Exploring Demographic Properties and Amplification Efficiency of N and ORF-1ab Gene in Suspected COVID-19 Patients in Dhaka, Bangladesh

: Background: In late December 2019, an outbreak of pneumonia with an unknown cause occurs in Wuhan, China. Later, the causative agent was identified as SARS-CoV-2. Early laboratory diagnosis plays an important role in any outbreak by cutting the spread of infection by isolating the infected person. Nucleic acid detection via PCR remains the gold standard because no alternative scientific method for detecting SARS-CoV-2 is available. Objective : The goal of this study was to learn more about the demographic characteristics of SARS-CoV-2-affected people and the sensitivity pattern of the N & ORF-1ab gene among them. Methods: A swab from the nasopharynx for nucleic acid detection was collected from 3183 SARS-CoV-2 suspected cases attending outdoors and indoors at Aichi Hospital and Japan East-West Hospital. Results: 649 (20.4%) were found PCR positive. Among the positive cases, the male was 478 (73.7%) and the female was 171 (26.3%), mean age was 40.35 ± 14.551 years, ranging from 4 – to 90 years. The highest prevalence of SARS-CoV-2 was identified in the 21–40 years old group (50.7%), followed by the 41–60 years old group (33.1%). The N gene was found to be 100% positive and ORF-1ab was 22.03% positive. Conclusion: According to the findings of this study, males and younger generations are at an elevated risk of contracting the virus, and maybe a source of infection for other family members. According to this study, the N gene has higher sensitivity and amplification efficiency than ORF-1ab.


INTRODUCTION
On December 31, 2019, a novel coronavirus that causes pneumonia was discovered in Wuhan, China. 1 Since the first case was identified, 42,966,344 confirmed cases and 1,152,604 deaths have been reported to the World Health Organization (WHO). 2 Because the virus is highly contagious and spreads quickly in nature, WHO classified the sickness as a pandemic on March 11, 2020. 3 The first incidence was confirmed in Bangladesh on March 8, 2020. Following that, a total of 398,815 confirmed cases and 5,803 deaths were reported to WHO until October 26, 2020. 4 It was first known as 2019-nCoV, but the Coronavirus Study Group of the International Committee on Taxonomy of Viruses (ICTV) renamed it severe acute respiratory syndrome coronavirus 2 (SARS-Cov-2), and the condition caused by it was named COVID-19 by the WHO. 5,6 The coronavirus SARS-CoV-2 is the seventh coronavirus to infect humans. 6 Human coronavirus (HCoV)-229E, HCoV-NL63, HCoV-HKU1, HCoVOC43, Middle East respiratory syndrome coronavirus (MERS-CoV), and severe acute respiratory syndrome coronavirus were the previous six coronaviruses (SARS-CoV). 7 MERS-CoV, SARS-CoV, and SARS-CoV-2 are three beta coronaviruses that are more pathogenic and cause more severe respiratory tract sickness, as well as affect other systems such as the cardiovascular and renal systems. 8,9 SARS-CoV-2 is a virus that is enveloped. The nucleic acid is a positive sense, nonsegmented, and ss RNA. Among RNA viruses, it has a genomic size of about 30 kb, which is quite large. This virus displays a high frequency of mutation 28 | P a g e Website: https://www.banglajol.info/index.php/UpDCJ (deletion) & recombination which is unusual for an RNA virus with a non-segmented genome. With the complete genome sequencing, it was discovered that SARS-CoV-2 shares the most homology with Bat SL-CoVZC45 and Bat SL-CoVZXC21 coronaviruses, but differs from SARS and shares 79.7% similarities. 10 A 5' untranslated region (UTR), a conserved replicase domain (ORF-1ab), four genes S, E, M, and N that encode structural proteins spike, envelope, membrane, and nucleocapsid proteins, a 3' UTR, as well as other unidentified nonstructural ORFs make up the genome. 8,11 SARS-CoV-2 has highly conserved ORF-1ab, E, and N genes. The ORF-1ablocated RNA-dependent RNA polymerase (RdRp; nsp12) plays a critical function in RNA synthesis and is prone to frequent mutation and recombination. 9,12,13 As a result, the conserved domains (ORF-1ab, E, and N genes) are frequently selected as standard target genes for molecular detection of SARS-CoV-2. ARS-CoV-2 has a 3% case fatality rate, compared to 10% and 34.5 percent for SARS-CoV and MERS-CoV, respectively. However, the fast-increasing number of cases is a reason for concern. 14, 15 The management of any emerging infectious illness requires timely and accurate laboratory examination of samples from cases under investigation. Because there are presently no effective antiviral medicines or vaccines against SARS-CoV-2, early diagnosis, isolation, and supportive care are essential for the prevention of COVID-19. Molecular methods have been used to identify any pathogens for several years successfully. Although, due to negative results in certain suspected cases of the disease based on their clinical presentation and exposure history, the sensitivity and reliability of polymerase chain reaction (PCR) have been questioned. The gold standard for diagnosing SARS-CoV-2 is currently reverse transcription-polymerase chain reaction (RT-PCR) detection of viral nucleic acid. In the same sample, the sensitivity patterns of the N and ORF-1ab genes are varied. The sensitivity pattern of the N gene was found to be roughly 10 times greater than that of the ORF-1ab gene, according to Chu et al. 16 As a result, the current research studied the demographic features and sensitivity pattern of the N and ORF-1ab genes in SARS-CoV-2 infected cases.

MATERIALS AND METHODS
This cross-sectional study was done from June 2020 to August 2020. Samples were collected from all suspected cases of COVID-19 patients attending the outdoor & indoor at Aichi Hospital, Uttara, and Japan East-West Medical College Hospital, Turag, Dhaka. A total of 3183 suspected cases of COVID-19 patients were enrolled in this study after taking consent. 17 The nucleic acid of the SARS-CoV-2 virus was detected from nasopharyngeal swabs collected in viral transport media. The work was carried out in Aichi Hospital's PCR laboratory. Nucleic acid detection: Sansure Biotech Novel Coronavirus (2019-nCoV) Nucleic Acid Diagnostic Kit was used to detect ORF-1ab and N genes of SARS-CoV-2 using rRT-PCR (real-time reverse transcription polymerase chain reaction). The viral RNA was firstly extracted using the releasing reagent that came with the PCR kit. A 200 µL specimen was pipetted into a 1.5 ml Eppendorf tube, centrifuged for 5 minutes at 12,000 rpm, and the supernatant fluid was carefully discarded, leaving the precipitation at the bottom. After that, 50 µL of Sample Release Reagent was added to each tube and vortexed for 5 seconds. Then, in a PCR reaction tube, 30 µL of PCR-Mastermix was mixed with 20 µL of processed material, and amplification was carried out. All the procedures were conducted inside the Biosafety cabinet II. A typical S-shape amplification curve or Ct ≤ 40 of the N and/or ORF-1ab genes were considered positive. If there was no characteristic S-shape amplification curve or Ct ≤ 40 of N and/or ORF-1ab gene and Ct value ≤ 40 or no amplification curve for internal control, the test was considered negative. If there was no characteristic S-shape amplification curve or Ct > 40 of N, ORF-1ab, and the internal control gene, the result was deemed invalid. The data was analyzed using SPSS. A descriptive analysis of all relevant variables was done by using proportion, central tendency, and dispersion.

Figure 2: Covid-19 infection rates in various age categories among SARS-CoV-2 positive individuals (n= 649).
The frequency of positivity for SARS-CoV-2 was high in July among the 3 months ( Figure 3).

SARS-CoV-2's N gene was found in all cases positive for SARS-
CoV-2, while ORF-1ab was found in 143 (22.03%) patients. (Table III).  (Table  IV).  18 But in another study done in Bangladesh, the rate of positivity is higher than in our study. 19 This finding is also much lower than the study done by Rui et al. 20 In this study, most of the suspected cases were males (75.9%) and also SARS-CoV-2 positive cases (73.7%). This finding is very much closer to the study done by Akram et al. (71%), Italy (60%), and the United States (63%), but more than the other studies done in Bangladesh and China. 18,19,[21][22][23][24][25] Male predominance is concerning because male predominance has been linked to an increased risk of mortality. 26 SARS-CoV-2 positive individuals had a mean age of 40.35 ± 14.551 years, while suspected cases had a mean age of 36.94 ± 14.015 years. The lowest and highest age was found to be positive for SARS-CoV-2 was 4 years and 90 years, respectively. This observation is similar to those of others. 18,19,27 The majority of positive cases (50.7%) were detected in the age category of 21 to 40 years, followed by 41 to 60 years (33.1%). This finding is in agreement with the findings of other studies. 18,19,27,28 But in studies in China and Italy, the frequency of positivity was found to be high in the age group of 41 to 70 years. 21,22,24,27 Most affected cases within the age group 21 to 40 may be due to the youth age group in Bangladesh is more  29 This study included data only for 3 months, June to August. Among 3 months the highest cases with positive PCR test was found in July. The N and ORF-1ab genes of SARS-CoV-2 were detected in this study. In all of the positive cases, the N gene was found. But the positivity of ORF-1ab is only 22.03%. Some other researchers also found almost similar findings. In their research, Chu et al. discovered that the N gene assay is about 10 times more sensitive than the ORF-1ab, while Liu et al. discovered that the ORF-1ab positivity rate was about 62.5 %. 16,30 The mean Ct value of the N and ORF-1ab genes was 27.84 ± 4.61 and 37.00 ± 3.35, respectively, among the cases positive for both genes. In most cases, the N gene's Ct value is lower than that of the ORF-1ab gene in the same sample. It signifies that the N gene has a higher sensitivity and amplification efficiency than ORF-1ab. It is comparable to the findings of Liu et al. study's 30 . It could be because samples containing infected cells express subgenomic mRNA, resulting in more N gene copies, are being tested for SARS-CoV-2. 31 Again the low positivity of SARS-CoV-2 in this study may be related directly to the quality of the collected sample. As a result, other genes must be detected to confirm clinically suspected cases. Clinical parameters, additional laboratory findings, comorbidities, and mortality status were not included in this study. As the most positive cases found within the youth group, another study including those parameters is needed. As this is qualitative PCR, we cannot detect the viral load. However, research into the relationship between viral load and affected people's morbidity and mortality is required.

Conclusions
Emerging and reemerging infectious illnesses are easily transferred across the world as a result of global migration. In any outbreak, it is very much important to know the causative agent and source of infection. In this case, laboratory testing is quite important. Until and unless additional diagnostic procedures for SARS-CoV-2 are approved, rRT-PCR remains the gold standard. The low positive result of SARS-CoV-2 and the variation of sensitivity and amplification efficiency in this study raise the need to test other samples, i.e., urine and stool, for clinically suspected cases because the virus is shaded for a significantly longer time than respiratory samples to avoid a false-negative result. As the most positivity was found among the male and young generation, it is an alarming situation because they are the most mobilized person in the family. So further study is required to know whether these young people play the role of the source of infection to their other family members.

ACKNOWLEDGEMENT
We highly appreciate the contribution and help of Japan East-West Medical College Hospital and Aichi Hospital in the identification of suspected cases of COVID-19 patients. We are also thankful to Aichi Hospital for giving laboratory support to perform the rRT-PCR. We wish to thank all the suspected cases of COVID-19 patients for their cooperation and help that made this study possible. FUNDING SOURCE