key: cord-0988646-mb5dlixl authors: Hemida, Maged G. title: The next‐generation coronavirus diagnostic techniques with particular emphasis on the SARS‐CoV‐2 date: 2021-03-26 journal: J Med Virol DOI: 10.1002/jmv.26926 sha: ee6e2e427d0436a4601d49defa4a69bf12074caa doc_id: 988646 cord_uid: mb5dlixl The potential zoonotic coronaviruses (SARS‐CoV, MERS‐CoV, and SARS‐CoV‐2) are of global health concerns. Early diagnosis is the milestone in their mitigation, control, and eradication. Many diagnostic techniques are showing great success and have many advantages, such as the rapid turnover of the results, high accuracy, and high specificity and sensitivity. However, some of these techniques have several pitfalls if samples were not collected, processed, and transported in the standard ways and if these techniques were not practiced with extreme caution and precision. This may lead to false‐negative/positive results. This may affect the downstream management of the affected cases. These techniques require regular fine‐tuning, upgrading, and optimization. The continuous evolution of new strains and viruses belong to the coronaviruses is hampering the success of many classical techniques. There are urgent needs for next generations of coronaviruses diagnostic assays that overcome these pitfalls. This new generation of diagnostic tests should be able to do simultaneous, multiplex, and high‐throughput detection of various coronavirus in one reaction. Furthermore, the development of novel assays and techniques that enable the in situ detection of the virus on the environmental samples, especially air, water, and surfaces, should be given considerable attention in the future. These approaches will have a substantial positive impact on the mitigation and eradication of coronaviruses, including the current SARS‐CoV‐2 pandemic. This protein plays an important role in virus replication, pathogenesis, and immune response. The coronaviruses-S protein is one of the most important targets for vaccine development, therapy as well as in the development of various diagnostic assays. [9] [10] [11] [12] [13] [14] [15] [16] The S protein is composed of two subunits, S1 and S2. The S1 is much more variable while the S2 is conserved. 17 The S protein is usually inert until it infects its host then utilizes the host cell's enzymes to cleave the S into S1 and S2. This step is the most crucial in the coronavirus replication cycle. The S1 gene contains two hypervariable regions (HVR) as well as the receptor-binding domains. 18, 19 Some sequences within the HVRs are the primary determinants of the tropism and immunogenicity of many coronaviruses. 20 The coronavirus genomes are prone to changes at short intervals due to different factors. These changes may result in the emergence of new viruses, new virus strains, and clades or subclades of the same virus. The main reasons behind these changes are the accumulated mutations because of the poor proofing capabilities of their viral polymerases. This is in addition to the possibility of viral genome recombination and the fast adaptation to a new host, particularly animals. 21 In addition to the ongoing changes in the genetic makeup of coronaviruses, many factors hampered the development of diagnostic assays and vaccines and antiviral therapy, particularly for the SARS-CoV-2. Among these factors, the erratic immune response, the kinetics of the pathophysiology, the existence of asymptomatic patients. [22] [23] [24] [25] There are many laboratory techniques for the diagnosis of coronaviruses in general, including the SARS-CoV-2. There are several approaches to the laboratory diagnosis of coronaviruses. The direct approaches, which mainly based on the detection of the virus particles, isolation of the virus, antigens, or viral nucleic acids (NAT). The indirect techniques, which mainly depend on the detection of viral-specific antibodies in sera of infected/recovered patients. Another approach is the identification of some specific immunological markers for SARS-CoV-2 in the sera and blood of infected/recovered patients. These markers include some differentially displayed immunology markers, such as CD4+-T, CD8+-T cells, and interleukin 6 (IL6). 26, 27 This article will discuss the urgent need for the development of novel next-generation laboratory diagnostic assays for coronaviruses with special emphasis on the currently available assays, their advantages, and pitfalls. Figure 1 (Figure 2 ). Some generalized samples can be considered, such as the whole blood, the sera, the conjunctival (ocular) swabs, tears, and urine ( Figure 2 ). This is in addition to some autopsy or biopsy specimens from some tissues ( Figure 2 ). The selection of the right sample at the right time is the milestone in the identification of cases and for the follow-up during viral infection. In some COVID-19 cases, the URT showing negative reverse-transcriptase polymerase chain reaction (RT-PCR) testing at the beginning of the infection. In that case, it is recommended to repeat the test for both the URT and LRT samples to monitor the progress of the diseases. 28 Since the Table 1 is showing a comparison between the coronavirus diagnostic assays based on the coast, the reaction time, high-throughput potential, specificity, and sensitivity. Each diagnostic assay requires the presence of certain equipment, facilities, and can be done on a limited number of samples or can be adjusted to screen a large number of samples to cope up with the high workflow of testing a large number of samples as in the case of the current pandemic. Table 2 is showing a comparison of the suitability of the coronavirus diagnostic assays to certain a facility, the possibility of their conduction under the rural and urban regions or in high biosecurity laboratories. During the early phase of the SARS-CoV-2 pandemic, there was a high demand for the laboratory diagnosis of patients admitted with signs of pneumonia using real-time PCR. With this high number of cases admitted to the hospitals at a certain place, some hospitals were not able to meet the screening of a large number of cases per day. Based on this situation, some health authorities decided to rely on some clinical examination of the suspected cases, particularly using chest radiography. Patients who showed signs of pneumonia by chest X-ray and CT scanning were considered positive and started to receive the course of treatment available at that time immediately to save more lives. 29 This procedure has many pros and cons. The main advantage of this approach is the low cost of the technique since one machine can perform lab screening of a large number of patients per day. It is usually very quick and can be performed in a remarkably short time. It can be used to monitor the progress of the health condition of some patients and the success of the course of certain treatments. On the other hand, this clinically based diagnosis is not pathogen-specific. The signs of pneumonia and lung consolidation could be due to many different pathogens and allergic conditions; thus, it requires further laboratory-supporting techniques specific for the target causative agent, particularly SARS-CoV2 in this situation. The unique crown or solar morphology of coronaviruses gives a very good privilege for the detection of the virus particles under the electron microscope (EM) through the negative staining technique. 30 The EM was used to detect coronaviruses in the stool of children suffering from acute enteritis caused by the HCoV-229E, HCoV-OC43, HCoV-NL63, and HCoV-HKU1. 31 The ultra-resolution electron microscope was also used to detect SARS-CoV. 32 This technique was used as a routine diagnostic assay for several viruses, including coronaviruses, in the early 1980s. This technique was used as routine work for the detection of some enteric viruses of humans, animals, and birds, particularly the coronaviruses, rotaviruses, and enteroviruses. The sample of choice is usually the fecal or cloacal swabs from animals and birds, respectively, and stool samples from humans. Although this method is simple and very specific, this technique requires the availability of some expensive equipment, such as the high-speed centrifuge and the availability of electron microscope units. Moreover, the SEM is not suitable for screening a large number of samples and requires expert technical personnel. [33] [34] [35] It may also take a long time for sample preparation to be able to detect the virus particles under the EM. 35 The classical plaque purification assay is recently used to purify the SARS-CoV-2 for further testing. The plaque purified virus is suitable for some downstream applications, especially the development of the specific vaccine as well as diagnostic assays. [36] [37] [38] Having pure virus particles is the ideal scenario for downstream virus research, including vaccine preparation, antiviral therapy, and diagnostic assays development. This approach was recently used to purify the SARS-CoV-2 to study virulence in a purified virus population in vitro in the Vero cell line. 38 The plaque assay was used for the infectivity titration of many coronaviruses, including SARS-CoV, MERS-CoV, and SARS-CoV-2. [38] [39] [40] This approach using some mathematical calculations to demonstrate the infectivity of some coronaviruses in a given sample. Although this technique was developed a long time ago, 41, 42 it is still one of the best methods for the titration of the virus infectivity for viruses that grow well in the cell culture and able to F I G U R E 2 Schematic representations of different types of samples for the diagnosis of coronaviruses, especially SARS-CoV-2. Summary of the potential samples in case of SARS-CoV-2 infection, as suggested by the WHO and CDC. This diagram showing the type of samples to be collected from various body organs and locations, including the upper/lower respiratory tracts, the digestive tract as well as some generalized samples HEMIDA | 4221 induce cytopathic effects, including some coronaviruses. 43, 44 The plaque assay has many applications in the diagnostic corona-virology as the titration of the virus infectivity and the evaluation of the efficacy of some antiviral compounds and vaccines for coronaviruses. [45] [46] [47] [48] 3 | ISOLATION OF CORONAVIRUSES Isolation of any pathogen is the milestone of the identification and characterization of this pathogen. After the emergence of SARS-CoV-2, there are well-known seven human coronaviruses identified in the past 60 years. 49, 50 The first reported human coronavirus was the HCoV-229E was identified through the isolation of the causative agent of a common cold in some patients in the United States. 51, 52 They used the human embryonic tracheal organ cultures to isolate this virus, which was initially called B814. 51, 52 They examined this virus under the electron microscope and confirmed its morphology to belong to the coronaviruses. In a similar approach, the HCoV-OC-43 was discovered in 1967 by isolation of the virus in the tracheal cultures. 53 One of the advantages of both viruses is their ability to grow on cell cultures, which allows their characterization in more detail. In late 2002, the SARS-CoV was first identified. 54, 55 The virus was isolated using the fetal rhesus kidney (FRhK-4) cells. 54, 55 In 2004, another human coronavirus was identified in Netherland called HCoV-NL-63. They used the tertiary monkey kidney cells and the monkey kidney LLC-MK2 cell line to isolate the virus from a young child. 56 Another coronavirus similar to the HCoV-NL-63 was identified in a group of children in Hong Kong who suffered from typical respiratory distresses, including fever, coughing, and shortening of breath. 57 One study used the human-ciliated airway epithelial cell cultures (HAE) for isolating the HCoV-HKU-1 virus. 39 T A B L E 1 Comparison of the coronavirus diagnostic assays based on the coast, the reaction time, high-throughput potential, specificity, and sensitivity More recently, the SARS-CoV-2 was identified in late 2019, which was initially isolated on human airway epithelium from the broncoalveolar lavage of some patients. 1 An interesting study showed that the HAE cell culture is a universal system for the cultivation and isolation of the most common human coronaviruses. 59 Although isolation of coronaviruses is one of the gold standard diagnostic assays, it is time-consuming and requires the availability of certain cell types of cell cultures and, in most cases, a high biosafety contaminant laboratory, particularly (BSL-3) facilities. The antigen-based detection tests are usually the simplest diagnostic assays because they can do in a very short time. The test can be conducted in as fast as a few minutes. It does not require skilled personnel to be conducted; meanwhile, in most cases, it does not require expensive machines to be carried out. However, the specificity and sensitivity of the antigen-based assays are usually less in comparison to most NAB assays. Meanwhile, these assays require known specific antibodies to be carried out. Thus, the antigen-based detection assays could be useful initial screening tests that require further confirmation by other NAB techniques. 60 One study was conducted to compare the performance of some antigen detection assays to that of the RT-qPCR assay in the detection of SARS-CoV-2 Abbreviations: (+), criteria fulfilled; (-), criteria not fulfilled; (±), criteria fulfilled in some cases. | 4223 thio-nicotinamide adenine dinucleotide (thio-NAD) cycling in association with the double antibody sandwich ELISA. In this assay, the thio-NAD cycling allowed a high sensitivity starting 10 min of the application of the assay. This assay is considered one of the ultrasensitive antigen detection assays, characterized by high specificity and ultrasensitivity for the detection of SARS-CoV-S antigen. 63 3.2 | Immunofluorescence assay (IFA) The IFA is one of the old techniques that is still in use and valid for many pathogens, including a large number of coronaviruses. Some recent studies used the IFA to detect the SARS-CoV-N antigen in the throat wash of some infected patients. 64, 65 Figure 3 is showing a summary of the common FAT used in the diagnosis of various types of coronaviruses. The immunochromatographic-based assays have been recently developed to detect the SARS-CoV-2 in the saliva of infected patients. 66 These techniques should be coupled with another confirmatory NAT test to achieve an accurate diagnosis of coronavirus infection in clinical samples. Although these techniques are simple and convenient, their specificity and sensitivity still require further evaluation. The AST used some bio-dot microfiltration units to detect the antigens of some coronaviruses, such as the bovine coronaviruses (BCoV). This technique may be handled as the ELISA-based assays for antigen detection. 67 The ELISA technique is used for the detection of some coronavirus antigens, such as IBV, BCoV, and MERS-CoV. [67] [68] [69] In this approach, The NAT assays are widely used for diagnostic and research purposes for coronaviruses. The rationale behind these assays is the amplification of certain target regions of the coronavirus genome. The success of these assays depends on many factors, such as the selection of a unique amplification target, the collection of the representative sample at the right time, the processing of the specimens, the amplification parameters, and the interpretation of each reaction. Table 3 is showing a comparison between the classical and the recent techniques for the diagnosis of various coronaviruses, especially SARS-CoV-2. The RT-PCR, the real-time quantitative PCR (qRT-PCR), is the most common technique widely used not only for coronaviruses but also for large numbers of viruses. 7, 70, 71 Theoretically, the qRT-PCR technology should detect a minimal quantity of the viral-NA, even one copy of the genome. 72 Despite the high sensitivity and specificity and broad application of the qRT-PCR in the process of coronaviruses diagnosis, this technique requires the availability of expensive real-time PCR machines, which may not be available at many laboratories or point of care settings. 73 Meanwhile, this technique also required high-trained personnel to experiment. This is in addition to the time required to conduct the experiment and to ship the samples from some small hospitals or clinics to be tested in a larger facility apart from the site of collection. This will increase the time required to get the test A known antibody is allowed to adsorb to the solid-phase ELISA. The tested antigen is added and allowed to react at appropriate conditions and time. A second antibody specific to the antigen is allowed to react at appropriate conditions. Conjugated antibodies with enzymes showed to be added and react at appropriate conditions. The substrate is added for the standard time then the reaction stopped using the appropriate solution. The plates are read using a special spectrophotometer or ELISA reader. (B) Indirect ELISA: a known antigen is attached to the solid phase and allowed to adsorb for appropriate time and conditions. The unknown antibody is added to the plates and allowed to react at appropriate conditions. The conjugated antibodies are added and allowed to react with the mixture for appropriate time and conditions. The substrate is added and allowed to react for an appropriate time. The reaction is stopped using the stop solution. The optical densities of each reaction are read using the ELISA reader at the appropriate wavelength. (C) Competitive ELISA. The known antibodies are allowed to adsorb to the solid phase. The enzyme-labeled antigens plus the unknown antigens are allowed to react in some wells, while the enzyme-labeled antigens are added to other sets of wells. The substrate is added and allowed to react. The reactions are read at the appropriate wavelength using the ELISA reader T A B L E 3 Comparison between the newer next-generation assays over traditional methods 89 Another privilege for the LAMP assays in the context of viral diagnosis is the ability to perform the test with the RNA extraction approach or without RNA extraction (directly on the sample). The latter approach is called direct RT-LAMP. 87 It is also known that the performance of the PCR is usually affected by the presence of some specific inhibitors in the tested samples. 90 The high efficiency of the enzymes used in the LAMP techniques makes it relatively less affected by the presence of inhibitors. 91 The detection limit of this assay is as low as 1-10 copies of the RNA molecules of the SARS-CoV-2. 87 The RT-LAMP reactions usually contain a mixture of several chemicals and enzymes, such as the high thermal tolerance DNA polymerase enzyme used in the regular PCR techniques, a set of up to six specific primers, and aliquots of the RNA of the tested samples. This reaction is usually exposed to a fixed temperature for 20 min at 65°C before the actual reaction. 92 The protein-based assays have been developed to enable the detection of some coronaviruses in the sera of humans and animals. 106, 107 An earlier study developed a recombinant-based SARS-CoV-N recombinant ELISA that had high specificity and sensitivity for the detection of antibodies against SARS-CoV. 107 One study developed a novel MERS-CoV-S1-based assay, mainly targeting two different regions across the MERS-CoV-S gene. The first is designed to target the receptor-binding domains that enable the viral attachment to its cellular receptors, the dipeptidyl peptidase-4 (DPP4), and the second one is designed to target the N-terminal domains of the MERS-CoV-S1 gene. 106 The assay that used the first approach showed high specificity for the detection of MERS-CoV antibodies and showed a high degree of consistency to the PPNT discussed earlier for the detection of MERS-CoV neutralizing antibodies. 106 This study mapped and designed two important epitopes within the SARS-CoV-S, and N proteins then used them to develop IFA-based assays that can be conducted in BSL-2 laboratories. 109 The main advantage of the newly developed assay was its safety, which does not require the handling or manipulation of the SARS-CoV. 109 Those assays showed similar sensitivity and specificity to that of the whole-virus-based IFA assay. Meanwhile, the IFA techniques were used to study the molecular pathogenesis and intracellular localization of some other coronaviruses, such as MERS-CoV. 110 Some other studies compared the performance of some ELISAs and IFAs techniques in the detection of the SARS-CoV-2-IgG antibodies in the moderate and severe cases of infections during 10-18 days postinfection. 111 Initially, the LFIA was used to detect several biomarkers, especially cortisol in the saliva, with great success. 112 Using the LFIA, saliva was an ideal biological sample when fast results are needed compared to other biological fluids, such as urine, tears, and blood. 112 This approach was designed to detect either the viral antigen or its antibodies by using portable strips or dipstick, which carry the required reagents to conduct the assay. 100 Usually, one drop of human plasma is placed on a strip containing monoclonal antibodies (mAb) against SARS-CoV-2. If the patient were exposed to an active SARS-CoV-2 infection, the mAbs would recognize the counter antigen, and the reaction is developed. 100 Although this technique is very simple, it requires the preparation of some expensive mAb; meanwhile, this technique requires validation by using positive SARS-CoV-2-infected blood. Meanwhile, the LFIA can be used to detect the antigens in the saliva and the secretions of the URT of the infected patients. 113 Saliva is proved to be one of the more convenient samples to be collected for screening a large number of people in short time. It is very simple and easy to collect, store, and transfer to the laboratory. Meanwhile, saliva collection is a noninvasive technique, which does not harm the patient during the collection compared to other samples, such as the bronco alveolar lavage and the nasopharyngeal swabs. 113 The LIP assay is a new technique based on the luciferase and the immunoprecipitation activities in the detection of many viruses, such as RSV as well as SARS-CoV-2 antibodies. 114, 115 It is a sensitive technique used for the quantitative estimation of antibodies against the SARS-CoV-2-N and S proteins. 115 One of the privileges of this approach is its ability to detect the epitopes in their native conformational form as well as the linear epitopes. 116 Another privilege that the LIPs assay provides is the detection of the immunological profile of various classes of SARS-CoV-2 infected people with various clinical outcomes. 116 3.20 | The gold-immunochromatography assay (GICA) The colloidal gold Immunochromatography assay has been used for the detection of many pathogens, including bacteria, parasites, and viruses, in addition to many other hormones in the milk or sera. [117] [118] [119] [120] The GICA was showing a promising trend in the diagnosis of some high influenza virus strains, including influenza type B and some of the highly pathogenic influenza subtypes, such as H7N7. 121, 122 The GIA has many advantages as a rapid, sensitive technique that do not re- Although serological assays are really important in the diagnosis and epidemiological studies of many coronaviruses, they do have several pitfalls that require fine-tuning to achieve maximum performance. 133 The problem is serological techniques are usually used to measure the immune response, not the causative agent/s. Thus, we cannot rely on a single serum sample to assess the disease condition of some patients or diseased animals. 134 The more practical way is to test two serum samples collected during the acute febrile stage of the diseases and another sample collected about four weeks later. If the antibody level in the second (convalescent) sample is greater than the first (acute) sample, this considers an infection with the pathogen under study. This is the common concept for seroconversion, not only for coronaviruses but also for other pathogens. 135 various coronaviruses, such as MERS-CoV, which infect dromedary camels, and the BCoV. 128 To overcome the problem of crossreactivity, the development of very specific serological techniques for each coronavirus candidate should be adopted. Both the microneutralization and the PPNT assays were used to overcome the problem of the cross-reactivity of various coronaviruses. 103, 108, 128 Further studies are required to improve the performance of various serological techniques and improve their performance in the field of coronaviruses research and diagnosis. The most common theme of the coronavirus antigen detection assays is the application of known and specific antibodies against the tested antigen. The cross-reactivity among human coronaviruses was reported on many occasions. 138, 139 In many techniques, the used antibodies are polyclonal; thus, they may pick up several antigens of closely related viruses 138,139 that may hamper both the specificity and sensitivity of these antigen-based assays. To overcome this problem, the known antibody used in these assays should be produced against a single producing clone or monoclonal antibody against the unknown antigen. Sometimes improper sampling techniques may be reflected in the outcomes of the antigen detection tests. A low concentration of the viral antigen in the tested sample may be prone to a false-negative. Based on the frequent emergence of new coronavirus candidates, the N-CoV-DA has to cope-up with this diverse group of viruses. There should be some novel next-generation diagnostic assays for coronaviruses. These assays should provide new features that do not exist or are weakly presented in the current techniques. As there is a growing number of coronavirus due to the emergence of new candidates, there is a mandate for the screening of one serum sample for a large number of coronavirus targets in one reaction simultaneously. This will have a high impact on monitoring the dynamics and distribution of coronaviruses in specific communities. Meanwhile, it will help in the monitoring and assessment of the im- Most diagnostic assays, especially those based on the NAT-based approaches or those based on the recombinant proteins, depend on specific sequences across the coronavirus genomes. The continuous mutations and changes of the coronavirus genomes render some diagnostic assays outdated. Thus, frequent monitoring of the coronaviruses on their genomic levels is highly suggested, and to upgrade the diagnostic tests especially those NAB assays to match the circulating strains. The coronaviruses' NAT based on specific primers and probes should be prepared based on the most conserved regions of the genomes. Nevertheless, regular assessment of the specificity and sensitivity of these assays should be practiced. The same concept should be adopted from any upcoming recombinant vaccines. These potential coronavirus vaccines should be prepared from the closely related circulating strains of the target coronavirus. This approach is the widely used strategy for the preparation of seasonal influenza virus vaccines. 168 The proper sampling procedure and technique are the milestones for the diagnosis of coronaviruses. The WHO and the CDC provided detailed interim guidelines for the collection of samples from COVID-19 patients. 169 There are many types of suggested samples (nasopharyngeal, oropharyngeal, nasal midturbinate swab, the nasal swab, and the nasopharyngeal wash/aspirate) for the diagnosis of respiratory viruses in general, especially the SARS-CoV-2. These samples should be collected under complete medical supervision. Each type of sample has a standard protocol, as suggested earlier. 169 Failure to apply the guidelines per each sample may result in falsenegative for the downstream testing. One common error is the col- The timing of the coronavirus sample collection is a very crucial step in the success of some diagnostic assays. 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Development and performance assessment of a luminex xMAP (R) direct hybridization assay for the detection and identification of indoor air fungal contamination How to cite this article: Hemida MG. The next-generation coronavirus diagnostic techniques with particular emphasis on the SARS-CoV-2 The authors declare that there are no conflict of interests.