Molecular characterization and prenatal diagnosis for beta-thalassemia can be carried out using the Amplification Refractory Mutation System (ARMS). The ARMS is a rapid and direct molecular technique in which beta-thalassemia mutations are visualized immediately after DNA amplification by gel electrophoresis. In the University of Malaya Medical Center, molecular characterization and prenatal diagnosis for beta-thalassemia is carried out using ARMS for about 96% of the Chinese and 84.6% of the Malay patients. The remaining 4% and 15.4% of the uncharacterized mutations in the Chinese and Malay patients respectively are detected using DNA sequencing. DNA sequencing is an accurate technique but it is more time-consuming and expensive compared with the ARMS. The ARMS for the rare Chinese beta-mutations at position -29 (A-->G) and the ATG-->AGG base substitution at the initiator codon for translation in the beta-gene was developed. In the Malays, ARMS was optimized for the beta-mutations at codon 8/9 (+G), Cap (+1) (A-->C) and the AATAAA-->AATAGA base substitution in the polyadenylation region of the beta-gene. The ARMS protocols were developed by optimization of the parameters for DNA amplification to ensure sensitivity, specificity and reproducibility. ARMS primers (sequences and concentration), magnesium chloride concentration, Taq DNA polymerase and PCR cycling parameters were optimized for the specific amplification of each rare beta-thalassemia mutation. The newly-developed ARMS for the 5 rare beta-thalassemia mutations in the Chinese and Malays in Malaysia will allow for more rapid and cost-effective molecular characterization and prenatal diagnosis for beta-thalassemia in Malaysia.
Phylogenetic analyses of the envelope (E) gene sequence of five recently isolated dengue virus type 4 (DENV-4) suggested the emergence of a distinct geographical and temporal DENV-4 subgenotype IIA in Malaysia. Four of the isolates had direct ancestral lineage with DENV-4 Indonesia 1973 and showed evidence of intra-serotypic recombination with the other recently isolated DENV-4, MY01-22713. The E gene of isolate MY01-22713 had strong evidence of an earlier recombination involving DENV-4 genotype II Indonesia 1976 and genotype I Malaysia 1969. These results suggest that intra-serotypic recombination amongst DENV-4 from independent ancestral lineages may have contributed to the emergence of DENV-4 subgenotype IIA in Malaysia.
The effects of Enterovirus 71 (HEV71) infection on African green monkey kidney cells (Vero) were investigated. It was found that the infected cells showed progressive cellular morphological changes characteristic in apoptotic cells within 10 hours post-infection. The number of apoptotic cells correlated significantly with the number of HEV71 antigen positive cells when cells were labeled using terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) and stained for HEV71 antigen. Approximately 11, 26, 45 and 50% of the infected cells were apoptotic at 12, 24, 48 and 72 hours post-infection, respectively. Internucleosomal DNA fragmentation, characteristic in the late stage of apoptosis was noted beginning on day 2 post-infection. The DNA fragmentation, however, was absent in cells treated with the heat- and ultraviolet light-inactivated virus inocula. These results demonstrate the capacity of HEV71 to induce apoptosis in the infected cells. The induction, however, requires high level of HEV71 infectivity and the presence of live virus particles, suggesting the need for the presence of specific viral proteins for apoptosis to occur.
Hand, foot and mouth disease (HFMD) is a common illness of infants and young children <10 years of age. It is characterized by fever, ulcers in the oral cavity, and rashes with blisters that appear on the palm and sole. The most common causal agents of HFMD are coxsackievirus A16 (CV-A16) and human enterovirus 71 (HEV71), but other enteroviruses, including CV-A5 and CV-A10, can also cause it. When caused by CV-A16 infection, it is usually a mild disease, and patients normally recover without requiring any special medical attention.
The efficacy of Virkon S, a commercial disinfectant as a virucidal spray against human enterovirus 71 (HEV71), the causative agent of the fatal form of hand, foot and mouth disease was examined. At least one log10 reduction of HEV71 titer was achieved when one spray of Virkon (1% or 2%) with ten minutes of contact time was applied. The infectivity was completely lost when four sprays of 1% or 2% Virkon were applied, suggesting that at least four sprays of 1% Virkon to the surface bound HEV71 was necessary to completely inactivate the virus. These findings suggest that Virkon S at the proper concentration is suitable to be used as an effective and easy to use disinfectant against HEV71.
At least three different EV-71 subgenotypes were identified from an outbreak in Malaysia in 1998. The subgenotypes C2 and B4 were associated with the severe and fatal infections, whereas the B3 virus was associated with mild to subclinical infections. The B3 virus genome sequences had >= 85% similarity at the 3' end to CV-A16. This offers opportunities to examine if there are characteristic similarities and differences in virulence between CV-A16, EV-71 B3 and EV-71 B4 and to determine if the presence of the CV-A16-liked genes in EV-71 B3 would also confer the virus with a CV-A16-liked neurovirulence in mice model infection.
Varicella-zoster virus (VZV) infections are a particular problem in healthcare settings. A survey of chickenpox was carried out amongst healthcare workers (HCWs) following potential ward exposures. A prior history of chickenpox was given by 61/98 (62.2%). Of 64 HCWs tested for VZV IgG, 10 (15.6%) were seronegative, indicating susceptibility. The sensitivity, specificity, positive predictive value, and negative predictive value of a history of prior chickenpox were 57.4%, 90%, 96.4%, and 31.0%, respectively. VZV screening of HCWs without a history of chickenpox, and vaccination of susceptible HCWs should be undertaken in this hospital.
The 1st 9 clinical isolates of multisensitive community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) from Malaysia carry SCCmec type IV and predominantly cause skin and soft-tissue infections. Seven were classified as nosocomially acquired. There was considerable clonal diversity, with both pandemic and novel multilocus sequence types detected. CA-MRSA rates appear to be increasing in our hospital, warranting close surveillance.
BACKGROUND: Chikungunya virus (CHIKV) of the Central/East African genotype has caused large outbreaks worldwide in recent years. In Malaysia, limited CHIKV outbreaks of the endemic Asian and imported Central/East African genotypes were reported in 1998 and 2006. Since April 2008, an unprecedented nationwide outbreak has affected Malaysia.
OBJECTIVE: To study the molecular epidemiology of the current Malaysian CHIKV outbreak, and to evaluate cross-neutralisation activity of serum from infected patients against isolates of Asian and Central/East African genotypes.
STUDY DESIGN: Serum samples were collected from 83 patients presenting in 2008, and tested with PCR for the E1 gene, virus isolation, and for IgM. Phylogenetic analysis was performed on partial E1 gene sequences of 837bp length. Convalescent serum from the current outbreak and Bagan Panchor outbreak (Asian genotype, 2006) were tested for cross-neutralising activity against representative strains from each outbreak.
RESULTS: CHIKV was confirmed in 34 patients (41.0%). The current outbreak strain has the A226V mutation in the E1 structural protein, and grouped with Central/East African isolates from recent global outbreaks. Serum cross-neutralisation activity against both Central/East African and Asian genotypes was observed at titres from 40 to 1280.
CONCLUSIONS: The CHIKV strain causing the largest Malaysian outbreak is of the Central/East African genotype. The presence of the A226V mutation, which enhances transmissibility of CHIKV by Aedes albopictus, may explain the extensive spread especially in rural areas. Serum cross-neutralisation of different genotypes may aid potential vaccines and limit the effect of future outbreaks.
Human enterovirus 71 (EV-71) is genotyped for molecular epidemiological investigation mainly using the two structural genes, VP1 and VP4. Based on these, EV-71 is divided into three genotypes, A, B and C, and within the genotypes B and C, there are further subgenotypes, B1-B5 and C1-C5. Classification using these genes is useful but gives incomplete phylogenetic information. In the present study, the phylogenetic relationships amongst all the known EV-71 and human enterovirus A (HEV-A) isolates with complete genome sequences were examined. A different tree topology involving EV-71 isolates of subgenotypes, C4 and B5 was obtained in comparison to that drawn using VP1. The nucleotide sequence divergence of the C4 isolates was 18.11% (17-20%) when compared to other isolates of subgenotype C. However, this positions the C4 isolates within the cut-off divergence value of 17-22% used to designate the virus genotypes. Hence, it is proposed here that C4 should be designated as a new genotype D. In addition, the subgenotype B5 isolates had an average nucleotide divergence of only 6.14% (4-8%) when compared to other subgenotype B4 isolates. This places the B5 isolates within the subgenotype B4. It is proposed here that the B5 isolates to be redesignated as B4. With the newly proposed genotype D and inclusion of subgenotype B5 within B4, the average nucleotide divergence between genotypes was 18.99% (17-22%). Inter- and intra-subgenotype average divergences were 12.02% (10-14%) and 3.92% (1-10%), respectively. A phylogenetic tree built using the full genome sequences is robust as it takes into consideration changes in the sequences of both the structural and non-structural genes. Similar nucleotide similarities, however, were obtained if only VP1 and 3D RNA polymerase genes were used. Furthermore, addition of 3D RNA polymerase sequences will also show recombination events. Hence, in the absence of full genome sequences, it is proposed here that a combination of VP1 and 3D RNA polymerase gene sequences be used for initial genotyping of EV-71 isolates.
Chikungunya virus (CHIKV) is a mosquito-borne alphavirus which causes fever, rash, and arthralgia. In the past, life-threatening complications were very rarely reported. However, during the recent worldwide outbreaks, there have been several reports of unusually severe complications and deaths. Malaysia is experiencing a nationwide outbreak of CHIKV, with over 10 000 patients affected since April 2008. We report the first case of culture-confirmed CHIKV-associated death in Malaysia, in a patient with fever, rash, acute exacerbation of pre-existing heart failure, rhabdomyolysis, and multiple organ failure. CHIKV infections may cause atypical, severe or fatal presentations.
OBJECTIVES: The clinical impact of seasonal influenza is understudied in tropical countries. The aim of this study was to describe the clinical features and seasonal pattern of influenza in children hospitalized in Malaysia, and to identify predictors of severe disease.
METHODS: Children hospitalized with community-acquired, laboratory-confirmed influenza at a teaching hospital in Kuala Lumpur, Malaysia during 2002-2007 were identified retrospectively. Clinical data were collected, and predictors of severe disease were identified by multivariate logistic regression. All influenza cases from 1982 to 2007 were also analyzed for seasonal patterns.
RESULTS: A total of 132 children were included in the study, 48 (36.4%) of whom had underlying medical conditions. The mean age was 2.5 years and 116 (87.9%) were <5 years old. The most common presenting features were fever or history of fever, cough, rhinitis, vomiting, and pharyngitis. Severe influenza was seen in 16 patients (12.1%; nine previously healthy), including 12 (9.1%; eight previously healthy) requiring intensive care. There were three (2.3%) deaths. Severe disease was associated with age <12 months, female sex, and absence of rhinitis on admission. Influenza was seen year-round, with peaks in November-January and May-July.
CONCLUSIONS: Seasonal influenza has a considerable impact on children hospitalized in Malaysia, in both the healthy and those with underlying medical conditions.
Study site: University Malaya Medical Centre (UMMC)
In 2006, an outbreak of Chikungunya virus (CHIKV) of the Asian genotype affected over 200 people in Bagan Panchor village in Malaysia. One year later, a post-outbreak survey was performed to determine attack rate, asymptomatic rate, and post-infection sequelae. Findings were compared with recent CHIKV outbreaks of the Central/East African genotype. A total of 180 residents were interviewed for acute symptoms and post-infection physical quality of life and depressive symptoms. Sera from 72 residents were tested for CHIKV neutralizing antibodies. The estimated attack rate was 55.6%, and 17.5% of infected residents were asymptomatic. Arthralgia was reported up to 3 months after infection, but there were no reports of long-term functional dependence or depression. Symptomatic and seropositive residents were significantly more likely to live in the area with the most dense housing and commercial activities. CHIKV had a high attack rate and considerable clinical impact during the Bagan Panchor outbreak.