On-Site Sequencing Of Sars-Cov-2 Genomes Identify Two Variant Of Concern Clusters In Pahang, Malaysia

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INTRODUCTION
The coronavirus has been responsible for multiple worldwide outbreaks throughout human history, such as H1N1 in 2009 and the Middle East Respiratory Syndrome (MERS) caused by MERS-CoV in 2012. In the latter month of 2019, the WHO China Country Office received reports of pneumonia cases with unclear causes, ultimately linked to a novel coronavirus identified as SARS-CoV-2 that causes COVID-19 [1]. The World Health Organization has declared 627,573,579 confirmed cases of COVID-19, including 6,570,363 deaths as of October 2022 [2]. The pandemic is regarded as a worldwide crisis creating severe disruptions across the economy and health system, including Malaysia.
Malaysia was one of the first countries in Southeast Asia to implement the Movement Control Order (MCO) to prevent the spread of the coronavirus during the early outbreak [3]. Malaysia eventually faced a much more difficult task in combating the COVID-19 pandemic in its third wave, which began on 8th September, 2020, due to the Benteng LD cluster in Sabah [4,5]. Since then, the highest lineage contributor appeared to be from Variant of Concerns (VOCs) and Variant of Interests (VOIs) with D614G mutations in the spike protein [6]. Two large clusters reported in Pahang, Malaysia, includes Taman Tanah Putih Baru and Pasar Kemunting cluster. Taman Tanah Putih Baru, Pahang, was previously under an Enhanced Movement Control Order (EMCO) by the National Security Council (NSC) of Malaysia on 5th May 2021 after 11 residents tested positive for COVID-19. The state government had to 'appeal' for free screening tests after 200 residents ran away from their houses before the EMCO. A total of 4795 surveillance test for COVID-19 was done, and a total of 385 individual was reported positive from this cluster. The Pasar Kemunting cluster, on the other hand, is a workplace cluster linked to a market. The cluster started with one case reported on 21st June 2021 in Kuantan District. The name of the cluster refers to the locality where the outbreak is suspected of having occurred at the workplace located at Jalan Seri Kemunting 2, Kemunting, Kuantan. The index case for this cluster is a Malaysian citizen working there. The index case started experiencing fever and sore throat symptoms on 14th June 2021. The results of the investigation found that 5 other colleagues tested positive for COVID-19. The transmission of the infection is suspected of having occurred in the workplace due to social gatherings, and there were violations of SOPs. As of 20th August 2021, a total of 5135 individuals had been screened in this cluster, 1123 cases were detected positive for COVID-19, and 16 deaths had been reported.
Herein, we report 2 variants of concern -ß and ∆ variants in these 2 large clusters in Pahang, Malaysia. The representative sample of Taman Tanah Putih Baru cluster patient genome surveillance showed that he contracted with ABSTRACT -Timely variant identification is required in respond to effective public health measures. It has been demonstrated that comprehending genetic epidemiology can be aided by a combination of public health expertise and through viral genomic sequencing using Oxford Nanopore Technologies (ONT). The purpose of this study is to determine the utility of using Oxford Nanopore Sequencing to elucidate the genetic epidemiology in two significant clusters in Pahang, Malaysia. The combined oropharyngeal and nasopharyngeal swabs of clinical specimens from two significant clusters in Pahang, Malaysia, were retrieved for long-read sequencing. Results: We identify Beta and Delta variants as 2 variants of concern from our analysis. We found that the B.1.351 (ß) and B.1.617 () variants were responsible for the Taman Tanah Putih Baru and Pasar Kemunting clusters, respectively. In conclusion, ONT long-read sequencing tools offer a practical solution with a number of advantages. ONT devices are compact, affordable, and need little technical hands-on or laboratory equipment to prepare samples. They can also be used to quickly and adaptably execute sequencing analyses and to understand genomic epidemiology. journal.ump.edu.my/cst ◄ B.1.351 lineage, which had been identified previously in October 2021 in Eastern Cape Province, South Africa. This cluster ended on 23rd May 2021. In addition, the representative sample of Pasar Kemunting cluster patient genome surveillance showed to be related to B.1.617 lineage, which was first detected in India in late 2020, and this cluster ended on 20th August 2021. Moreover, the utility of long-read sequencing devices from Oxford Nanopore Technologies (ONT) was tested, showing as a promising diagnostic tool in investigating COVID-19 for disease surveillance.

Sample Collection, Patients Background and Ethical Approval
Clinical specimens were collected directly from combined oropharyngeal and nasopharyngeal swab specimens from two individuals with diverse clinical presentations representing both clusters. Both were COVID-19 positive and diagnosed via reverse transcriptase PCR (RT-PCR) with a threshold cycle (CT) value below 30. The study was approved by the International Islamic University Malaysia Research Ethics Committee (IREC 2021-080).

DNA Extraction, Nanopore Library Preparation and Sequencing
The genomic RNA of SARS-CoV-2 was isolated using Maxwell HT simplyRNA kit (Promega, USA) according to the manufacturer's recommended procedures with some modifications. The concentration and quality of gDNA were measured by Nanodrop spectrophotometer (Thermo Scientific, USA) and agarose gel electrophoresis, respectively, for quality and control measurement prior library preparation. The library preparation was performed using NEBNext ® ARTIC SARS-CoV-2 Companion Kit (ONT ® , UK) according to the manufacturer's instructions. Briefly, the extracted total RNA was converted into cDNA using SuperScript™ IV Reverse Transcriptase (Invitrogen, US) with some modifications, a hexamer annealing and extension step of 25 o C for 2 minutes was performed followed by cDNA synthesis at 42 o C for 50 minutes. The purified PCR products were quantified using DeNovix ® dsDNA High Sensitivity Assay (DeNovix Inc., US) and The prepared CDNA library was then loaded onto a MINION flow cell and a 72 hr sequenced for 72 hours.

Whole-Genome Sequencing and Phylogenetic Tree Analysis
The SARS-CoV-2 genome was reconstructed from the raw reads using a combination of several bioinformatic tools enlisted in https://github.com/CDCgov/SARS-CoV-2_Sequencing/tree/master/protocols/BFX-UT_ARTIC_Illumina. Genome sequences from other studies related to humans and animal coronaviruses were mined from the GISAID (https://www.gisaid.org) and NCBI GenBank (https://www.ncbi.nlm.nih.gov/genbank/). The multiple sequence alignment was performed using Clustal X [7] and observed in BioEdit [8] and finalized using MEGA XI [9]. Evolutionary analysis was conducted in MEGA XI by reconstructing bootstrap consensus tree of sequences employing Neighbor-Joining (NJ) method with 1000 bootstrap replicates to represent the evolutionary history of the taxa analyzed.

Data Availability
The sequences deposited in the GISAID database are EPI_ISL_3221234 and EPI_ISL_2622089.

Case 1.
A 72-year-old man, an active smoker with underlying hypertension, presented with a fever history and loose stools for 4 days. His symptoms were also associated with a productive cough and loss of appetite. He denied shortness of breath, chest pain, vomiting, anosmia or ageusia. His chest radiograph showed small patchy peripheral consolidations in both lungs, predominantly at the lower zones ( Figure 1). Initial blood investigation revealed a white cell count of 7.3 x 109/L, haemoglobin of 14.1 g/dL and platelet of 298 x 109/L. journal.ump.edu.my/cst ◄ His renal and liver profile was normal. His real-time PCR showed positive result and he was treated for COVID-19 Category 3. On day 5 of hospitalization, he clinically deteriorated, and he became tachypneic and required oxygen supplementation. His clinical diagnosis was revised to COVID-19 Category 4 and he was started on IV Dexamethasone 6 mg daily. A repeated chest radiograph showed worsening bilateral peripheral lung consolidation during his clinical deterioration, particularly on the right side. Scattered ground glass opacities are also noted within both lungs. A CT-pulmonary angiogram was also ordered to look for the possibility of a pulmonary embolism. However, it was none and showed a typical feature of COVID-19 ( Figure 2).

Case 2.
A 17-year-old man who worked at Pasar Kemunting market presented with headache and fever for 1 week associated with anosmia and loose. He was treated as COVID-19 Category 2 and quarantined at PKRC Gambang for nearly 11 days. He was discharged well. No blood or radiological investigation was done. His genome surveillance showed that he contracted B.1.617 (delta variant).

Phylogenetic Tree Analysis
The pandemic of COVID-19 is devastating, and Malaysia is currently affected by the 5 th wave. Taman Tanah Putih Baru and Kemunting clusters are the two most significant clusters of COVID-19 reported in Pahang, Malaysia, during the 4 th wave.
Reverse transcriptase PCR (RT-PCR) remains the gold standard for diagnosing COVID-19, despite its inability to distinguish variant types. On the other hand, it is anticipated that the combination of real-time whole-genome sequence analysis and epidemiology will aid in controlling SARS-CoV-2 transmission in the population and aiding early clinical decision-making. Taman Tanah Putih Baru and Kemunting clusters are the two most significant clusters of COVID-19 reported in Pahang, Malaysia. Using the local population of Pahang, Malaysia, as an example, we describe a substantial chain of transmission that began with an initial local infection with SARS-CoV-2 Beta and Delta variant.
The representative sample of Taman Tanah Putih Baru cluster patient genome surveillance showed that he contracted with B.1.351 (beta) variant, and the representative sample of Pasar Kemunting cluster patient genome surveillance showed that he contracted with B.1.617 (delta variant). Both variants had been classified as variants of concern (VOC). This mutation was reported previously and will not impact diagnostic testing [11], and our patient is shown to have a positive PCR test. journal.ump.edu.my/cst ◄ We highlighted this case to show that the potential transmission of this VOC is greater, and a large population was affected. It has been proven previously that this variant had caused a rapid spread of disease due to multiple spike mutations. It was predicted that B.1.351.5 resulted in significant selection pressure in a population with high seroprevalence and immunity [12]. Multiple data is still needed to ascertain the disease severity, and early reports show that there is no considerable difference in the severity of infection [13].
The D614G variation is a precursor that accounts for 90.30 percent of all COVID-19 infections in Malaysia, and this mutation is found in all new variants under development [6]. Because of positive natural selection, D614G improves the infectivity, viral fitness, transmission rate, and efficiency of cellular entry for the SARS-CoV-2 virus across a wide range of human cell types [5]. Despite this, the D614G mutation hasn't been proven to cause an increase in COVID-19 mortality or clinical severity, nor has it been shown to alter the efficacy of existing laboratory diagnoses, medicines, immunizations, or public health preventative efforts [14]. The Delta variation is thought to have a greater viral replication rate, causing viral loads in Delta infected individuals to be 1000 times higher than other strain infections on the day the testing is positive [15]. This indicates that the Delta variation is more infectious during the early stages of infection, and the frequency of population screening should be maximized [15]. Surveillance data suggest that the Delta variant swiftly overtook the previously successful Alpha (B.1.1.7) strain in other countries. The Delta variant has the fastest growth rate of all the variants found, reflecting its biological features and the setting in which it is transmitted [16]. The Delta variant has also been linked to a higher risk of hospitalization than the Alpha variant [17,18], however, research suggests that the current COVID-19 vaccinations provide almost similar protection against the Delta variant after two doses as they do against the Alpha variant [19].
Because of the increased prevalence of the Beta and Delta variants globally, disease control strategies such as population testing, quarantine during the pre-symptomatic period, and genetic surveillance should be updated. Because of the higher infectivity rate of Delta variant infections in the pre-symptomatic phase, suspected individuals or close contacts should be quarantined immediately. Despite a single case, it can eventually become dominant in the population if the pandemic is not successfully contained.
Rapid identification of variants is required to implement public health actions promptly. It has been demonstrated that public health expertise and Oxford Nanopore Technologies (ONT) can aid in comprehending genomic epidemiology. Furthermore, combining short and long reads dramatically enhanced the assembly of the SARS-CoV-2 genome and established a novel method for correcting erroneous frame shifts from a single sequencing effort [20].

CONCLUSION
Oxford Nanopore Technologies (ONT) long-read sequencing devices offer a viable alternative with many advantages. ONT devices are tiny, affordable, and need minimal laboratory infrastructure and technical skills for sample preparation. In addition, they can be used to undertake flexible, rapid sequencing analyses that aid in the comprehension of genomic epidemiology.