key: cord-0860341-mqvdzele authors: Sam, I‐Ching; Chong, Yoong Min; Abdullah, Azwani; Fu, Jolene Yin Ling; Hasan, M. Shahnaz; Jamaluddin, Fadhil Hadi; Kamarulzaman, Adeeba; Lim, Koo Koon; Mohd Nor, Mohd Afiq; Pang, Yong Kek; Ponnampalavanar, Sasheela; Shahib, Muhammad Fadzil; Syed Omar, Sharifah Faridah; Chan, Jonathan Chia Jui; Perera, David; Chan, Yoke Fun title: Changing predominant SARS‐CoV‐2 lineages drives successive COVID‐19 waves in Malaysia, February 2020 to March 2021 date: 2021-11-16 journal: J Med Virol DOI: 10.1002/jmv.27441 sha: 42dc17ebdabb88c0866a2f3f78993fa224c0b5f2 doc_id: 860341 cord_uid: mqvdzele Malaysia has experienced three waves of coronavirus disease 2019 (COVID‐19) as of March 31, 2021. We studied the associated molecular epidemiology and SARS‐CoV‐2 seroprevalence during the third wave. We obtained 60 whole‐genome SARS‐CoV‐2 sequences between October 2020 and January 2021 in Kuala Lumpur/Selangor and analyzed 989 available Malaysian sequences. We tested 653 residual serum samples collected between December 2020 to April 2021 for anti‐SARS‐CoV‐2 total antibodies, as a proxy for population immunity. The first wave (January 2020) comprised sporadic imported cases from China of early Pango lineages A and B. The second wave (March–June 2020) was associated with lineage B.6. The ongoing third wave (from September 2020) was propagated by a state election in Sabah. It is due to lineage B.1.524 viruses containing spike mutations D614G and A701V. Lineages B.1.459, B.1.470, and B.1.466.2 were likely imported from the region and confined to Sarawak state. Direct age‐standardized seroprevalence in Kuala Lumpur/Selangor was 3.0%. The second and third waves were driven by super‐spreading events and different circulating lineages. Malaysia is highly susceptible to further waves, especially as alpha (B.1.1.7) and beta (B.1.351) variants of concern were first detected in December 2020/January 2021. Increased genomic surveillance is critical. The coronavirus disease 2019 (COVID- 19) pandemic has now entered its second year, having caused over 4.5 million deaths worldwide as of September 2021. The cause, SARS-CoV-2, is a positive-sense RNA betacoronavirus with a~30 kb genome. Unprecedented efforts have been expended for global genomic surveillance. This utilizes whole genome sequencing to identify genetic lineages and variants of concern (VOC) carrying mutations that may increase transmission, enable immune escape, or impact vaccine responses or diagnostic tests. 1 Malaysia is a southeast Asian country comprising 13 states and 3 federal territories, with a population of about 32 million. The first wave of COVID-19, caused by SARS-CoV-2, consisted of 22 mainly imported cases from China and lasted for 3 weeks from late January 2020. 2 A second, much larger wave occurred between March and June, mainly driven by a religious mass gathering linked to at least 3375 confirmed cases, a third of national cases at the time. 3 A nationwide movement control order and other public health measures led to a considerable reduction in numbers. 2 However, an ill-timed election in the state of Sabah in September led to an even larger, nationwide third wave extending into 2021. 4 As of March 31, 2021, there have been 345 500 confirmed cases and 1272 deaths. 5 In this study, we performed whole-genome sequencing from 60 SARS-CoV-2 cases from the recent third wave in Kuala Lumpur and Selangor. We analyzed them with other complete genome sequences from Malaysia available on the GISAID database (www.gisaid.org) from samples collected before March 31, 2021. Our objective was to relate the molecular epidemiology of circulating SARS-CoV-2 to the waves of reported cases in Malaysia. Additionally, having previously reported seroprevalence of 0.4% in Kuala Lumpur/Selangor after the second wave, 6 we carried out a follow-up study to determine seroprevalence progression during the third wave. Generated reads were first imported into Geneious Prime 2020 (Biomatters) and trimmed using a BBDuk trimmer plugin (version 1.0) and mapped to reference genome Wuhan-Hu-1 (GenBank accession number: MN908947). The threshold to generate consensus sequences was set at "highest quality (adjusted)," with other settings at default, including calling an N if coverage depth is <2. We aimed to produce sequences that would fulfill GISAID inclusion criteria as complete sequences, that is >29 000 nucleotides with <50% Ns. The To estimate the seroprevalence of the Kuala Lumpur/Selangor population, we tested residual serum samples from UMMC inpatients collected between December 2020 to April 2021 for diagnostic testing for nonrespiratory infections. Residual serum has been an adequate proxy for general population serosurveys for COVID-19. 13 The sample size was calculated with an expected seroprevalence of 5% (95% CI, 3%-7%), and found to be 457. A total of 653 archived samples were tested, including 43-103 samples from every 10-year age group (<10, 10-19, 20-29, 30-39, 40-49, 50-59, 60-69, and ≥70 years). The serum samples were screened using a SARS-CoV-2 total antibody enzyme-linked immunosorbent assay (ELISA) (Beijing Wantai Biological Pharmacy Enterprise). This assay has received emergency use authorization by the US Food and Drug Administration and has high reported sensitivity (96.7%) and specificity (99.5%). 14 Samples testing indeterminate (optical density/cut-off ratio: 0.9-1.1) were retested and considered positive if the retest was reactive. Crude seroprevalence rates are given with 95% exact binomial confidence intervals (CIs). Daily SARS-CoV-2 case numbers in Malaysia from January 2020 to Table S1 ). All were successfully accepted in GISAID, and all were considered as "high coverage" with <1% Ns except for 1 sequence (hCoV-19/Malaysia/8827/2021) with 9.52% Ns. All cases were acquired locally. The Pango lineages were When the lineages were analyzed over time (Figures 2 and 3 Serosurveys provide better tracking of the extent of population infection and immunity, as previously undiagnosed cases can also be identified. A total of 653 serum samples collected between December 2020 to April 2021 from UMMC inpatients were tested for total anti-SARS-CoV-2 antibodies (Table 1) , and the crude seropositive rate was 4.1% (95% CI: 2.7-6.0). The highest rates were seen in those aged 30-49 years and >60 years. No seropositives were seen in the 123 samples from patients aged 0-19 years. Using age-stratified population data for Kuala Lumpur and Selangor, a direct age-standardized seroprevalence rate was calculated as 3.0%. F I G U R E 2 Total reported daily cases (above) and circulating lineages of SARS-CoV-2 (below) in Malaysia, from sequences available on GISAID as of March 31, 2021. Lineages are named using the Pango system There were clear changes in circulating lineages over the course of the pandemic. The first wave cases were from early lineages A and B, as most were imported from China. 12 We previously showed that the second wave from March to June was driven mainly by lineage B. We previously estimated an age-standardized seroprevalence of 0.4% for Kuala Lumpur/Selangor during and after the second pandemic wave ending June 2020. 6 A national, population-wide seroprevalence study carried out between August and October 2020 showed a compatible rate of 0.5%. 21 In this study, we estimated that the rate in Kuala Lumpur/Selangor had increased to 3.0% during the third wave, exceeding the cumulative incidence of reported cases of 1.9% as of March 31, 2021. Seropositive rates were lowest among children, consistent with the relatively low rate of pediatric cases reported here. 22 The overall very low seropositive rate points to population susceptibility to further disease, which is concerning amidst reports of extreme pressures on the healthcare system. 23 A recent review of population-based studies up to March 2021 estimated a global seroprevalence of 9.5%, with the lowest regionspecific seroprevalence of 1.6% in the east and southeast Asia. 24 Differences in sample population, testing method and study timing make direct comparisons between countries difficult. However, in keeping with the overall lower regional seroprevalence rate and the perception that east and southeast Asian countries have been relatively less impacted by the pandemic, our seroprevalence finding was lower than the global average. 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Institute for Public Health Clinical and epidemiological characteristics of children with COVID-19 in Negeri Sembilan, Malaysia Idris AN PM: nation's healthcare system is at breaking point Update on SARS-CoV-2 seroprevalence -regional and worldwide Rapid and simultaneous identification of three mutations by the Novaplex SARS-CoV-2 variants I assay kit The authors declare no conflict of interests.