Changing predominant SARS-CoV-2 lineages drives successive COVID-19 waves in Malaysia, February 2020 to March 2021.

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.

Influenza in Malaysian adult patients hospitalized with community-acquired pneumonia, acute exacerbation of chronic obstructive pulmonary disease or asthma: a multicenter, active surveillance study.

BACKGROUND: Available data on influenza burden across Southeast Asia are largely limited to pediatric populations, with inconsistent findings. METHODS: We conducted a multicenter, hospital-based active surveillance study of adults in Malaysia with community-acquired pneumonia (CAP), acute exacerbation of chronic obstructive pulmonary disease (AECOPD) and acute exacerbation of asthma (AEBA), who had influenza-like illness ≤10 days before hospitalization. We estimated the rate of laboratory-confirmed influenza and associated complications over 13 months (July 2018-August 2019) and described the distribution of causative influenza strains. We evaluated predictors of laboratory-confirmed influenza and severe clinical outcomes using multivariate analysis. RESULTS: Of 1106 included patients, 114 (10.3%) were influenza-positive; most were influenza A (85.1%), with A/H1N1pdm09 being the predominant circulating strain during the study following a shift from A/H3N2 from January-February 2019 onwards. In multivariate analyses, an absence of comorbidities (none versus any comorbidity [OR (95%CI), 0.565 (0.329-0.970)], p = 0.038) and of dyspnea (0.544 (0.341-0.868)], p = 0.011) were associated with increased risk of influenza positivity. Overall, 184/1106 (16.6%) patients were admitted to intensive care or high-dependency units (ICU/HDU) (13.2% were influenza positive) and 26/1106 (2.4%) died (2.6% were influenza positive). Males were more likely to have a severe outcome (ICU/HDU admission or death). CONCLUSIONS: Influenza was a significant contributor to hospitalizations associated with CAP, AECOPD and AEBA. However, it was not associated with ICU/HDU admission in this population. Study registration, NMRR ID: NMRR-17-889-35,174.

Propagation of a hospital-associated cluster of COVID-19 in Malaysia.

BACKGROUND: Hospitals are vulnerable to COVID-19 outbreaks. Intrahospital transmission of the disease is a threat to the healthcare systems as it increases morbidity and mortality among patients. It is imperative to deepen our understanding of transmission events in hospital-associated cases of COVID-19 for timely implementation of infection prevention and control measures in the hospital in avoiding future outbreaks. We examined the use of epidemiological case investigation combined with whole genome sequencing of cases to investigate and manage a hospital-associated cluster of COVID-19 cases. METHODS: An epidemiological investigation was conducted in a University Hospital in Malaysia from 23 March to 22 April 2020. Contact tracing, risk assessment, testing, symptom surveillance, and outbreak management were conducted following the diagnosis of a healthcare worker with SARS-CoV-2 by real-time PCR. These findings were complemented by whole genome sequencing analysis of a subset of positive cases. RESULTS: The index case was symptomatic but did not fulfill the initial epidemiological criteria for routine screening. Contact tracing suggested epidemiological linkages of 38 cases with COVID-19. Phylogenetic analysis excluded four of these cases. This cluster included 34 cases comprising ten healthcare worker-cases, nine patient-cases, and 15 community-cases. The epidemic curve demonstrated initial intrahospital transmission that propagated into the community. The estimated median incubation period was 4.7 days (95% CI: 3.5-6.4), and the serial interval was 5.3 days (95% CI: 4.3-6.5). CONCLUSION: The study demonstrated the contribution of integrating epidemiological investigation and whole genome sequencing in understanding disease transmission in the hospital setting. Contact tracing, risk assessment, testing, and symptom surveillance remain imperative in resource-limited settings to identify and isolate cases, thereby controlling COVID-19 outbreaks. The use of whole genome sequencing complements field investigation findings in clarifying transmission networks. The safety of a hospital population during this COVID-19 pandemic may be secured with a multidisciplinary approach, good infection control measures, effective preparedness and response plan, and individual-level compliance among the hospital population.

Evaluation of rapid influenza diagnostic tests for influenza A and B in the tropics.

Rapid diagnosis of influenza is important for early treatment and institution of control measures. In developing tropical countries such as Malaysia, influenza occurs all year round, but molecular assays and conventional techniques (such as immunofluorescence and culture) for diagnosis are not widely available. Rapid influenza diagnostic tests (RIDTs) may be useful in this setting. A total of 552 fresh respiratory specimens were assessed from patients with respiratory symptoms at a teaching hospital in Kuala Lumpur, Malaysia from November 2017 to March 2018. Two digital immunoassays (DIAs), STANDARD F Influenza A/B Fluorescence Immunoassay (STANDARD F) and Sofia Influenza A + B Fluorescence Immunoassay (Sofia) and one conventional RIDT (immunochromatographic assay), SD Bioline Influenza Ag A/B/A(H1N1) Pandemic rapid test kit (SD Bioline) were evaluated in comparison with a WHO-recommended reverse transcription quantitative PCR (RT-qPCR). Of the 552 samples, influenza A virus was detected in 47 (8.5%) and influenza B virus in 7 (1.3%). The digital immunoassays STANDARD F and Sofia had significantly higher overall sensitivity rates (71.7% and 70.6%, respectively) than the conventional RIDT SD Bioline and immunofluorescence/viral culture (55.8% and 52.8%, respectively). Sensitivity rates were higher for influenza A than influenza B, and specificity rates were uniformly high, ranging from 98% to 100%. Digital readout RIDTs can be used in tropical settings with year-round influenza if PCR is unavailable.

Andrographolide is neither a human organic anion transporter 1 (hOAT1) substrate nor inhibitor.

Andrographolide, a major bioactive compound isolated from Andrographis paniculata (Burm. F.) Nees, was evaluated for its effects on the hOAT1 membrane transporter. Substrate determination and inhibition of hOAT1-mediated uptake transport assay was carried out using recombinant CHO-hOAT1 cells. The results showed that the uptake ratio of andrographolide was less than 2.0 at all concentrations tested, indicating that andrographolide is not a hOAT1 substrate. Andrographolide has no significant effects on the p-aminohippuric acid uptake and on the mRNA and protein expression of hOAT1. In conclusion, andrographolide may not pose a drug-herb interaction risk related to hOAT1.

Mitragynine and its potential blocking effects on specific cardiac potassium channels.

Mitragyna speciosa Korth is known for its euphoric properties and is frequently used for recreational purposes. Several poisoning and fatal cases involving mitragynine have been reported but the underlying causes remain unclear. Human ether-a-go-go-related gene (hERG) encodes the cardiac IKr current which is a determinant of the duration of ventricular action potentials and QT interval. On the other hand, IK1, a Kir current mediated by Kir2.1 channel and IKACh, a receptor-activated Kir current mediated by GIRK channel are also known to be important in maintaining the cardiac function. This study investigated the effects of mitragynine on the current, mRNA and protein expression of hERG channel in hERG-transfected HEK293 cells and Xenopus oocytes. The effects on Kir2.1 and GIRK channels currents were also determined in the oocytes. The hERG tail currents following depolarization pulses were inhibited by mitragynine with an IC50 value of 1.62µM and 1.15µM in the transfected cell line and Xenopus oocytes, respectively. The S6 point mutations of Y652A and F656A attenuated the inhibitor effects of mitragynine, indicating that mitragynine interacts with these high affinity drug-binding sites in the hERG channel pore cavity which was consistent with the molecular docking simulation. Interestingly, mitragynine does not affect the hERG expression at the transcriptional level but inhibits the protein expression. Mitragynine is also found to inhibit IKACh current with an IC50 value of 3.32µM but has no significant effects on IK1. Blocking of both hERG and GIRK channels may cause additive cardiotoxicity risks.

SARS-CoV-2 multiplex RT-PCR to detect variants of concern (VOCs) in Malaysia, between January to May 2021.

Emerging SARS-CoV-2 variants of concern (VOC) have been associated with enhanced transmissibility and immune escape. Next-generation sequencing (NGS) of the whole genome is the gold standard for variant identification for surveillance but is time-consuming and costly. Rapid and cost-effective assays that detect SARS-CoV-2 variants are needed. We evaluated Allplex SARS-CoV-2 Master Assay and Variants I Assay to detect HV69/70 deletion, Y144 deletion, E484K, N501Y, and P681H spike mutations in 248 positive samples collected in Kuala Lumpur, Malaysia, between January and May 2021. Spike variants were detected in 78/248 (31.5 %), comprising 60 VOC B.1.351 (beta) and 18 B.1.1.7 (alpha). With NGS as reference for 115 samples, the sensitivity for detecting the spike mutations was 98.7 % with the Master Assay and 100 % with the Variants I Assay. The emergence of beta variants correlated with increasing COVID-19 infections in Malaysia. The prevalence of alpha VOC and lineage B.1.466.2 was low. These assays detect mutations present in alpha, beta and gamma VOCs. Of the VOCs which have subsequently emerged, the assays should detect omicron (B.1.1.529) but not B.1.617.2 (delta). In conclusion, spike variant PCR assays can be used to rapidly monitor selected SARS-CoV-2 VOCs in resource-limited settings, but require updates as new variants emerge.

Rhinovirus/enterovirus was the most common respiratory virus detected in adults with severe acute respiratory infections pre-COVID-19 in Kuala Lumpur, Malaysia.

BACKGROUND: Severe acute respiratory infections (SARI) pose a great global burden. The contribution of respiratory viruses to adult SARI is relatively understudied in Asia. We aimed to determine viral aetiology of adult SARI patients in Kuala Lumpur, Malaysia. METHODS: The prevalence of 20 common (mainly viral) respiratory pathogens, and MERS-CoV, SARS-CoV and 5 bacterial select agents was investigated from May 2017 to October 2019 in 489 SARI adult patients in Kuala Lumpur, Malaysia, using molecular assays (Luminex NxTAG-RPP kit and qPCR assays). Viral metagenomics analysis was performed on 105 negative samples. RESULTS: Viral respiratory pathogens were detected by PCR in 279 cases (57.1%), including 10 (2.0%) additional detections by metagenomics analysis. The most detected viruses were rhinovirus/enterovirus (RV/EV) (49.1%) and influenza virus (7.4%). Three melioidosis cases were detected but no SARS-CoV, MERS-CoV or other bacterial select agents. Bacterial/viral co-detections and viral co-detections were found in 44 (9.0%) and 27 (5.5%) cases respectively, mostly involving RV/EV. Independent predictors of critical disease were male gender, chronic lung disease, lack of runny nose and positive blood culture with a significant bacterial pathogen. Asthma and sore throat were associated with increased risk of RV/EV detection, while among RV/EV cases, males and those with neurological disease were at increased risk of critical disease. CONCLUSIONS: Prior to the COVID-19 pandemic, the high prevalence of respiratory viruses in adults with SARI was mainly attributed to RV/EV. Continued surveillance of respiratory virus trends contributes to effective diagnostic, prevention, and treatment strategies.

Detection of respiratory viruses in adults with suspected COVID-19 in Kuala Lumpur, Malaysia.

BACKGROUND: Reports of co-circulation of respiratory viruses during the COVID-19 pandemic and co-infections with SARS-CoV-2 vary. However, limited information is available from developing countries. OBJECTIVES: We aimed to investigate the incidence of respiratory viruses in adult patients with suspected COVID-19 in Kuala Lumpur, Malaysia. STUDY DESIGN: We collected 198 respiratory samples from adult patients hospitalized with suspected COVID-19 in a single teaching hospital in Kuala Lumpur in February-May 2020 and tested combined oro-nasopharyngeal swabs with the NxTAG Respiratory Pathogen Panel (Luminex) and Allplex RV Essential (Seegene) assays. Forty-five negative samples further underwent viral metagenomics analysis. RESULTS: Of the 198 samples, 74 (37.4%) had respiratory pathogens, including 56 (28.3%) with SARS-CoV-2 and 18 (9.1%) positive for other respiratory pathogens. There were five (2.5%) SARS-CoV-2 co-infections, all with rhinovirus/enterovirus. Three samples (6.7%; 3/45) had viruses identified by metagenomics, including one case of enterovirus D68 and one of Saffold virus genotype 6 in a patient requiring ICU care. Most of the COVID-19 patients (91.1%; 51/56) had mild symptoms but 5.4% (3/56) died. CONCLUSION: During the early COVID-19 period, common respiratory viruses other than SARS-CoV-2 only accounted for 9.1% of hospitalization cases with ARI and co-infections with SARS-CoV-2 were rare. Continued surveillance is important to understand the impact of COVID-19 and its associated public health control measures on circulation of other respiratory viruses. Metagenomics can identify unexpected or rare pathogens, such as Saffold virus, which is rarely described in adults.

Serology surveillance of SARS-CoV-2 antibodies among healthcare workers in COVID-19 designated facilities in Malaysia.

BACKGROUND: Asymptomatic severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infections are well documented. Healthcare workers (HCW) are at increased risk of infection due to occupational exposure to infected patients. We aim to determine the prevalence of SARS-CoV-2 antibodies among HCW who did not come to medical attention. METHODS: We prospectively recruited 400 HCW from the National Public Health Laboratory and two COVID-19 designated public hospitals in Klang Valley, Malaysia between 13/4/2020 and 12/5/2020. Quota sampling was used to ensure representativeness of HCW involved in direct and indirect patient care. All participants answered a self-administered questionnaire and blood samples were taken to test for SARS-CoV-2 antibodies by surrogate virus neutralization test. FINDINGS: The study population comprised 154 (38.5%) nurses, 103 (25.8%) medical doctors, 47 (11.8%) laboratory technologists and others (23.9%). A majority (68.9%) reported exposure to SARS-CoV-2 in the past month within their respective workplaces. Adherence to personal protection equipment (PPE) guidelines and hand hygiene were good, ranging from 91-100% compliance. None (95% CI: 0, 0.0095) of the participants had SARS-CoV-2 antibodies detected, despite 182 (45.5%) reporting some symptoms one month prior to study recruitment. One hundred and fifteen (29%) of participants claimed to have had contact with known COVID-19 persons outside of their workplace. INTERPRETATION: Zero seroprevalence among HCW suggests a low incidence of undiagnosed COVID-19 infection in our healthcare setting during the first local wave of SARS-CoV-2 infection. The occupational risk of SARS-CoV-2 transmission within healthcare facilities can be prevented by adherence to infection control measures and appropriate use of PPE.

SARS-CoV-2 lineage B.6 was the major contributor to early pandemic transmission in Malaysia.

Malaysia had 10,219 confirmed cases of COVID-19 as of September 20, 2020. About 33% were associated with a Tablighi Jamaat religious mass gathering held in Kuala Lumpur between February 27 and March 3, 2020, which drove community transmission during Malaysia's second wave. We analysed genome sequences of SARS-CoV-2 from Malaysia to better understand the molecular epidemiology and spread. We obtained 58 SARS-CoV-2 whole genome sequences from patients in Kuala Lumpur and performed phylogenetic analyses on these and a further 57 Malaysian sequences available in the GISAID database. Nine different SARS-CoV-2 lineages (A, B, B.1, B.1.1, B.1.1.1, B.1.36, B.2, B.3 and B.6) were detected in Malaysia. The B.6 lineage was first reported a week after the Tablighi mass gathering and became predominant (65.2%) despite being relatively rare (1.4%) globally. Direct epidemiological links between lineage B.6 viruses and the mass gathering were identified. Increases in reported total cases, Tablighi-associated cases, and community-acquired B.6 lineage strains were temporally linked. Non-B.6 lineages were mainly travel-associated and showed limited onward transmission. There were also temporally correlated increases in B.6 sequences in other Southeast Asian countries, India and Australia, linked to participants returning from this event. Over 95% of global B.6 sequences originated from Asia Pacific. We also report a nsp3-C6310A substitution found in 47.3% of global B.6 sequences which was associated with reduced sensitivity using a commercial diagnostic real-time PCR assay. Lineage B.6 became the predominant cause of community transmission in Malaysia after likely introduction during a religious mass gathering. This event also contributed to spikes of lineage B.6 in other countries in the Asia-Pacific. Mass gatherings can be significant causes of local and global spread of COVID-19. Shared genomic surveillance can be used to identify SARS-CoV-2 transmission chains to aid prevention and control, and to monitor diagnostic molecular assays. Clinical Trial Registration: COVID-19 paper.

Real-time reverse transcription loop-mediated isothermal amplification for rapid detection of SARS-CoV-2.

BACKGROUND: Highly sensitive real-time reverse transcription polymerase chain reaction (RT-qPCR) methods have been developed for the detection of SARS-CoV-2. However, they are costly. Loop-mediated isothermal amplification (LAMP) assay has emerged as a novel alternative isothermal amplification method for the detection of nucleic acid. METHODS: A rapid, sensitive and specific real-time reverse transcription LAMP (RT-LAMP) assay was developed for SARS-CoV-2 detection. RESULTS: This assay detected one copy/reaction of SARS-CoV-2 RNA in 30 min. Both the clinical sensitivity and specificity of this assay were 100%. The RT-LAMP showed comparable performance with RT-qPCR. Combining simplicity and cost-effectiveness, this assay is therefore recommended for use in resource resource-limited settings.

Selective media and real-time PCR improves diagnosis of melioidosis in community-acquired pneumonia in a low-incidence setting in Kuala Lumpur, Malaysia.

Introduction. Burkholderia pseudomallei (melioidosis) is an important cause of community-acquired pneumonia (CAP) in the tropics. Selective medium is recommended for laboratory diagnosis with non-sterile respiratory samples, while PCR is not routinely used due to variable reported performance. The effectiveness of these diagnostic modalities varies by site.Aim. To compare selective media and real-time PCR (qPCR) with routine media in detecting B. pseudomallei in CAP respiratory samples in a low-incidence setting in Kuala Lumpur, Malaysia.Methodology. Respiratory samples were routinely cultured on blood, chocolate and MacConkey agar (RESP-ROUTINE), and compared to culture on selective Ashdown medium (RESP-SELECTIVE) and qPCR. The gold standard was routine culture of B. pseudomallei from any site (ALL-ROUTINE).Results. B. pseudomallei was detected in 8/204 (3.9 %) samples. Overall sensitivity rates differed (P=0.03) for qPCR (100%), RESP-SELECTIVE (87.5%) and RESP-ROUTINE (50%). There was a trend towards lower median days to positive culture for RESP-SELECTIVE (1 day) compared to RESP-ROUTINE (2 days, P=0.08) and ALL-ROUTINE (2 days, P=0.06). Reagent costs for each additional detection were USD59 for RESP-SELECTIVE and USD354 for PCR.Conclusions. In a low-incidence setting, selective culture of respiratory samples on Ashdown was more sensitive and allowed quicker identification than routine media, at reasonable cost. Blood cultures are critical, confirming four cases missed by routine respiratory culture. Selective medium is useful in early pneumonia (pre-sepsis) and resource-limited settings where blood cultures are infrequently done. Real-time PCR is costly, but highly sensitive and useful for high-risk patients with diabetes, cancer or immunosuppressants, or requiring ventilation or intensive care.

Complete Genome Sequences of SARS-CoV-2 Strains Detected in Malaysia.

We sequenced four severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genomes from Malaysia during the second wave of infection and found unique mutations which suggest local evolution. Circulating Malaysian strains represent introductions from different countries, particularly during the first wave of infection. Genome sequencing is important for understanding local epidemiology.