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Current Diagnostic Approach for COVID-19

Nitika Thakur1 and Rachit Sood1

1Shoolini University of Biotechnology and Management Sciences, Faculty of Applied Sciences and Biotechnology, Department of Biotechnology, Solan, Himachal Pradesh, India

1.1 Introduction

The ongoing spread of coronavirus has presented a threatening scenario globally because of the non-availability of accurate and rapid detection methods. However, on 30 January 2020, World Health Organization (WHO) has declared "COVID-19" (coronavirus disease 2019) as the largest threat under "public health emergency of global concern," as it is alone responsible for 250?000-260?000 deaths worldwide and across 3-3.5?million positive cases [1].

The detection and analysis procedure for this threatening virus started initially with a virus detection method, which somewhat has an advantage of non-detection of long culture cycles. Another way of detection is through the use of "nucleic acid profiling," which [2] can rapidly, sensitively, and accurately detect the pathogens in confirmed COVID patients, but large amounts of genetic variations, mismatches in primers, probes, and some target sequences may result in interpretation of false results. Detection via genomic sequence analysis and the point-of-care diagnosis have become popular in the detection of emerging viruses for finally detecting the specific antibodies IgM and IgG related to COVID [3].

Section 1.2 describes and highlights the current diagnostics and treatment strategies for COVID-19.

1.2 Recommended Laboratory Diagnosis for COVID-19

1.2.1 SARS-CoV-2 Testing: Detection Approach by Screening Suitable Specimen Cultures

The first and foremost step in diagnosis and identification is related to the appropriate collection of suitable specimens, which [4] are being collected from the upper and lower respiratory tracts, WBC's, and serum specimens. Furthermore, it has been mostly detected and screened from the swabs pertaining to nasopharyngeal area, oropharyngeal, sputum, stool samples, urine, saliva, conjunctival area, and rectal swabs [5].

It is recommended that the samples and swabs should be strictly collected from the lower respiratory tract, for confirmatory diagnosis, even if the upper respiratory swab analysis is negative for COVID-19, as the receptor "AEC 2" is actively distributed in the alveolar lining of epithelial cells. Various studies compared [6] the viral loads from the lower respiratory tract specimen for the suspected and confirmed COVID patients. The study further stated that the average viral load differed in different collected samples [7], as the viral load detected in sputum was higher around 17?420?±?6925?copies/test than the nasal swabs (655?±?502?copies/test) and throat swabs (2555?±?1965?copies/test). In addition, high viral load was also recorded in swabs collected from [8] the lower respiratory tract. Most of the cases were examined and confirmed positive through isolation and culturing techniques from oral swab on the first day, followed by a five [9-11] day diagnosis of anal swabs, indicating a shift from early period diagnosis to late period diagnosis. However, in asymptomatic conditions, it can be detected by analysis of urine sample, with no urinary irritation symptoms. Recently, it has also been detected in samples of saliva. In addition, it has been detected in nasopharyngeal swab, conjunctival tear swabs, and [12] oropharyngeal swabs. However, there still exist glitches in terms of monitoring and isolation process to screen conjunctival secretions for confirmatory diagnosis. Currently, the [13] virus has not been traced in many samples such as cerebrospinal fluid, semen, pericardial effusion, female reproductive tract, etc.

1.2.2 SARS-CoV-2 Detection: The Nucleic Acid Approach

For successful diagnostic strategies, identification of some specific primers and probes is important to screen out the target sequences. These target sequences for COVID-19 involve the "envelope - E," "the nucleocapsid - N," "spikes - S," "RNA-dependent RNA polymerase," and "open reading frame - ORF." WHO further recommends [14] reverse transcription polymerase chain reaction (RT-PCR) as a routine recommendation but lacks suitability in terms of time consumption, requirement of expensive equipment and biosafety conditions.

1.2.2.1 COVID-19 Detection Approach Through Real-Time PCR

The target gene sequences for detecting CoV-2 vary globally from China (ORF's), the United States (3?N gene), Germany (RdRp, N, and E genes) to France (two targets in RdRp). Center for Disease Control and Prevention (CDC) established a RT-PCR process for the detection and analysis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), with three specific primer sets to detect ß forms of CoV-2 and the other two for SARS-CoV-2.

Different countries have a [15-18] large number of qRT-PCR (quantitative reverse transcription polymerase chain reaction) protocols provided by the official WHO website, which play the principal role in the detection of SARS-CoV-2. In recent time, different countries are following different protocols of gene targeting for the detection of SARS-CoV-2, for example, France (two targets in RdRp aka RNA-dependent RNA polymerase), Japan (pancorona and numerous targets, spike protein), the United States (three targets in N gene), China (N genes and ORF1ab), Thailand (N gene), and [19-21] Germany (RdRp, N, and E genes). Different institutes use different RT-PCR primers or tests for the detection of SARS-CoV-2. A new RT-PCR panel has been rooted by the CDC for the universal detection of SARS-like ß-CoVs and specific detection of SARS-CoV-2. For the N gene [22-25], three sets of distinct primers were devised - two sets of probes or primers were specific for identifying SARS-CoV-2 and the last set was universally used for detecting all ß-CoVs. COVID-19 must be confirmed as positive for all the three individual targets. The Charite (Germany) developed two nucleic acid tests for the detection of E genes of the bat-like ß-CoVs, SARS-CoV-2, and [26] SARS-CoV. If both of the tests are positive, only then it could enter the next level/step of detection, which is for the RdRp gene and is called the SARS-CoV-2-specific RT-PCR test [27].

Despite the various protocols developed by numerous institutions for SARS-CoV-2 testing, it is still not crystal clear whether the outcomes of the [28-31] nucleic acid tests based on the different targets can be compared or not. Various RNA transcripts that were extracted from a COVID-19 patient by Chantal et al. were used to study the detailed analytical sensitivities of the four qRT-PCR assays rooted in Hong Kong, Germany, China, and the United States. According to a study, in all the primer-probe sets enforced in the qRT-PCR tests, SARS-CoV-2 could be identified; however, there was a significant disparity in the ability to find the positives and negatives with a lesser viral load and in the detection limit. HKU-ORF1 (Hong Kong), 2019-nCV_N1 (United States), and E-Sarbeco (Germany) were found to have the highest sensitivity primer-probe sets, while RdRp-SARSr (Germany) had the lowest sensitivity, which can be due to the mismatching in the reverse primer. Also, the sputum samples or nasopharyngeal swab from the [32-34] COVID-19 patients (Germany) were used for comparing the qRT-PCR tests in a commercial reagent and different polymerase chain reaction (PCR) systems. A clear discrepancy in the analytical sensitivities among different PCR systems was detected when the same probes and primers were used. The results concluded that when a one-step qRT-PCR system was used, the RdRp [35] target was less sensitive than the E gene target. However, the test evaluation was not crystal clear as it was disturbed by the high background nature of the E gene target. The sensitivity may be improved by the additional optimization of the E gene assay [36].

1.2.2.2 Detection Approach Through Nested RT-PCR

To detect the low-copy-number SARS-CoVs present in the early stage of the disease, real-time nested RT-PCR assay [37] is the perfect choice as it bridges the real-time instruments (time-saving) with the high sensitivity of the nested PCR. The identification of the SARS-CoV-2 with the help of nested RT-PCR has already [38] been verified in countries like Japan during the initial days of the pandemic. This technique had already detected 20-25...