests and POC kits for emergency use against COVID-19. IgM, Immunoglobulin M; IgG, Immunoglobulin G; RDT, Rapid Diagnostic Test; N, Nucleocapsid; ELISA, Enzyme-linked Immunosorbent Assay; ECLIA, Electrochemiluminescence immunoassay; MIRA, Methylated-CpG island recovery assay; CMIA, Chemiluminescent Microparticle Immunoassay; FIA, Fluorescent Immunoassay. References as cited within the textCSVDisplay Table Diagnostic approaches in COVID-19: clinical updatesAll authorsPurva Asrani, Mathew Suji Eapen, Collin Chia, Greg Haug, Heinrich C. Weber , Md. Imtaiyaz HassanSukhwinder Singh Sohalhttps://doi.org/10.1080/17476348.2021.1823833Published online:30 September 2020Figure 4. Result interpretation of immunodiagnostic POC test kits. The test measures the presence of IgG and IgM antibodies in the patient’s serum sample. The results are obtained in the form of colored lines. Control (C) line should always be present however; line specific to IgG and IgM may vary depending upon the results. No colored lines appearing for IgG and IgM but presence of C, indicates a negative test in contrast to a IgG and IgM positive test where lines are observed for all the three parameters. If lines only appear for C and IgG but not IgM then the patient is said to be IgG positive in nature. Similarly, lines appearing only for IgM and C but not for IgG, the test is said to be IgM positive in nature. If colored lines are not observed for C, the test is invalid irrespective of the presence of IgG- and IgM-colored linesDisplay full sizeFigure 4. Result interpretation of immunodiagnostic POC test kits. The test measures the presence of IgG and IgM antibodies in the patient’s serum sample. The results are obtained in the form of colored lines. Control (C) line should always be present however; line specific to IgG and IgM may vary depending upon the results. No colored lines appearing for IgG and IgM but presence of C, indicates a negative test in contrast to a IgG and IgM positive test where lines are observed for all the three parameters. If lines only appear for C and IgG but not IgM then the patient is said to be IgG positive in nature. Similarly, lines appearing only for IgM and C but not for IgG, the test is said to be IgM positive in nature. If colored lines are not observed for C, the test is invalid irrespective of the presence of IgG- and IgM-colored lines4. Laboratory and point of care devicesThe main basis of diagnostic devices could be either identification of viral RNA sequence through molecular instruments or either detection of viral-specific antigens/host antibodies through serological testing devices [48]. The purpose of introducing POC devices relies upon their fast and accurate testing that generates results within hours or minutes of performing the test [49]. These devices mainly work upon molecular and serology-based mechanisms as discussed earlier. Molecular devices work by identifying the specific sequence of the RNA genome and then amplifies it to the point it becomes easily detectable [50,51]. Similarly, serology-based devices depend upon ELISA, a technique for the identification of host antibodies and viral proteins [41].During the emergence of a novel coronavirus in China, 11 molecular lab-based devices were approved for emergency testing of the viral infections. The different approaches were used by these devices such as next-generation sequencing (NGS), isothermal amplification accompanied by chip detection and the most commonly used was rRT-PCR through probe hydrolysis [52]. The urgent use of serological devices was also provided by the National Medical Products Administration (NMPA) in China [53]. Similarly, the Government of Canada has approved several devices (lab-based and POC) for fast and accurate testing of the virus (Table 5). Diagnostic approaches in COVID-19: clinical updatesAll authorsPurva Asrani, Mathew Suji Eapen, Collin Chia, Greg Haug, Heinrich C. Weber , Md. Imtaiyaz HassanSukhwinder Singh Sohalhttps://doi.org/10.1080/17476348.2021.1823833Published online:30 September 2020Table 5. Emergency authorized laboratory and point of care devices for diagnosis of COVID-19. rRT-PCR, Real-Time Reverse Transcriptase-Polymerase Chain Reaction; IgM, immunoglobulin M; IgG, Immunoglobulin G; FIA, Fluorescent Immunoassays; NGS, Next Generation Sequencing. References as cited within the text. https://www.abbott.com/corpnewsroom/product-and-innovation/detect-covid-19-in-as-little-as-5-minutes.htmlCSVDisplay Table Recently, FDA has approved Abbott ID now COVID-19 test that runs on POC device- Abott’s ID NOWTM platform – a lightweight box that can be fit and adjusted in different locations. It is portable and easy to handle. The most remarkable feature it possesses is the ability to give positive results in as less than five minutes and negative results in thirteen minutes. Approximately, 50,000 tests can be performed through this instrument daily with utmost accuracy.5. Radiology-based diagnosisThe radiological diagnosis is based upon the identification of clinical changes in the patient’s respiratory system as it is the primary target of SARS-CoV-2 infection upon entry. This test can be used for early diagnosis of viral exposure to the patient [54]. It is a medical imaging technique that uses a chest computed tomography (CT) scan and chest X-ray (CXR) for giving a greater view of internal organs, bones, and soft tissues [55]. It is also required for an understanding of the pathophysiology of viruses and in elucidating the progression of viral infection inside the human body [56,57]. The targeted organs and their clinical symptoms could be easily studied through this approach [58].Initially, when the symptoms appeared in the patients of Wuhan, basic clinical tests started which later on modified by several advancements in series to understand the physiological changes and in the diagnosis of the virus [55]. The earlier reports from Hubei province showed 75% of people had bilateral pneumonia while 25% exhibited unilateral pneumonia. Ground-glass opacities (GGO) and multiple mottling were observed in around 14% of the patients. The illness was mainly peripheral and was seen in the right lower lobes [59]. Several medical imaging techniques were used to conduct further studies on evaluating the affected organs [60]. CT scan was found to be more appropriate in providing insights about pneumonia but the results were variable in different patients highlighting the different stages the virus might have been at that time [56]. The most commonly observed clinical features were GGO lesions (56.4%), local patchy shadowing (28.1%), bilateral patchy shadowing (51.8%), and interstitial abnormalities (4.4%). Severe cases showed more prominent of these symptoms than nonsevere cases [61,62]. The analysis of the clinical changes associated with the disease progression revealed that the early stage of infection is marked by a GGO lesion [63]. The later formation of multifocal GGO lesions occurs and in some cases, these lesions undergo septal thickening in interlobular and intralobular as the disease progresses [64]. At last, more consolidated lesions are formed causing severe lung injuries [65].6. Viral cell cultureThe main basis of viral culture diagnosis depends upon isolation of SARS-CoV-2 on cell lines promoting growth and replication of the virus. It is required for confirmation when vaccine testing is being done [66]. The virus isolated from the specimens of suspected patients is grown on primary monkey cells and cell lines including Vero E6 and LLC-MK2. Vero cells are obtained from the kidney of an African green monkey whereas LLC-MK2 is derived from the kidney of rhesus monkey [42,43,67]. Vero cells are widely used for research purposes mainly in testing the vaccines against some viruses such as rotavirus, poliovirus, influenza virus, and smallpox [68]. There are different cell lines derived from Vero cells such as Vero E6 which was found to be very sensitive with the viral replication of SARS-CoV [69]. Upon virus replication, cell death occurred due to apoptosis, and the cells which survived the replication process showed morphology similar to the uninfected cells supporting the production of these infectious agents [70].SARS-CoV2 used to get activated by proteolytic cleavage by TMPRSS2 under both in vitro and in vivo conditions [71,72]. Sharing the similarities with SARS-CoV, Matsuyama, and his team [71] engineered a Vero E6 cell line expressing TMPRSS2 for culturing of SARS-CoV-2. Results have shown that using cell lines with TMPRSS2 expression, more than 100 times greater production of viral RNA copies than Vero E6 cells alone was supported [73,74]. These engineered cell lines constitutively express TMPRSS2, and thus their mRNA expression is ̴ 10-fold higher than normal human lung tissues suggesting the importance of these proteases in early viral replication through proteolytic mechanisms [75]. SARS-CoV-2 is also easily cultivable and shows similar mechanisms when grown on these cell lines [8,76].6.1. Diagnosis of histopathological changes in COVID-19In general, the histopathological changes in the COVID-19 patient ranges from vascular complications, different patterns of epithelial damage, fibrosis and inflammation of lungs but these changes may be microscopic or macroscopic under observation [77]. Microscopic findings are usually nonspecific and may be associated with the asymptomatic patients as well. However; autopsy results from 50-year-old man who was severely infected with COVID-19 indicated early changes in the acute lung injuries such as multiple ground glass opacities in the CXR, pneumocyte hyperplasia, edema, mostly lymphocytic focal inflammation and multinucleated giant cells alongside atypical pneumocytes [11,78]. These clinical features were similar to the histopathological changes associated with previsously known SARS and MERS infections [79,80]. Macrofindings includes more prominent changes in the infected lung such as pericarditis, pleurisy, lung consolidation, and pulmonary edema. An increased lung weight may also be observed than the normal [81]. Sometimes, this viral infection gets superimposed by the presence of other secondary bacterial infections leading to purulent inflammation, making the condition more severe for the patient to sustain [82]. Identification of histopathological changes in the COVID-19 patients could be either done by genotyping or through clinical, serological, radiological, and histopathological phenotyping for timely and accurate detection [77]. Different biomarkers that may be used for the detection purposes include reduced levels of albumin, lymphocytes and prolonged prothrombin time [83,84]. Increased levels of ferritin, C-reactive protein, D-dimer, and lactate dehydrogenase may also serve as a potential biomarker for detection of SARS-CoV-2 in the earlier stages of infection [62,85].7. Future direction and tests on horizonThe innovation of researchers in bringing out the best diagnostic tools is not restricted to the ones mentioned above rather there are many more tests that are waiting to get green signals. A brief information of some of the future prospects of diagnostics is covered in this section (Table 6). Diagnostic approaches in COVID-19: clinical updatesAll authorsPurva Asrani, Mathew Suji Eapen, Collin Chia, Greg Haug, Heinrich C. Weber , Md. Imtaiyaz HassanSukhwinder Singh Sohalhttps://doi.org/10.1080/17476348.2021.1823833Published online:30 September 2020Table 6. Future diagnostic tests for COVID-19 detection. Abbreviations: CRISPER, Clustered Regularly Interspaced Short Palindromic Repeats; LAMP, Loop-mediated Isothermal Amplification; POC, Point of Care. References as cited within the textCSVDisplay Table 7.1. CRISPER in diagnosis of SARS-CoV-2The use of CRISPER based technology is gaining wide importance for the construction of diagnostic kits where it works by recognition of a specific genetic sequence followed by cutting of the reporter molecule added into the reaction mixture. This process is highly specific and can reveal the presence of genetic sequence of the virus in 5–10 minutes [35]. This CRISPER-based approach is called DETECTR for coronaviruses and is coproduced by a Biotechnology company in San Francisco known as Mammoth Biosciences and by a pioneer of CRISPER technology, Jennifer Doudna at University of California [86]. Similar inputs from Keith Jerome, a virologist at University of Washington and Feng Zhang at Broad Institute of MIT and Harvard in codevelopment of a kit referred to as SHERLOCK has been accepted recently for emergency use authorization (EUA) by FDA [87–89].7.2. Algorithm based coronavirus detectionHologic Panther Fusion (PF) is an algorithmic method which emphasis on random accessing rather than batch-wise testing [90,91]. Two PCR protocols were adapted and then processed for PF program. This process permitted immediate and fully automated diagnostic processes such as extraction of RNA, amplification of the target sequences and real time detection of PCR amplicons in 3.5 hours [40]. No any sort of random accessing diagnostic tool existed before this and the batch-wise testing systems is often tedious with long response time [92]. After further validation on large clinical specimens it has been approved for introduction into the laboratories for testing, suggesting a scope of more algorithmic approaches in shaping the new future of diagnosis [93]. Similar algorithm-based testing has been under trials in many parts of the world to come up with faster diagnostic approaches after its approval. One of them is proposed by Iranian experts to diagnose and provide effective treatment to the children suffering from COVID-19 [94]. It is prepared after the study on patient symptoms, their response to treatments and the surveillance data obtained from their country. However; close monitoring must be done for this approach if any exceptional cases of infection arises or the parameters on which the model is based changes among the patients [94].7.3. LAMP based detection of SARS-CoV-2Loop-mediated isothermal amplification (LAMP) with colorimetric detection is proposed by Zhang and his team. This technique provided results in par with the available molecular RT-qPCR tests and facilitated the viral detection without being dependent upon complex infrastructures [95]. A professor from University of Pennsylvania, Jinzhao Song has suggested a further modification of LAMP technique along with a use of recombinase polymerase amplification (RPA) for a two stage amplification in a single tube [96]. Using this strategy, he has further suggested the production of a POC device where diagnosis could be done in homes and no clinical set-ups are required. Such devices combines a paper-based technology along with LAMP assay technique [97]. One of the remarkable features of this device is its potential to be integrated with a smartphone where highly sensitive, reliable and rapid results could be obtained by quarantined and self-isolated individuals on their own. This could be done by collection of nasal swabs, addition of LAMP-specific reagents on a paper and visualization of a color change. The pictures of the paper-based test results can be uploaded on the cloud internet which will be accessible to the doctors for immediate information about the health status of an individual and further can be transferred to the government [98]. Hence, it reduces traveling to the hospitals for a checkup, the chances of spreading the infection also gets significantly reduced [65].The tests that measures the ability of antibody responses toward SARS-CoV-2 infection is critical in development of vaccines and when combined with surveillance data, adds to our knowledge about the number of patients that should be tested for a possible infection [46]. The current situation demands the application of lessons learnt during the SARS-CoV outbreak that had happened previously to fight against recent COVID-19 pandemic with a greater response [99].8. SummaryCoronaviruses have been remerging after every decade since 2003 when first known virus SARS-COV to infect humans was identified. Later, MERS-CoV emerged in 2012 and presently, world is witnessing a third viral outbreak caused by a novel strain of coronavirus, SARS-CoV-2 sharing 80% genome similarity to SARS- CoV. This virus has spread to 213 countries and has been declared as a COVID-19 pandemic by WHO. Mutations in S gene is responsible for its higher transmission rate than the earlier counterparts and hence is problem of global concern.Different technology-based diagnostic approaches are currently being employed in detection of SARS-CoV-2. Molecular strategies focuses upon the identification and amplification of specific viral sequences through rRT-PCR approach. There may be different viral genes which may be targeted for primer binding and detection, e.g. N, E, S, RdRP, and ORF 1ab genes. Another important approach is serological-based testing that relies upon antigen-antibody iteractions based upon principles like ELISA, chemiluminescent immunoassay and neutralization assay. Various rapid diagnostic tests are made on the similar principles providing efficient results within a matter of few minutes to hours. Radiological tests indicate the clinical changes in patient’s lungs as the disease progresses and therefore, are considered as important means of studying the affect on internal organs upon encounter of the virus to the human host. This feature distinguishes radiological detection with other means of diagnosis. Viral cell culturing techniques has also a relevant role when it comes to viral detection. Vero E6 cell lines are primarily used for the culturing purposes however; certain advancements such as Vero E6 cell line expressing TMPRSS2 for culturing of SARS-CoV-2 has shown more than 100 times greater production of viral RNA copies than Vero E6 cells alone. This suggests the importance of these proteases in early viral replication through proteolytic mechanisms. Laboratory and POC devices; immunodiagnostic and molecular diagnostic kits not only provide rapid and fast test results but are accurate and portable. However, these tests should always be performed by professionals and the chance of misinterpreting the results are high indicating one of the most common limitations of these techniques. rRT-PCR and NGS sequencing is the best approach in diagnosis followed by serological testing. Radiological tests may generate varied results in different patients under CXR and CT scan; therefore, it should be combined with molecular strategies for confirmation of results.Many ongoing trials are currently happening across the world. Some of them relies upon the use of CRISPER technology to detect the viral sequence while some focuses upon algorithmic approach for diagnosis. Colorimetric LAMP assay and smartphone mediated paper-based LAMP assay technology is also being studied. Human intervention has always been successful in improvisation of the medical system to respond back emerging challenges in health care settings; similar such opportunities are provided by the recent outbreak of Coronaviruses. Therefore, joining hands of hospitals, clinical laboratories, public and private sectors on a global platform would bring more glory to the advancements in medical sciences.9. ConclusionIn this paper, we have highligthed different diagnostic procedures that are currently available for detecting COVID-19 with a special focus on their mechanisms and recent advancements. Various POC kits and devices that are authorized for emergency use by FDA are also enlisted which may provide a wide knowledge to the readers and keep them updated about the latest advancements in the diagnostic field. However; not all emergency authorized POC kits and devices could be explained in detail, indicating one of the limitation of this study. Such narrtiave and critical review holds importance to the clinicals in selecting the best possible method with maximum accuracy for COVID-19 detection and in conducting a timely and efficient diagnosis. It may also help researchers in identification of the suitable viral targets for diagnosis and for therapeutic reasons in future and in addressing the limitations associated with the diagnostic procedures. This article concludes with the research questions such as Are we prepared enough to conduct as many diagnostic tests of COVID-19 as its growing demand? How well we have strategically developed and modified the existing diagnostic methods for improvising the COVID-19 detection recently? How strongly prepared we are in the filed of diagnosis if such zoonosis outbreak would occur in future? These questions must be addressed should we want to be prepared for any other zoonotic outbreaks in future. Therefore, this paper has a direct impact in the scientific community where it may serves as an intial point in influencing how the diagnostic tests are conducted and should be conducted in future for advancing the clinical strategies and strengthening the health systems across the globe.10. Expert opinionThe continued arisen of novel coronavirus infections since past 17 years cauing severe respiratory illnesses has led to believe in the hypothesis about possibility of its remergence in the coming future. Diagnois of an infection remains the first and the most crucial aspect when an outbreak occurs. It can pave a way to limit the spread and most importantly helps in identifying the extent to which it has already been spread in a particular area. It also serves as an intial point for the researchers to study about the pathogen characteristics, understanding the diasease prognosis and in estimating the incubation period, R0 value and other important parameters required for prevention from the epidemic. Altough, advanced diagnostic strategies are available today, there is still a need to develop more specific strategies when a new emerging infection arises whose little information is available to the scientific community. A similar such crises happened recently and still ongoing, when a novel coronavirus strain emerged and spread to 213 countries leaving a great impact on the social and economic status. Owning to the higher transmission rate of the virus, all the countries where advancements in the medical sciences is not updated, suffered a huge loss because of delay in diagnosing the infection at an earlier stage. By the time first case of the infection was identified, already the virus had spread to a miserable level.Diagnosis of SARS-CoV-2 is challenging in many aspects, since it represents normal flu like symptoms, SARS-CoV-2 can often be confused with other CoV infections and thus, the detection of viral antigen can provide false-positive results. Different diagnostic strategies are available from basics to advanced but more specificity is required by understanding the mechanisms of SARS-CoV-2 to counteract the effect of generating the misleading results with other coronavirus infections. rRT-PCR technique focusing upon the particular viral genes responsible for the pathogensis is the most reliable when it comes to identification but it should always be done by professionals. NGS although providing accurate results but is not feasible to get it done for each and every sample received in a laboratory from a suspected patient. The second most reliable approach is to undergo testing for antigen-antibody interactions through serological detection mechanism however; the concern lies within the late onset of antibody titers in the patient’s specimen. Sometimes, it may takes 1–3 weeks for antibodies to be produced making this technique not very reliable for an early detection. Radiological approach is important to study the pathogenesis caused by the virus but the result varies among individuals depending upon several factors such as age, stage of infection, severiety of infection and preexisting combordities. Viral cell culture techniques could only be performed under BSL-3 which is not supported by most of the laboratories. Only specified labs have such access restricting the quantity of research in many countries. Also, these culturing mechanisms are more suitable when vaccine trials are to be done. The technical limitations associated with the approaches discussed so far could be solved by producing more researchers with professional skills so that no risk of mishandling and misinterpretation of data may exist. Also, more fundings are required in setting up of advanced research laboratories with infrastructure for supporting high quality research across the world. Standardization of specimen collection procedures, tissue selection, their time of collection after onset of symptoms, storage and handling of samples is required to ensure better results while working on serological tests.The research in this area has direct impact in influencing how the diagnostic tests are conducted and should be conducted in future for advancing the clinical strategies and strengthening the health systems across the globe. There is no definitive end point to this area of research as more advanced diagnostic strategies would bring us more closer in controlling the infection even if we do not hold any authorized drugs and vaccines toward this virus presently. The future results hold promising development in the field of medical and clinical sciences not just for diagnosis of coronaviruses but for any other infections that may arise in the future. There could be multiple other techniques and areas which can be explored for producing better and faster results. CRISPER and isothermal amplification are one such area where work is being done for improving the diagnostic procedures through molecular based gene targeting. More research is required in the area where less interaction of patients with the health professionals could be done during a diagnostic test which further limits the spread of virus. Smartphone assisted survelliance and paper based-LAMP assay is currently under trials which would ensure less contact of patients and would ensure updates to the hospitals and government agencies about their health status from their homes. Such kind of innovations would bring more glory to the field of medical sciences.In the coming five years, we will be more closer in producing a pathogen specific diagnostic test and this would change the way how currently we respond to an emerging pandemic. If such situation arises in the future, we may be prepared by learning the lessons from this pandemic.A five year view of this article shows that a clear understanding of all avalaible diagnostic approaches of coronaviruses allows production of more efficient and advanced strategies in future that would help in early identification of the virus. Without availability of vaccines and drugs at the moment, diagnosis is the key element in identifying, isolating and treating the suspected patients to prevent the spread of virus. Therefore, a better understanding of the viral targets and diagnostic strategies would open gates for the researchers to bring out the best possible mechanisms in diagnosis and reducing the worldwide burden caused from coronaviruses after every decade. By having gained sufficient knowledge of basic diagnostic strategies, now more research is required to bring out an innovation and advancement in the existing mechanisms focusing upon robust, inexpensive, efficient, reliable, and quick testing results. The reemergence of coronaviruses after every decade hints about the possibility of its arrival in the future as well therefore, diagnostic advancements would be more important in the coming years when such situation would arise. It is hypothesized that by five years, clinical sciences would become more powerful in understanding and diagnosing the threat caused by coronaviruses which is burdensome to the global population in the current times.Table 1. Interpretation of rRT-PCR results of SARS-CoV-2 mediated COVID-19. SARS-CoV N1 and N2 markers – two regions with nucleocapsid (N) gene; RP, human RNase P. Abberevations: CDC, Centre of Disease Control and Prevention; rRT-PCR, Real-Time Reverse Transcriptase-Polymerase Chain Reaction. References as cited within the textSARS-CoV-2 (N1 marker)SARS-CoV-2 (N2 marker)RP gene (humans) ControlResultImmediate actions+++Positive test result. SARS-CoV-2mediated infection in specimens.Report the test results to government officials, CDC, and sender.-++Inconclusive resultsRepeat the diagnostic process from RNA extraction to amplification in rRT-PCR. If repeatedly same results are obtained contact CDC for further guidance.+-+Inconclusive results--+Negative test result. No SARS-CoV-2 mediated infection is found.Check for presence of other respiratory infections.++-Invalid test results.Repeat the diagnostic process from RNA extraction to amplification in rRT-PCR. New specimen to be collected from the patients if repeated invalid results are obtained.+--Invalid test results.-+-Invalid test results.Table 2. FDA approved molecular-diagnostic tests and POC kits for emergency use against COVID-19. Abbervations: ND, not disclosed; N, Nucleocapsid; E, Envelope; S, Spike; RdRP, RNA-dependent RNA Polymerase; M, Matrix; rRT-PCR, Real-Time Reverse Transcriptase-Polymerase Chain Reaction; CRISPER, Clustered Regularly Interspaced Short Palindromic Repeats; ORF, Open Reading Frame; IL, Interleukin; KL, Krebs von den Lungen; pp1ab, nonstructural polyprotein 1ab; NGS, Next generation Sequencing. 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Inc.ChinaORF 1ab and N geneBio-Rad SARS-CoV-2 ddPCR TestBio-Rad Laboratories, Inc.USATwo regions within N gene (N1 and N2)BioFire Respiratory Panel 2.1BioFire Diagnostics, LLCUSAND*LabGun COVID-19 RT-PCR KitLabGenomics Co., Ltd.USARdRP gene and E geneRheonix COVID-19 MDx AssayRheonix, Inc.USAN1 region within N geneU-TOP COVID-19 Detection KitSESSUN BIOMATERIALSKoreaORF 1ab and N geneSTANDARD M nCoV Real-Time Detection KitSD Biosensor, Inc.KoreaORF 1ab and E geneRealStar SARS-CoV-2 RT-PCR Kits U.S.altona Diagnostics GmbHGermanyE gene and S geneAllplex 2019-nCoV AssaySeegene, Inc.KoreaE, RdRP, and N genesPhoenixDx 2019-CoVTrax Management Services Inc.USAE, RdRP, and N genesGeneFinder COVID-19 Plus RealAmp KitOSANG HealthcareSouth KoreaE, RdRP, and N genesFosun COVID-19 RT-PCR Detection KitFosun Pharma USA Inc.USAORF1ab, N, and E geneGS COVID-19 RT-PCR KITGenoSensor, LLCUSAND*Curative-Korva SARS-CoV-2 AssayKorvaLabs Inc.USATwo regions within N gene (N1 and N2)SARS-CoV-2 Fluorescent PCR KitMaccura Biotechnology (USA) LLCChinaORF1ab, N, and E geneiAMP COVID-19 Detection kitAtila BioSystems, Inc.USATargets biomarkers such as IL-6 (inflammation). KL-6 epithelial lung injury), PCT, and ferritin (infection)BD SARS-CoV-2 Reagents for BD MAX SystemBecton, Dickinson CompanyUSATwo regions within N gene (N1 and N2)QuantiVirus SARS-CoV-2 Test KitDiaCarta, Inc.USAORF1ab, N, and E geneSmart Detect SARS-CoV-2 rRT-PCR KitInBios International, Inc.USAORF1ab, N, and E genesGnomegen COVID-19 RT-Digital PCR Detection KitGnomegan LLCUSATwo regions within N gene (N1 and N2)ARIES SARS-CoV-2 AssayLuminex CorporationUSAORF1ab, N, and E geneScienCell SARS-CoV-2 Coronavirus Real-time RT-PCR (RT-qPCR) Detection KitScienCell Research LaboratoriesUSATwo regions within N gene (N1 and N2)Logix Smart Coronavirus Disease 2019 (COVID-19)Co-Diagnostics, Inc.USARdRPBioGX SARS-CoV-2 Reagents for BD MAX SystemBecton, Dickinson Company (BD)USATwo regions within N gene (N1 and N2)CoV-19 IDx assayIpsum Diagnostics, LLCUSAN geneNeuMoDx SARS-CoV-2 AssayNeuMoDx Molecular, Inc.USANsp2 gene and N geneQlAstat-Dx Respiratory SARS-CoV-2 PanelQIAGEN GmbHGermanyORF 1b and E geneID NOW COVID-19Abbott Diagnostics Scarborough, Inc.USARdRP geneNxTAG CoV Extended Panel AsaayLuminex Molecular Diagnostics, Inc.USAORF1ab, N, and E genesReal-Time Fluorescent RT-PCR Kit for Detecting SARS-CoV-2BGI Genomics Co. LtdChinaORF1abAvellinoCoV2 testAvellino Lab USA, Inc.USAN genePerkinElmer New Coronavirus Nucleic Acid Detection KitPerkinElmer, Inc.USAORF1ab and N geneBioFire COVID-19 TestBioFire Defense, LLCUSAND*Accula SARS-CoV-2 TestMesa Biotech Inc.USAN genePrimerdesign Ltd COVID-19 genesig Real-Time PCR assayPrimerdesign Ltd.UKND*Xpert Xpress SARS-CoV-2 testCepheidUSAN2 and ESimplexa COVID-19 DirectDiaSorin Molecular LLCUSAORF1ab and S geneePlex SARS-CoV-2 TestGenMark Diagnostics, Inc.USAND*Abbott Real Time SARS-CoV-2 assayAbbott MolecularUSARdRP and N-genesLyra SARS-CoV- AssayQuidel Corp.USANonstructural polyprotein (pp1ab) geneQuest SARS-CoV-2 rRT-PCRQuest Diagnostics Infectious Disease, Inc.USANucleocapsid gene (N1 and N3)Panther Fusion SARS-CoV-2 AssayHologic, Inc.USAORF1ab Region 1ORF1ab Region 2COVID-19 RT-PCR TestLaboratory Corporation of AmericaUSAN geneThermo fisher Scientific, Inc.TaqPath COVID-19 Combo KitUSAS protein and N proteinRoche Molecular Systems, Inc.Cobas SARS-CoV-2USAORF1/a and E-geneWadsworth Center, NYSDOHNew York SARS-CoV-2 Real Time Reverse Transcriptase (RT)-PCR Diagnostic PanelUSAN geneCDCCDC 2019-Novel Coronavirus Real-Time RT-PCR Diagnostic PanelUSAN geneTable 3. Serological-based testing of COVID-19. (https://www.cdc.gov/coronavirus/2019-ncov/lab/serology-testing.html)Type of testResponse timePrincipleLimitationsNeutralization assay3–5 daysIdentifies the presence of antibodies in the patient’s serum on the basis of their ability to block replication of SARS-CoV-2 on Vero E6 cell lines.The detection of antibodies specific to virus proteins that are not primarily involved in replication might get missed by this approach.ELISAQualitative detection2–5 hoursIdentifies the presence of antibodies through formation of colored product obtained after binding of secondary labeled antibodies to primary antigen-antibody complex.It doesn’t give information about whether the virus growth can be inhibited with these antibodies or not.Chemiluminescent immunoassayQualitative detection1–2 hoursIdentifies the presence of antibodies through formation of luminescence activity obtained by a chemical reaction that occurs by binding of secondary labeled antibodies to primary antigen-antibody complex.It doesn’t give information about whether the virus growth can be inhibited with these antibodies or not.Rapid diagnostic testQualitative detection10–30 minutesIdentifies the presence or absence of antibodies against virus in patient’s blood serum specimens. Works on the principle of lateral flow assay where antigen-antibody complex moves by capillary action across a membrane and gets immobilized by capture antibodies producing a color change.The number of antibodies in the patient’s specimen cannot be determined. Also, it does not tell if these antibodies are able to inhibit the growth of virus either in cell culture or in vivo.Table 4. FDA approved immunodiagnostic tests and POC kits for emergency use against COVID-19. IgM, Immunoglobulin M; IgG, Immunoglobulin G; RDT, Rapid Diagnostic Test; N, Nucleocapsid; ELISA, Enzyme-linked Immunosorbent Assay; ECLIA, Electrochemiluminescence immunoassay; MIRA, Methylated-CpG island recovery assay; CMIA, Chemiluminescent Microparticle Immunoassay; FIA, Fluorescent Immunoassay. References as cited within the textName of testManufacturerType of testCountry of developmentSCoV-2 Detect IgM ELISAInBios International, Inc.IgM-based detectionUSAAccess SARS-CoV-2 IgGBeckman Coulter, Inc.IgG-based detectionUSABabson Diagnostics aC19G1Babson Diagnostics, Inc.IgG-based detection (indirect sandwich chemiluminescent assay)USALYHER Novel Coronavirus (2019-nCoV) IgM/IgG Antibody Combo Test Kit (Colloidal Gold)Hangzhou Laihe Biotech Co., Ltd.IgM/IgG-based detection (immunochromatography)USABiohit SARS-CoV-2 IgM/IgG Antibody Test KitBiohit Healthcare (Hefei) Co. 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Emergency authorized laboratory and point of care devices for diagnosis of COVID-19. rRT-PCR, Real-Time Reverse Transcriptase-Polymerase Chain Reaction; IgM, immunoglobulin M; IgG, Immunoglobulin G; FIA, Fluorescent Immunoassays; NGS, Next Generation Sequencing. References as cited within the text. https://www.abbott.com/corpnewsroom/product-and-innovation/detect-covid-19-in-as-little-as-5-minutes.htmlName of testManufacturerDeviceTechnologyType of testAbbott Realtime SARS-CoV-2Abbott Molecular Inc. (USA)Abbott m2000 systemNucleic acid detectionLab-based testMultiple Respiratory Virus Nucleic Acid IVD KitShanghai ZJ Bio-Tech (China)RT thermocycler, e.g. ABI 7500 Fast Dx RT-PCR InstrumentNucleic acid detection (Fluorescence RT-PCR)Lab-based testBD SARS-CoV-2 Reagents for BD Max systemBecton Dickinson and company (Canada)BD Max systemNucleic acid detectionLab-based testAbbott ID NOW COVID-19 testAbbott Diagnostics Scarborough, Inc.Abbott’s ID NOWTM platformNucleic acid detectionPoint of care testBiofire COVID-19 Test Biofire COVID-19 External Control Test KitBiofire Defense LLC (USA)FilmArray 2.0 FilmArray Torch Instrument SystemNucleic acid detectionLab-based testCobas SARS-CoV-2Roche (USA)Cobas 6800 Cobas 8800 systemNucleic acid detectionLab-based testRespiratory Virus Nucleic Acid Detection KitCapitalBio (Chengdu) (China)RTisochip™-A (20,173,401,354)Nucleic acid detection (Isothermal amplification and microarray)Lab-based testDiaSorin Simplexa COVID-19 Direct Molecular AssayDiaSorin Molecular LLC. (USA)LIAISON MDX InstrumentNucleic acid detectionLab-based testLiaison SARS-CoV-2 S1/S2 IgG, Liaison Control SARS-CoV-2 S1/S2 IgGDiasorin Inc. (USA)LIASION XL analyserSerological detectionLab-based testSARS-CoV-2 Antibody Detection KitXiamen Wantai Kairui Biotechnology (China)Caris 200 Automatic Chemiluminescence AnalyserSerological detection (Chemiluminescence immunoassay)Lab-based testLYRA SARS- CoV-2 AssayDiagnostic Hybrids, Inc./Quidel Corporation (USA)Applied Biosystems 7500 fast Dx RT-PCR.Nucleic acid detectionLab-based testNxTAG CoV Extended PanelLuminex Molecular Diagnostics, Inc. (Canada)SYNCT software and MAGPIX instrument.Nucleic acid detectionLab-based testSARS-CoV-2 AssayHologic (USA)Panther Fusion SystemNucleic acid detectionLab-based testSpartan Cube COVID-19 SystemSpartan Bioscience Inc. (Canada)Spartan Cube SystemNucleic acid detectionPoint of care testMGI’s Laboratory Detection Total Solution for COVID-19MGI Tech (China)Genetic sequencer (DNBSEQ-T7)Nucleic acid detection (NGS)Lab-based testTaqPathTM COVID-19 Combo KitThermo Fisher (USA)Applied Biosystems 7500 fast Dx RT-PCR.Nucleic acid detectionLab-based testXpert Xpress SARS-CoV-2Cepheid (USA)GeneXpert PX GeneXpert Infinity SystemsNucleic acid detectionLab-based testPoint of care testSARS-CoV-2 IgM Antibody Detection KitBioscience (Chongqing) (China)Automated magnetic analyser: Axceed 260Serological detection (Magnetic particle chemiluminescence)Lab-based testSofia 2 SARS-CoV-2 FIAQuidel CorporationSofia 2 analyzerSerological detection (N viral protein detection based on FIA).Point of care testTable 6. Future diagnostic tests for COVID-19 detection. Abbreviations: CRISPER, Clustered Regularly Interspaced Short Palindromic Repeats; LAMP, Loop-mediated Isothermal Amplification; POC, Point of Care. References as cited within the textName of testManufacturerTechnologyPrincipleType of testCountry of originReferencesDETECTRCo-produced by University of California and Mammoth Biosciences, San Francisco.Molecular (CRISPER-based diagnosis)Qualitative detection of nucleic acid from SARS-CoV-2 in specimens from suspected patients of COVID-19.Molecular diagnostic POC kit.USA[86]Algorithmic approach to diagnosis and treatment of COVID-19 in children.Research Institute for Children Health, IranAlgorithmic approach-based on available diagnostic and preventive strategies against COVID-19.Suspected children would be diagnosed via a set of defined protocols made from consensus obtained from large clinical testing.Protocols based testing by consensus statements obtained from Iranian experts in medicine.Iran[94]Detection of SARS-CoV-2 RNA using colorimetric-LAMP.New England labs and Wuhan Institute of Virology.Colorimetric-LAMP assayIsothermal nucleic acid amplification and colorimetric detection for diagnosis.Lab based test without use of complex infrastructure. Open for development of POC devices and kits with this approach.England and China[95]Paper-based LAMP assay for COVID-19 detection.National Tsing Hua University, Tri-Service General Hospital, National Defence Medical Center, Taipei and National Cheng Kung University Hospital.Paper-based technology along with LAMP assay techniqueAddition of LAMP specific reagents on a paper can produce a color change which can be uploaded by the patients on internets via smartphones at their homes.POC-RNA-based diagnostic deviceTaiwan[97]Article highlightsCoronavirus are positive sense single stranded RNA viruses enveloped in nature. A total of three viral outbreak happened in humans, one of them is a recent novel strain, SARS-CoV-2. Higher transmission rate has resulted in its spread to 213 countries causing a major impact on their socio-economic status.The virus is known to cause acute lung injuries and pneumonia. People with existing comorbidities and higher age are at increased risk of catching the viral infection. There are 12.8 million confirmed cases and 0.5 million deaths across the globe so far.Different technology based tests such as molecular, serological, radiological, and microbiological techniques are currently being employed for faster and efficient testing of the virus.rRT-PCR based molecular diagnosis is considered as one of the best approach in diagnosing the infection that aims to identify the genetic sequence of the viral genome however; each time a highly professional approach is required for interpretation of the results. Different viral proteins can be targeted for the identification purpose including N, E, S, RdRP, and ORF 1ab.Serological tests such as ELISA, chemiluminescent assay, and neutralization assay are followed on the principles of antigen antibody detection and various rapid diagnostic tests are made as an advancement in diagnosis.Radiological detection through use of chest-X-ray (CXR) and computed tomography scan (CT) aids in studying the COVID-19 affected lung lessions but the varied results obtained under different stages and condition of infection, suggests the diagnosis to be performed by combining molecular and radiological testing together.U.S. Food and Drug Administration (FDA) has provided emergency authorization for the use of various point-of care kits based on molecular and serological detection.Future research will aim at development of point of care devices and kits that can help in identification of the suspected patients from their homes so that visits to the hospitals could be minimized and more information could reach to health professionals and government agencies.The potential of techniques like loop-mediated isothermal amplification (LAMP), next generation sequencing, and CRISPER needs to be more extensively researched for bringing about the efficiency in diagnostic processes.Declaration of interestThe authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. 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