Studies on COVID-19

In March 2020, the World Health Organization (WHO) declared the coronavirus disease 2019 (COVID-19), which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a global pandemic (WHO, 2020). Surprisingly, within a few months, the viral infection had spread to many countries of the world, affecting millions of people worldwide. Therefore, it became essential to identify the source of transmission of such a deadly virus, as this would help prevent its further transmission and allow the development of an effective vaccine.

It is discovered that the genome of the COVID-19 has a 96.3% similarity to the genome of SARS-CoV-2. Further research proves that the main routes of transmission of COVID‐19 and SARS‐CoV‐2 are strongly associated with respiratory droplets and human to human contacts which further allows the diagnosis and laboratory testing of respiratory tract specimens (Coronavirus, 2020). In order to tackle this virus, laboratory practices for the COVID-19 were divided into three stages: pre-analytic, analytic, and post-analytic. The pre-analytical stage involves the collection of the proper respiratory tract specimen at the right time from the right anatomic site that plays an essential role for accurate molecular diagnosis of COVID-19. In the analytic stage, real-time RT-PCR assays remain the molecular test of choice for the etiologic diagnosis of SARS-CoV-2 infection. Finally, the post-analytical stage requires testing results that should be carefully interpreted by using both molecular and serological findings.

This paper is mostly focused on the clinical diagnosis of COVID-19 that was later confirmed by detecting SARS-CoV-2 RNA in molecular diagnostic laboratories by using the real-time reverse transcription-polymerase chain reaction (rRTPCR) (Shirato et al., 2020). In the first study,  researchers developed and compared the performance of three novel, real-time reverse transcription-PCR (RT-PCR) assays targeting the RNA-dependent RNA polymerase (RdRp)/helicase (Hel), spike (S), and nucleocapsid (N) genes of SARS-CoV-2 with that of the reported RdRp-P2 assay (Gao et al., 2020). Whereas, the second study used a multiplex rRT-PCR methodology for the simultaneous detection of two regions of the SARS-CoV-2 genome (E and N genes), and the human ABL1 gene (Beillard et al., 2003).

Hence, the main aim of the study was to understand the development of the diagnostic methodology for use in public health laboratory settings. A very rapid and accurate diagnosis of COVID-19 is confirmed by detecting SARS-CoV-2 RNA by molecular diagnostic laboratories using the real-time reverse transcription-polymerase chain reaction (rRT-PCR). The SARS-CoV-2 is a positive, single-stranded RNA virus, which means that it

directly translates its viral machinery from the RNA. In the first study, researchers developed specific COVID-19 real-time RT-PCR assays that were novel and highly sensitive that, essentially targetted RdRp/Hel, S and N genes of SARS-CoV-2. Then, they compared their performance-level by using both in-vitro and patient specimens to that of established RdRp-P2 assay.

The analytical sensitivity of these assays was determined by evaluating Limit-of-Detection (LOD) using viral genomic RNA extracted from culture lysates and clinical specimens. Therefore, the LOD of COVID-19-RdRp/Hel and COVID-19-N was found to be 1 log unit lower than that of COVID-19-S and RdRp-P2. Furthermore, COVID-19-RdRp/Hel and COVID-19-N assays were selected for evaluation and further determination of LOD by using in-vitro viral RNA transcripts. Lastly, the detection of SARS-CoV-2 RNA in clinical specimens was done for RdRp/Hel assay in comparison to that of the RdRp-P2 assay.

The analysis was performed on both the respiratory tract, using nasopharyngeal aspirates/swabs, throat swabs, saliva, and sputum specimens and on non-respiratory tract specimens such as plasma and urine specimens and feces/rectal swabs. The results showed that the COVID-19-RdRp/Hel assay was significantly more sensitive than the RdRp-P2 assay for the detection of SARS-CoV-2 RNA in nasopharyngeal aspirates/swabs or throat swabs, saliva, and plasma specimens while the sensitivity of the two assays was almost similar for sputum specimens and feces/rectal swabs.

Therefore, COVID-19-RdRp/Hel assay was considered to be highly specific and thus, exhibited no cross-reactivity with other common respiratory pathogens in vitro and nasopharyngeal aspirates. Also, the positive controls used in the experiment were made from in-vitro RNA transcripts by using the MEGAscript T7 transcription kit while the reagent used was Light Cycler Multiplex RNA Virus Master along with the LightMix Modular SARS. The Wuhan CoV E-gene kit was also used for an additional test that would detect SARS-CoV-2 coronavirus without cross-reactivity with other human-pathogenic coronaviruses.

Above all, the experimental protocols involved in this study followed the approved standard operating procedures of the biosafety level 3 facility. Therefore, the COVID-19-RdRp/Hel assay to test saliva specimens from suspected cases of COVID-19 could be a simple and rapid way to avoid the need for aerosol-generating procedures during the collection of nasopharyngeal aspirates and suction of sputum, especially in regions where there are insufficient supplies of full personal protective equipments. (Chan et al., 2020). Another study allowed researchers to compare Simplex rRT-PCR methodology to that of multiplex rRT-PCR. The Simplex rRT-PCR involved three reagents, named Taq Path 1-Step Multiplex Master Mix, Quanti Tect Probe RT-PCR Kit, and Light Cycler Multiplex RNA Virus Master. It also involves two probes, named, FAM/ TAMRA a single-quenching probe, and FAM/ZEN/IBFQ a double-quenching probe. However, the Taq Path 1-Step Multiplex Master Mix reagent and the double-quenching probe were selected because of low Cq value and improved signal-to-noise ratio.

Moreover, this procedure involved analysis of Limit-of- Detection by using serially diluted synthetic control RNA samples where Cq values and detection rates were calculated. The Multiplex rRT-PCR features simultaneous detection of two regions of the SARS-CoV-2 genome (E and N genes) and the human ABL1 gene. This system compared the three probe sets NIID-N, N_Sarbeco and E_Sarbeco where the Cq values for all three probes were almost similar. The NIID_N and E_Sarbeco sets were considered because of their sensitivities for the multiplex. However, the sensitivity of the multiplex RT-PCR could be affected by the nucleic acid extraction method, the one-step RT-PCR reagent, and the probe sets. For this reason, the human ABL1 gene was used as an internal control (IC) for checking the qualities of the specimen, nucleic acid extraction step, and RT-PCR amplification and monitoring for minimal residual disease detection.

Additionally, the Multiplex rRT-PCR system was used to obtain the results of the SARS-CoV-2 tests. This experiment used two controls where one was a synthetic RNA control with the N- gene which was taken from NIID. The second control was prepared in their laboratory by using a SARS-CoV-2 test positive total nucleic acid solution (extracted from the clinical specimen) and total RNA solution (extracted from the K562 cell line). The probes and sequences that were used in Multiplex rRT-PCR are shown in Appendix-1 (Ishige et al., 2020). There are also some common types of equipment and reagents that are required for establishing a laboratory for the testing of the COVID-19 virus through PCR.

The equipment and consumables include the following: a Biosafety cabinet (BSL-III), adjustable pipettes (ranging from 0.5 uL to 1000 uL), filtered pipette tips (ranging from 0.5 uL to 1000 uL), micro-centrifuge tubes (1.5 mL, and 2.0 mL), PCR strips with caps, 96 well PCR plates, adhesive seals for 96 well PCR plates, benchtop centrifuge machines, 8-strip PCR tubes, 96 well PCR plates, refrigerators (4°C, and -20°C), a clinically approved real-time PCR machine (Bio-Rad, Applied Biosystems, or Roche), an Autoclave machine, and computers. Additionally, reagents required for a laboratory include a Viral Transport Medium (VTM) for the collection of samples for COVID-19 testing, kits for the isolation of viral RNA, kits for real-time PCR reactions, molecular biology grade water  (Coronavirus, 2020).

In addition to this, testing COVID-19 accurately is extremely important so precise diagnosis occurs. Therefore, during the performing of this testing, prompt quality measures are required to be taken to maintain the diagnostic accuracy. However, there are some common sources of errors while performing COVID-19 testings. First, Sections of VTM receiving/sorting/labelling, RNA isolation, and PCR reaction setup must be conducted separately from each other. Second, the preparation of PCR reagents and mixing of RNA into PCR reagents must be performed in separate sections to avoid contamination. Third, although the VTM is stable at ambient temperature for hours, it is preferable to store it at -20°C so that the viral RNA is saved from degradation. Fourth, preferably, the PCR kit with the stable probe should be used to avoid false-positive results that otherwise arise from the breakage of probes at later cycles of PCR reaction. Fifth, the results of PCR must be interpreted strictly following the manufacturer’s guideline following extensive validation and calibration. Lastly, gloves should be changed frequently to minimize cross-lab contamination (Corman et al., 2020).

It is essential, in order to test the individuals within the class for infection with COVID-19, to adopt the standard procedure according to the World Health Organization (WHO) (Coronavirus, 2020). Upper respiratory tract specimens such as nasopharyngeal and oropharyngeal swab or wash in ambulatory patients would be collected in a viral transport medium. The swab will be allowed to remain dipped in VTM for about 1 hour for maximum transfer of the viral copies into the VTM. The viral RNA would then be extracted from a designated volume of VTM following the kit manufacturer guideline. The extracted RNA would then be transported to the PCR section, where the PCR reaction would be set-up according to the kit protocol along with the positive and negative controls. The PCR reaction would be conducted using the PCR machine following the recommended thermal cycling conditions.

If no observable threshold value (Cq value) is obtained in the  PCR result, the person would be considered as negative for COVID-19. If a Cq value of less than the threshold set by the kit is obtained, the person would be considered positive for COVID-19 infection. If a Cq value is obtained near the threshold value (±1 of the threshold), the testing would be repeated from the RNA isolation step. If a similar Cq value is obtained again, the person would be considered as positive for the COVID-19 infection with low viral load (Ishige et al., 2020).

This pandemic has also forced addressed current issues regarding such testing for SARS-CoV-2 to be addressed that were preventing successful assessment for the virus. For instance, a nasopharyngeal swab rather than an oropharyngeal swab was recommended for early diagnosis or screening because it provides higher diagnostic yields, is easily tolerated by the patient and is also safer for the operator. Also, the researchers used the COVID-19-RdRp/Hel assay came to be recommended in usage because it did not cross-react with other human-pathogenic coronaviruses and respiratory pathogens in cell culture and clinical specimens, whereas the RdRp-P2 assay cross-reacted with SARS-CoV in cell culture.

In a study with the multiplex- rRT-PCR, the WHO recommended the detection of at least two different targets on the COVID-19 virus genome because there were cases where NIID_N was weakly positive and N_Sarbeco was negative, so researchers found it hard to determine which results were true positives or false positives Thus, this indicated the need for another assay with sensitivity equal to the NIID-N set to detect two regions of the COVID-19 genome with high sensitivity. These assays are safe, simple, and fast and can be used for treatment in local clinics and hospitals that already have the needed instruments and who are responsible for identifying and treating patients with COVID-19.

In conclusion, the ongoing outbreak of COVID-19 infections worldwide has focused resulted in the urgency of the laboratory diagnosis of human coronavirus infections in order to limit the spread and the need to appropriately treat patients who have serious conditions related to the virus.