Performance of selected SARS-CoV-2 antibodies immunoassays.

No abstract available.

Environmental surface sampling of SARS-CoV-2 in selected hospitals in Malaysia.

COVID-19 has spread rapidly worldwide. The role of fomites in facilitating onward transmission is plausible. This study aimed to determine the presence of viable virus and its persistence on the surfaces of fomites in wards treating COVID-19 patients in Malaysia. This study was conducted in two stages. First, environmental sampling was performed on random days in the intensive care unit (ICU) and general wards. Then, in the second stage, samples were collected serially on alternate days for 7 days in two selected general wards. In Stage 1, a total of 104 samples were collected from the surfaces of highly touched and used areas by patients and healthcare workers. Only three samples were tested positive for SARS-COV-2. In Stage 2, three surface samples were detected positive, but no persistence of the virus was observed. However, none of the SARS-CoV-2 RNA was viable through tissue culture. Overall, the environmental contamination of SARS-CoV-2 was low in this hospital setting. Hospitals' strict infection control and the compliance of patients with wearing masks may have played a role in these findings, suggesting adherence to those measures to reduce occupational exposure of COVID-19 in hospital settings.

Phylogenomic analysis of SARS-CoV-2 from third wave clusters in Malaysia reveals dominant local lineage B.1.524 and persistent spike mutation A701V.

The emergence of a third wave of COVID-19 infection in Malaysia since September 2020 has led to imminent changes in public health prevention and control measures. As high as 96.2% of registered COVID-19 cases and 88.5% of confirmed deaths in Malaysia occurred during this third wave of infection. A phylogenomic study on 258 SARS-CoV-2 full genomes from February 2020-February 2021 has led to the discovery of a novel Malaysian lineage B.1.524. This lineage contains another spike mutation A701V that co-exists with the D614G spike mutation that was predominant in most of the third-wave clusters. The study provides vital genomic insights on the rapid spread of the SARS-CoV-2 variants in Malaysia in conjunction with the presence of a dominant SARS-CoV-2 lineage during the third wave of COVID-19 infection.

Environmental surface sampling of SARS-CoV-2 in selected hospitals in Malaysia

@#COVID-19 has spread rapidly worldwide. The role of fomites in facilitating onward transmission is plausible. This study aimed to determine the presence of viable virus and its persistence on the surfaces of fomites in wards treating COVID-19 patients in Malaysia. This study was conducted in two stages. First, environmental sampling was performed on random days in the intensive care unit (ICU) and general wards. Then, in the second stage, samples were collected serially on alternate days for 7 days in two selected general wards. In Stage 1, a total of 104 samples were collected from the surfaces of highly touched and used areas by patients and healthcare workers. Only three samples were tested positive for SARS-COV-2. In Stage 2, three surface samples were detected positive, but no persistence of the virus was observed. However, none of the SARS-CoV-2 RNA was viable through tissue culture. Overall, the environmental contamination of SARS-CoV-2 was low in this hospital setting. Hospitals’ strict infection control and the compliance of patients with wearing masks may have played a role in these findings, suggesting adherence to those measures to reduce occupational exposure of COVID-19 in hospital settings.

Phylogenomic analysis of SARS-CoV-2 from third wave clusters in Malaysia reveals dominant local lineage B.1.524 and persistent spike mutation A701V

@#The emergence of a third wave of COVID-19 infection in Malaysia since September 2020 has led to imminent changes in public health prevention and control measures. As high as 96.2% of registered COVID-19 cases and 88.5% of confirmed deaths in Malaysia occurred during this third wave of infection. A phylogenomic study on 258 SARS-CoV-2 full genomes from February 2020-February 2021 has led to the discovery of a novel Malaysian lineage B.1.524. This lineage contains another spike mutation A701V that co-exists with the D614G spike mutation that was predominant in most of the third-wave clusters. The study provides vital genomic insights on the rapid spread of the SARS-CoV-2 variants in Malaysia in conjunction with the presence of a dominant SARS-CoV-2 lineage during the third wave of COVID-19 infection.

Anti-inflammatory effect of mushrooms in dengue-infected human monocytes.

Pathogenesis of dengue fever has been associated with the activation of the cytokine cascade that triggered inflammatory responses. The inflammatory reactions in dengue haemorrhagic fever/dengue shock syndrome (DHF/DSS) are the main cause of haemorrhagic manifestations, coagulation disorders, vascular permeability, hypotension and shock which could exacerbate the condition of the disease. In an earlier study, extracts belonging to Lignosus rhinocerotis, Pleurotus giganteus, Hericium erinaceus, Schizophyllum commune and Ganoderma lucidium mushrooms were screened for antidengue virus activities. We found that hot aqueous extract (HAE) and aqueous soluble separated from ethanol extract (ASE) exhibited their potential to reduce dengue viral load which were observed in plaque reduction assay and real-time RT-PCR. In continuation of our previous findings, this study was initiated to further investigate the other aspect; the anti-inflammatory activities of HAE and ASE of L. rhinocerotis, P. giganteus, H. erinaceus, S. commune and G. lucidium on human monocytes infected with dengue virus-2 (DENV-2) New guinea C strain. Human monocytes infected with DENV-2 were treated with mushroom extracts for 48 hours. The cytokine profile coincides with dengue infection, i.e. IFN-γ, TNF-α, IL-1ß, IL-6, IL-8, and IL-10 were measured by BD OptEIATM Elisa Kit. The expression of these cytokines was significantly elevated in untreated infected cells two days after infection. However, after treated with mushroom extracts prominent anti-inflammatory effect were detected towards IFN-γ, IL-10, TNF-α, IL-6, and IL-1ß. The most significant anti-inflammatory effects were detected in HAE of G. lucidium, S. commune, P. giganteus and ASE of L. rhinocerotis and the effects were comparable with dexamethasone, the reference inhibitor. These results demonstrated that mushroom HAE or ASE could successfully have suppressed cytokine production in dengue-infected monocytes and has a great potential to develop an antiinflammatory agent from mushroom extract for the treatment of dengue infection.

Anti-inflammatory effect of mushrooms in dengue-infected human monocytes

@#Pathogenesis of dengue fever has been associated with the activation of the cytokine cascade that triggered inflammatory responses. The inflammatory reactions in dengue haemorrhagic fever/dengue shock syndrome (DHF/DSS) are the main cause of haemorrhagic manifestations, coagulation disorders, vascular permeability, hypotension and shock which could exacerbate the condition of the disease. In an earlier study, extracts belonging to Lignosus rhinocerotis, Pleurotus giganteus, Hericium erinaceus, Schizophyllum commune and Ganoderma lucidium mushrooms were screened for antidengue virus activities. We found that hot aqueous extract (HAE) and aqueous soluble separated from ethanol extract (ASE) exhibited their potential to reduce dengue viral load which were observed in plaque reduction assay and real-time RT-PCR. In continuation of our previous findings, this study was initiated to further investigate the other aspect; the anti-inflammatory activities of HAE and ASE of L. rhinocerotis, P. giganteus, H. erinaceus, S. commune and G. lucidium on human monocytes infected with dengue virus-2 (DENV-2) New guinea C strain. Human monocytes infected with DENV-2 were treated with mushroom extracts for 48 hours. The cytokine profile coincides with dengue infection, i.e. IFN-γ, TNF-α, IL-1β, IL-6, IL-8, and IL-10 were measured by BD OptEIATM Elisa Kit. The expression of these cytokines was significantly elevated in untreated infected cells two days after infection. However, after treated with mushroom extracts prominent anti-inflammatory effect were detected towards IFN-γ, IL-10, TNF-α, IL-6, and IL-1β. The most significant anti-inflammatory effects were detected in HAE of G. lucidium, S. commune, P. giganteus and ASE of L. rhinocerotis and the effects were comparable with dexamethasone, the reference inhibitor. These results demonstrated that mushroom HAE or ASE could successfully have suppressed cytokine production in dengue-infected monocytes and has a great potential to develop an antiinflammatory agent from mushroom extract for the treatment of dengue infection.

HIV-2 Infection in Malaysia: Current situation and the use of in-house real-time reverse transcription PCR for HIV-2.

HIV-2 surveillance has been carried out in Malaysia for more than 25 years ago. Tests to discriminate HIV-1 and HIV-2 are available but the options of test are limited and the need to develop a new in-house HIV-2 real-time reverse transcription PCR (RT-PCR) is crucial. A study was done on 29 samples from hospitals in Malaysia which were found to be positive screening for HIV-2 antibodies by the commercial Western Blot assay. These samples were further tested by a Western Blot assay that detects specific antibodies to HIV-2. Detection of HIV-2 genome was then performed by using a commercial kit. Fifteen samples were evaluated by using in-house real-time RT-PCR for HIV-2. Ninety-three percent (27/29) of samples have positive results for HIV-2 on HIV-2 Western Blot with only 2 samples showing indeterminate results. All samples showed negative results for HIV-2 genomes by using a PCR commercial kit and the 15 samples that were subjected to our in-house real-time RT-HIV-2 PCR were also tested negative for HIV-2 RNA. Results of HIV-2 Western Blot did not reflect the actual positivity as both HIV-1 and HIV-2 antibodies may cross-react with either viral proteins. None of the samples was confirmed positive for HIV-2 by the commercial and in-house real-time RTPCR. In-house real-time RT-HIV-2 PCR assay can be further used to confirm the presence of HIV-2 genome. Up to the year 2015, Malaysia is still free from HIV-2 infection.

HIV-2 Infection in Malaysia: Current situation and the use of in-house real-time reverse transcription PCR for HIV-2

@#HIV-2 surveillance has been carried out in Malaysia for more than 25 years ago. Tests to discriminate HIV-1 and HIV-2 are available but the options of test are limited and the need to develop a new in-house HIV-2 real-time reverse transcription PCR (RT-PCR) is crucial. A study was done on 29 samples from hospitals in Malaysia which were found to be positive screening for HIV-2 antibodies by the commercial Western Blot assay. These samples were further tested by a Western Blot assay that detects specific antibodies to HIV-2. Detection of HIV-2 genome was then performed by using a commercial kit. Fifteen samples were evaluated by using in-house real-time RT-PCR for HIV-2. Ninety-three percent (27/29) of samples have positive results for HIV-2 on HIV-2 Western Blot with only 2 samples showing indeterminate results. All samples showed negative results for HIV-2 genomes by using a PCR commercial kit and the 15 samples that were subjected to our in-house real-time RT-HIV-2 PCR were also tested negative for HIV-2 RNA. Results of HIV-2 Western Blot did not reflect the actual positivity as both HIV-1 and HIV-2 antibodies may cross-react with either viral proteins. None of the samples was confirmed positive for HIV-2 by the commercial and in-house real-time RTPCR. In-house real-time RT-HIV-2 PCR assay can be further used to confirm the presence of HIV-2 genome. Up to the year 2015, Malaysia is still free from HIV-2 infection.

Sequence analysis of E/NS1 gene junction of Dengue Type-1 viruses isolated in Klang Valley 2010 to 2012.

Dengue is a mosquito-borne viral disease caused by four serotypes of dengue virus, affecting the human population for decades in many tropical and subtropical regions of the world. In Malaysia, all four dengue serotypes co-circulates in a dengue season even though any one of the serotypes can predominate. In this study, serum samples were collected from dengue fever and severe dengue fever patients within Klang Valley from 2010-2012 to determine the prevailing dengue serotypes. In addition, sequencing of the envelope/nonstructural 1 (E/NS1) gene junction of the virus isolated was performed to identify the presence of any mutations that are suggestive of increased virulence in the virus. The results showed that Dengue-1 (DEN-1) was the predominant circulating serotype. The E/NS1 gene sequences of the isolates were analysed to trace the evolutionary knowledge of the strains. All sequences of the isolates were compared with DEN-1 prototype Hawaii strain as the reference sequence. The E/NS1 sequences of other dengue strains from neighbouring regions as well as other parts of the world obtained from the GenBank database were also included in the phylogenetic tree analysis. Analyses showed that there was 97% to 100% similarity among the ten isolates at the nucleotide level. Similarly, the amino acid analogue also showed 98% to 100% homology. However, all five non-severe dengue isolates showed variation at position 780, resulting in an amino acid change from valine to alanine as compared to severe dengue isolates. A rooted phylogenetic tree was performed using neighbour-joining method with DEN-2 and DEN-3 as the outgroups. Results showed that all ten isolates were classified as genotype I. In addition, the five isolates from severe dengue patients were found to be clustered together with JN697057 and JN697058, Malaysian DEN-1 strains from the 2005 outbreak.

Drug resistance mutations among virological failure HIV-1 infected patients in Malaysia.

The determination of HIV drug resistance mutations (DRMs) towards antiretroviral (ARV) drugs among HIV-1 treated patients with virological failure is crucial for further management of the patient. This study aimed to assess the most common genomic mutation and to analyse subtypes among the HIV-1 patients with viral load level > 1,000 copies/mL. A total of 101 virological failure HIV-1 patients from four different regions of Peninsular Malaysia with a viral load measurement facility were included in the study. Majority of patients (89.1%) have at least 1 mutation associated with clinical resistance to either protease inhibitors (PIs), nucleoside reverse transcriptase inhibitors (NRTIs) or nonnucleoside reverse transcriptase inhibitors (NNRTIs). Major resistance mutations among the patients towards NRTIs and NNRTIs were 70.3% and 18.8%, respectively. The most common mutation for NRTIs was M184V while K103N mutation was detected in the majority of patients who were treated with NNRTIs. The most commonly observed mutations for major PI and minor PI seen among the study population were V82A/T and L10V, respectively. In HIV-1 subtype analysis, CRF33_01B was the most predominant HIV-1 subtype in this study group. The vast detection of DRMs in this study emphasized the importance of genotypic resistance test in the management of HIV patients as DRMs can alter patient's susceptibility towards ARV drugs. Further study on larger number of samples is essential for the development of a database on HIV-1 DRMs among patients that experience virological failure in Malaysia.

Drug resistance mutations among virological failure HIV-1 infected patients in Malaysia

The determination of HIV drug resistance mutations (DRMs) towards antiretroviral (ARV) drugs among HIV-1 treated patients with virological failure is crucial for further management of the patient. This study aimed to assess the most common genomic mutation and to analyse subtypes among the HIV-1 patients with viral load level > 1,000 copies/mL. A total of 101 virological failure HIV-1 patients from four different regions of Peninsular Malaysia with a viral load measurement facility were included in the study. Majority of patients (89.1%) have at least 1 mutation associated with clinical resistance to either protease inhibitors (PIs), nucleoside reverse transcriptase inhibitors (NRTIs) or nonnucleoside reverse transcriptase inhibitors (NNRTIs). Major resistance mutations among the patients towards NRTIs and NNRTIs were 70.3% and 18.8%, respectively. The most common mutation for NRTIs was M184V while K103N mutation was detected in the majority of patients who were treated with NNRTIs. The most commonly observed mutations for major PI and minor PI seen among the study population were V82A/T and L10V, respectively. In HIV-1 subtype analysis, CRF33_01B was the most predominant HIV-1 subtype in this study group. The vast detection of DRMs in this study emphasized the importance of genotypic resistance test in the management of HIV patients as DRMs can alter patient’s susceptibility towards ARV drugs. Further study on larger number of samples is essential for the development of a database on HIV-1 DRMs among patients that experience virological failure in Malaysia.

Sequence analysis of E/NS1 gene junction of Dengue Type-1 viruses isolated in Klang Valley 2010 to 2012

Dengue is a mosquito-borne viral disease caused by four serotypes of dengue virus, affecting the human population for decades in many tropical and subtropical regions of the world. In Malaysia, all four dengue serotypes co-circulates in a dengue season even though any one of the serotypes can predominate. In this study, serum samples were collected from dengue fever and severe dengue fever patients within Klang Valley from 2010-2012 to determine the prevailing dengue serotypes. In addition, sequencing of the envelope/nonstructural 1 (E/NS1) gene junction of the virus isolated was performed to identify the presence of any mutations that are suggestive of increased virulence in the virus. The results showed that Dengue-1 (DEN-1) was the predominant circulating serotype. The E/NS1 gene sequences of the isolates were analysed to trace the evolutionary knowledge of the strains. All sequences of the isolates were compared with DEN-1 prototype Hawaii strain as the reference sequence. The E/NS1 sequences of other dengue strains from neighbouring regions as well as other parts of the world obtained from the GenBank database were also included in the phylogenetic tree analysis. Analyses showed that there was 97% to 100% similarity among the ten isolates at the nucleotide level. Similarly, the amino acid analogue also showed 98% to 100% homology. However, all five non-severe dengue isolates showed variation at position 780, resulting in an amino acid change from valine to alanine as compared to severe dengue isolates. A rooted phylogenetic tree was performed using neighbour-joining method with DEN-2 and DEN-3 as the outgroups. Results showed that all ten isolates were classified as genotype I. In addition, the five isolates from severe dengue patients were found to be clustered together with JN697057 and JN697058, Malaysian DEN-1 strains from the 2005 outbreak.

Dengue serotype surveillance among patients admitted for dengue in two major hospitals in Selangor, Malaysia, 2010-2011.

Dengue serotype surveillance is important as any changes in serotype distribution may result in an outbreak or increase in severe dengue cases. This study aimed to determine circulating dengue serotypes in two hospitals in Selangor. Serum samples were collected from patients admitted for dengue at these two major public hospitals i.e. Hospital Sungai Buloh (HSB) and Hospital Tunku Ampuan Rahimah (HTAR) between November 2010 and August 2011 and subjected to real-time RT-PCR using SYBR® Green. All four dengue serotypes were detected in samples from both hospitals. The predominating serotype was dengue 1 in samples from both hospitals (HSB, DENV-1; 25.53 % and HTAR, DENV-1; 32.1 %).

Molecular typing of dengue viruses circulating on the East Coast of Peninsular Malaysia from 2005 to 2009.

From 2005 to 2009, the Institute for Medical Research (IMR), Kuala Lumpur, Malaysia received 488 serum and blood samples from hospitalized patients on the East Coast of Peninsular Malaysia, suspected of having dengue infection. In this study we determined the prevailing dengue serotypes using a real time polymerase chain reaction assay (RT-PCR). All 4 dengue virus serotypes were found circulating during the study period; however the predominant serotype varied. In 2005 and 2006, the predominant serotypes circulating were DENV-1 and DENV-3, in 2007, DENV-1 and DENV-2 were predominant, and in 2008 and 2009, DENV-3 was the predominant serotype.

Rapid detection and serotyping of dengue virus by multiplex RT-PCR and real-time SYBR green RT-PCR.

INTRODUCTION: Dengue fever and dengue haemorrhagic fever currently rank highly among the newly-emerging infectious diseases, and are considered to be the most important arboviral disease worldwide. The definitive diagnosis is culture analysis, but practical considerations limit its use. Also, the period for viral detection is limited. Within a day or two after fever subsides, rising levels of antibodies interfere with viral cultures. An alternative to this quandary is the use of viral RNA detection assays. In our laboratory, a reverse transcriptase polymerase chain reaction (RT-PCR) assay was developed using a set of degenerate primers. METHODS: This multiplex RT-PCR assay was evaluated with 280 samples collected during the year 2003. These groups include prototype dengue virus (serotypes 1-4), acute serum from which the dengue virus was isolated, seronegative acute samples (culture negative) but whose convalescent samples seroconverted, and sera positive for other microbial diseases. This assay was then modified into a real-time SYBR Green RT-PCR assay. Sensitivity and specificity of both assays were compared. RESULTS: The multiplex RT-PCR assay was able to detect 134 samples whereas SYBR Green RT-PCR assay was able to detect 178 out of 306 samples. Both assays were 100 percent specific. Further analysis of 53 samples showed that the virus could be amplified at IgM positive/negative values of up to 4.2, and up to six days after onset of fever. The viral detection rate was inversely proportional to the day of fever onset as well as IgM values. CONCLUSION: The sensitivity and specificity of the conventional multiplex RT-PCR assay are 98.18 percent and 100 percent, respectively, and for the real-time SYBR Green assay, 99.09 percent and 100 percent, respectively. The melting curve analysis allows all four dengue serotypes to be discriminated based on distinct melting temperature value. The accuracy and speed of this multiplex RTPCR assay makes it a suitable test for the diagnosis of dengue and for epidemiological surveillance.

Type-3 dengue viruses responsible for the dengue epidemic in Malaysia during 1993-1994.

To characterize the dengue epidemic that recently occurred in Malaysia, we sequenced cDNAs from nine 1993-1994 dengue virus type-3 (DEN-3) isolates in Malaysia (DEN-3 was the most common type in Malaysia during this period). Nucleic acid sequences (720 nucleotides in length) from the nine isolates, encompassing the precursor of membrane protein (preM) and membrane (M) protein genes and part of the envelope (E) protein gene were aligned with various reference DEN-3 sequences to generate a neighbor-joining phylogenetic tree. According to the constructed tree, the nine Malaysian isolates were grouped into subtype II, which comprises Thai isolates from 1962 to 1987. Five earlier DEN-3 virus Malaysian isolates from 1974 to 1981 belonged to subtype I. The present data indicate that the recent dengue epidemic in Malaysia was due to the introduction of DEN-3 viruses previously endemic to Thailand.

Comparison of sequences of E/NS1 gene junction of dengue type 3 virus following culture subpassage in C6/36 cells to study the possible occurrence of mutations.

The aim of this study was to determine whether mutations could occur in the dengue virus genome following three subpassages of the virus in a mosquito cell line. This was done because sources of virus isolates used for sequencing studies are usually maintained in cell lines rather than in patients' sera. Therefore it must be assured that no mutation occurred during the passaging. For this purpose, sequencing was carried out using the polymerase chain reaction (PCR) products of the envelope/non-structural protein 1 junction region (280 nucleotides) of dengue type 3 virus. Sequence data were compared between the virus from a patient's serum against the virus subpassaged three times in the C6/36 cell line. We found that the sequence data of the virus from serum was identical to the virus that was subpassaged three times in C6/36 cell line.

Genotypes of Japanese encephalitis virus isolated in three states in Malaysia.

Two hundred forty nucleotides from the pre-membrane gene region of 12 Japanese encephalitis virus (JEV) strains isolated from three different regions of Malaysia from 1993 to 1994 were sequenced and compared with each other and with the JEV strains from different geographic areas in Asia. These 12 Malaysian isolates were classified into two genotypes. The four JEV strains isolated from Sarawak in 1994 and the four JEV strains isolated from Sepang, Selangor in 1993 were classified into one genotype that included earlier isolated strains from Malaysia (JE-827 from Sarawak in 1968 and WTP/70/22 from Kuala Lumpur in 1970). The four JEV strains from Ipoh, Perak in 1994 were classified into another genotype that included JEV strains isolated from northern Thailand and Cambodia. In an earlier report, 10 JEV strains from Sabak Bernam, Selangor in 1992 were classified into the largest genotype that included strains isolated in temperate regions such as Japan, China, and Taiwan. The data indicate that at least three genotypes of JEV have been circulating in Malaysia.

The use of polymerase chain reaction (PCR) as a diagnostic tool for dengue virus.

This study describes the use of polymerase chain reaction as a diagnostic tool for detecting and typing of dengue virus. PCR was compared against virus isolation. First RT-PCR was done using dengue consensus primers after which positive samples were subjected to RT-PCR using type-specific primers. This study shows that the local strains of the dengue virus could be detected using the chosen primers. Furthermore, RT-PCR was found to be more sensitive than virus isolation in identifying the dengue positive samples.