Molecular identification, clinical assessment, characterization of SARS-CoV-2 RT-PCR results: Insights into twenty-one months of COVID-19 pandemic in a single center in Bangladesh

Introduction : The pervasiveness of SARS-CoV-2 infection over millions of people renders a variety of symptoms among group of people and ages. This study aimed to appraise the clinical assessment, characterization, and comparative study on suspected SARS-CoV-2 infected patients and further illuminate its transmission dynamics. Methods : It includes all the suspected SARS-CoV-2 infected cases from July 2020 to March 2022 in this retrospective single center study. All the cases confirmed by the golden method of detection (real-time RT-PCR) and analyzed for clinical and demographic features and laboratory data. Result : Out of 35,630 cases, 11.4% cases confirmed with SARS-CoV-2 infection. With 91.4% new and 8.6% follow-up suspected cases in total, SARS-CoV-2 infection confirmed with 85.5% new cases and 14.5% from follow-up cases. 86.1% male suspected cases found where SARS-CoV-2 infection confirmed for 73.8% with this gender group. 56.7% SARS-CoV-2 infection confirmed for age range 20-39 and this age group counts 59.3% from the total suspected cases. Most confirmed cases (28.3%) counted from Uttara, Dhaka and this area covers 34.9% suspected cases from the total. Conclusion : This study exhibited that, SARS-CoV-2 is more prone to male individual, individuals aged between 20-39 and occurs re infection hardly but not limited to. Symptoms observed for almost half of the confirmed cases.

Bangladesh has a history of becoming the hub for many transmissible diseases with its dense population (1,265 per square kilometer), where about 35.3% of its residents are below 14, about 60% are between 15 to 64 and just 4.7% are above 65 years of age 22 .It makes it more difficult to prevent or control, as SARS-CoV-2 has already thrived on several intermediates and final host before infecting Humans.Hence it dominates with higher transmissibility and infectivity.The infectivity, virulence, and severity of the outcome of SARS-CoV-2 may vary on factors such as age, sex, geographic locations, other chronic diseases, and so on 22,23 .Though SARS-CoV-2 varies no age, sex, or race; pregnant women, newborn, male (71%) individuals, and individuals over 20 years of age are prone to get infected by SARS-CoV-2 2,24,25 .Patients with SARS-CoV-2 infection may or may not develop any significant or chronic symptoms.About 18% to 31% of the SARS-CoV-2 infection observed asymptomatic with higher transmissibility and other patients found were minimally symptomatic, while they showed fever, cough, nasal congestion, anorexia, ageusia, dyspnea, rigors, pharyngeal erythema, malaise, sore throat, shortness of breath, headache, body ache, hemoptysis, fatigue, vomiting, diarrheal symptoms [25][26][27][28][29][30][31][32] .As SARS-CoV-2 utilizes the ACE2 (angiotensin-converting enzyme 2) receptor and latent CD147 (cluster of differentiation 147) to surplus invade towards cardiovascular, renal, endocrine, nervous, and gastrointestinal organ systems; associated viral invasion, dysregulated renin-angiotensin-aldosterone system (RAAS), hypoxia, hyper inflammation, cytokine storm, endotheliopathy, and thrombosis considered as a renowned pathophysiological mechanism of SARS-CoV-2 infection 33 .Immune response for confirmed SARS-CoV-2 infected patients is noticeable while mild to severe symptomatic patients develop significant IgM and IgA response, but antibody response of asymptomatic patients found less or unnoticed 34 .
This study focused on evaluating the clinical assessment, characterization of SARS-CoV-2 RT-PCR results on SARS-CoV-2 infected patients by molecular identification, logged on Novus Molecular Lab, Dhaka, Bangladesh.Following the guidance of the WHO digital documentation process of SARS-CoV-2 test results 35 , the availability of large disaggregated data such as sex, age, type of patients, the purpose of the test, geographical location, symptoms, etc stored on Novus database helped to reveal the obscured imbalances.

Development of a digital patient management system
The outbreak of corona virus disease-19 (COVID-19) is a public health emergency of international concern.During the COVID-19 outbreak Beximco Pharmaceuticals Ltd launched "BCD COVID-19 Medical Center" for the management of COVID-19 positive cases with symptoms.Any BCD cases and other beneficiaries (Permanent, Contractual, Temporary, Casual, Driver or DL) officially reports to the 24 hours HOTLINE number, if he/she or any of the following members residing with him/her shows any symptom and need any query related to COVID-19.After receiving a call, the Hotline doctor provides initial treatment and registers the patient on hotline software.If required, the Hotline doctor advises the cases to do Covid-19 test as per patient condition and send the recommendation to the testing team.Depending on the condition, hotline doctors sometimes advise the cases for hospitalization and provide necessary support also.
Testing team accumulates all the test recommendations and registers them in Novus software in-coordination with Novus Molecular lab.Testing team also provides an appointment date and time and sends appointment details to the individual's phone number through SMS.After completing the test, an automatic SMS with the test result shall be sent to the individual's phone no.Testing teams accumulate all the reports and send it to all responsible authorities including all respective HR and admin and patient management teams.
After receiving the report, doctors of patient management team talked to COVID positive employees and provide him/her a prescription as per his/her condition and send the copy of the prescription to the responsible department to send the medicines to the patient to all the COVID positive cases.Doctor of patient management team follow-up the patients regularly as per requirement and advised the patient to do some additional investigation and/or hospitalization if necessary.In the course of disease, the doctors of patient management team provided all types of medical and mental support to the patients.

Study Population
The study enrolled patients suspected of getting SARS-CoV-2 infection between July 2020 to March 2022 from Novus Molecular Lab.The clinical information for each patient recorded during the registration process either over a phone call or in person.Participants provided their oral informed consent to perform the SARS-CoV-2 test with their specimens collected.Ethical clearance was taken from NILMRC ethical board.

Specimen Collection and Storage
The specimen of COVID-19 suspected patients collected on a 2 ml STM (STANDARD TM Transport Medium; Cat.No. 90VTM10D) by following CDC and WHO specimen collection guidelines 36,37 .Both nasopharyngeal (Both sides of the nasal opening) and an oropharyngeal swab simultaneously collected and immersed in the STM to ensure proper specimen collection.All the STM with patient's specimens marked with the Novus ID, individual's name, date, and age in a printed sticker.All specimens collected were immediately transported to Novus Molecular Lab after collection and kept in 2-8 0 C 36,37 before the test.Specimens tested within six hours of collection by RT-PCR method.All the specimen collection, lab testing, storage, and waste disposal followed the standard guidance, health, and safety by CDC and WHO [36][37][38][39] .

Sample preparation and RNA extraction
After vortexing, 200 μl of the specimen from each STM transferred to individual 1.5 ml microcentrifuge tubes for SARS-CoV-2 RNA extraction.The RNA extraction of SARS-CoV-2 carried out via STANDARD TM M SPIN-X Viral RNA Extraction Kit (Cat.No. 11SPN10).After transferring 200 μl of specimen, a multistep extraction was carried out following the kit manual.The volume of eluted RNA of SARS-CoV-2 collected was 50 μl.

RT-qPCR for detection of SARS-CoV-2
Using STANDARD TM M nCoV Real-Time Detection kit (Cat.No. 11NCO10), master mix was prepared for the detection of ORF1ab (RdRp) gene, E gene, and IC (Internal Control).The RT-PCR was carried out in Quant-Studio-5 Real-Time PCR System (Thermo Fisher Scientific).After completion of the successful amplification of RNA, results were assessed.Ct (Cycle threshold) value equal to or below 36 considered a cut-off value for COVID-19 positive patients, and Ct value above 36 considered as COVID-19 negative.

Statistical analysis
All the data were statistically analyzed using IBM SPSS Statistics 25.0 software (SPSS Inc Chicago, IL, USA).Descriptive statistics like frequency and percentage used.

Figure 3 Predominant symptoms among study population
As the male (67.7-73%) individuals are more prone to the SARS-CoV-2 viral infection than female (32.3-27%) in Bangladesh [40][41][42] , current study manifests no exceptions.However, there is no clear gender bias of SARS-CoV-2 infection found from across the world, shows the data available so far.Even so, ages from all groups are prone to SARS-CoV-2 infection as the highest infection reported for middle-aged people ranging from 30-60 years according to Siam 22 .
Approximately more than 50% cases recorded with SARS-CoV-2 infection are aged between 21 and 40 in Bangladesh 2,41,43 which is a clear indication of a commendatory population for SARS-CoV-2.

Limitation and future scope of the study
In this study we also had some limitations as the collection and inclusion of Ct value data was a bit tough for this massive number of cases.We could not collect the vaccination history.Collection and analysis of COVID-19 associated biochemical data could enrich the potency of this research.We also could not obtain the genomic sequence of the confirmed SARS-CoV-2 specimen because of its massive sample size, but addition of sequenced data and its correlations could give us clearer etiology of SARS-CoV-2 found.Addition of vaccination or immunization history with quantitative immune protection could also make the research clearer in terms of understanding associated clinical manifestations.

Conclusion
This study encloses the situations of SARS-CoV-2 infection recorded in Novus Molecular Lab and only represents a portion of the country.It basically reflects the population that are more prone to getting SARS-CoV-2 infections living in capital Dhaka, a core of the country.Along with clinical and demographic data, this study provides us with a precise and clear vision of infection dynamics of SARS-CoV-2 during the period of twenty-one months.Similarities in prospective SARS-CoV-2 infected community with age range, gender, compared to the country, the study also reveals some unique finding such as the rate of infections towards the passengers and locals, follow-up and new cases, and the demographic overview in a monthly basis.For understanding the enormity, clinical spectrum, natural progression and to take proper control measure in case of SARS-CoV-2 infection all-round the Bangladesh or over any south Asian countries point of view, epidemiologist, virologists, or any other scientific professionals can get a sharp-edged apprehension.Foremost essence of this paper is an imbalanced risk on the extremely endangered population striving on SARS-CoV-2 infection and encouraging to confront SARS-CoV-2 outbreak with a better understanding and preparations.

Figure 1 Figure 2
Figure 1 Male and Female ratio and rate of positivity

Table 1
Demographic characteristics of study population However, the rate of SARS-CoV-2 infection was noticeable and they might escape the attention if not screened as most of the cases come along with no symptoms.InNovusMolecular lab, 32,579 (91.4%) cases were new with no history of SARS-CoV-2 infection before, of which 3,484 (85.5%) reported COVID-19 positive.A total of 3,051 (8.6%) cases were received for follow-up with confirmed COVID-19 history and 591 (14.5%) of them reported confirmed COVID-19 positive (Table1).Some of the follow-up cases found with COVID-19 for a longer period, found for more than 30 days.Most cases found were 21,136 (59.3%) with ages ranging from 20 to 39 with 2,308 (56.7%)COVID-19 positive cases.Following them, 1,227 (30.1%)COVID-19 positive cases recorded out of 11,142 (31.2%) suspected cases with ages ranging from 40 to 59 (Table1).Cases representing ages less than 20 and more than 60 are negligible in count and might indicate the laxity of infection by SARS-CoV-2.
In the case of test requirements, among 35,630 cases, a total of 11,022 (30.9%) cases required by passengers (for immigration purposes) of which only 308 (7.6%) reported COVID-19 positive.(Table:1).This might be the first ever study that revealed the type of passenger who performs COVID-19 screening test and clearly exhibits their condition during the immigration.

Table 2
Cross tabulation of symptoms