Sazaly AbuBakar

Abstract: Background: Brucella species are easily transmitted by aerosols and can be acquired in the laboratory. Aim: To report the management of a large exposure to Brucella melitensis that occurred over six days in a hospital diagnostic laboratory. Methods: Fifty-one exposed staff were managed according to Centers for Disease Control and Prevention guidelines. A further 96 non-exposed laboratory staff were tested for seroprevalence. Testing was carried out using the Brucella sp. serum agglutination test. Findings: Twenty-seven people had high-risk exposure and 24 had low-risk exposure. High-risk staff were offered post-exposure prophylaxis. Twelve (44.4%) agreed to this, of whom eight (66.7%) completed the course. Overall compliance with serological follow-up at baseline, 2, 4, 6 weeks and 8 months was 45.9%. Despite this poor compliance there were no clinical brucellosis cases and no seroconversion in the 47.1% of staff tested at 8 months. Brucella sp. seroprevalence among all staff tested was 3/147 (2.0%). Conclusion: Lack of experience with Brucella spp. and lack of policies for handling potentially hazardous organisms contributed to this prolonged exposure. As compliance with current recommendations may be poor, the optimum frequency of serological follow-up and target groups for prophylaxis should be reassessed. Laboratories in low- or non-endemic areas must prepare for potential isolation of Brucella spp. The impact of human brucellosis in Malaysia requires further study. (C) 2011 The Healthcare Infection Society. Published by Elsevier Ltd. All rights reserved.

Abstract: Dengue fever is a mosquito-borne viral disease estimated to cause about 230 million infections worldwide every year, of which 25,000 are fatal. Global incidence has risen rapidly in recent decades with some 3.6 billion people, over half of the world�s population, now at risk, mainly in urban centres of the tropics and subtropics. Demographic and societal changes, in particular urbanization, globalization, and increased international travel, are major contributors to the rise in incidence and geographic expansion of dengue infections. Major research gaps continue to hamper the control of dengue. The European Commission launched a call under the 7th Framework Programme with the title of �Comprehensive control of Dengue fever under changing climatic conditions�. Fourteen partners from several countries in Europe, Asia, and South America formed a consortium named �DengueTools� to respond to the call to achieve better diagnosis, surveillance, prevention, and predictive models and improve our understanding of the spread of dengue to previously uninfected regions (including Europe) in the context of globalization and climate change.The consortium comprises 12 work packages to address a set of research questions in three areas:Research area 1: Develop a comprehensive early warning and surveillance system that has predictive capability for epidemic dengue and benefits from novel tools for laboratory diagnosis and vector monitoring.Research area 2: Develop novel strategies to prevent dengue in children.Research area 3: Understand and predict the risk of global spread of dengue, in particular the risk of introduction and establishment in Europe, within the context of parameters of vectorial capacity, global mobility, and climate change.In this paper, we report on the rationale and specific study objectives of �DengueTools�. DengueTools is funded under the Health theme of the Seventh Framework Programme of the European Community, Grant Agreement Number: 282589 Dengue Tools.

Abstract: Bioactive compounds from the medicinal plant, Eurycoma longifolia Jack have been shown to promote anti-proliferative effects on various cancer cell lines. Here we examined the effects of purified eurycomanone, a quassinoid found in Eurycoma longifolia Jack extract, on the expression of selected genes of the A549 lung cancer cells. Eurycomanone inhibited A549 lung cancer cell proliferation in a dose-dependent manner at concentrations ranging from 5 to 20 mu g/ml. The concentration that inhibited 50% of cell growth (GI(50)) was 5.1 mu g/ml. The anti-proliferative effects were not fully reversible following the removal of eurycomanone, in which 30% of cell inhibition still remained (p < 0.0001, T-test). At 8 mu g/ml (GI(70)), eurycomanone suppressed anchorage-independent growth of A549 cells by >25% (p < 0.05, T-test, n = 8) as determined using soft agar colony formation assay. Cisplatin, a chemotherapy drug used for the treatment of non small cell lung cancer on the other hand, inhibited A549 cells proliferation at concentrations ranging from 0.2 mu g/ml to 15 mu g/ml with a GI(50) of 0.58 mu g/ml. The treatment with eurycomanone reduced the abundance expression of the lung cancer markers, heterogeneous nuclear ribonucleoprotein (hnRNP) A2/B1, p53 tumor suppressor protein and other cancer-associated genes including prohibitin (PHB), annexin 1 (ANX1) and endoplasmic reticulum protein 28 (ERp28) but not the house keeping genes. The mRNA expressions of all genes with the exception of PHB were significantly downregulated, 72 h after treatment (p < 0.05, T-test, n = 9). These findings suggest that eurycomanone at viable therapeutic concentrations of 5-20 mu g/ml exhibited significant anti-proliferative and anti-clonogenic cell growth effects on A549 lung cancer cells. The treatment also resulted in suppression of the lung cancer cell tumor markers and several known cancer cell growth-associated genes. (C) 2011 Elsevier GmbH. All rights reserved.

Abstract: This study investigates the effects of 2-phenyl-1-benzopyran-4-one (flavone) on DENV-2 infectivity in Vero cells. Virus adsorption and attachment and intracellular virus replication were investigated using a foci forming unit assay (FFUA) and quantitative RT-PCR, respectively. Addition of flavone (100 mu g/mL) significantly increased the number of DENV-2 foci by 35.66% +/- 1.52 and 49.66% +/- 2.51 when added during and after virus adsorption to the Vero cells, respectively. The average foci size after 4 days of infection increased by 33% +/- 2.11 and 89% +/- 2.13. The DENV-2 specific RNA copy number in the flavone-treated infected cells increased by 6.41- and 23.1-fold when compared to the mock-treated infected cells. Flavone (100 mu g/mL) did not promote or inhibit Vero cell proliferation. The CC50 value of flavone against Vero cells was 446 mu g/mL. These results suggest that flavone might enhance dengue virus replication by acting antagonistically towards flavonoids known to inhibit dengue virus replication.

Abstract: Larvae of Aedes albopictus Skuse typically inhabit natural and artificial containers. Since these larval habitats are replenished by rainfall, Ae. albopictus may experience increased loss of immature stages in areas with high levels of rainfall. In this study, we investigated the effects of rainfall and container water level on population density, and oviposition activity of Ae. albopictus. In field and laboratory experiments, we found that rainfall resulted in the flushing of breeding habitats. Excess rain negatively impacted larval and pupal retention, especially in small habitats. When filled with water to overflowing, container habitats were significantly repellent to ovipositing females. Taken together, these data suggest that rainfall triggers population loss of Ae. albopictus and related species through a direct detrimental effect (flushing out) and an indirect effect (ovipositional repellency).

Abstract: Objective: To identify the unusual breeding sites of two dengue vectors, i.e. Aedes albopictus (Ae. albopictus) and Aedes aegypti (Ae. aegypti). Methods: During the second half of 2010, we performed an occasional survey in rural (Teluk Tempoyak) and urban (Gelugor) areas ofPenang Island, Malaysia, to identify cryptic breeding sites. Results: In the rural area, we found heterogeneous immature stages of Ae. albopictus in the water bowl of an encaged bird. We also observed Ae. aegypti eggs deposited in the flush tank of a toilet in the urban area. Conclusions:It can be concluded that both breeding patterns can increase contact with hosts (humans and birds) and presumably population densities of Ae. albopictus and Ae. aegyp

Abstract: The In vitro susceptibility of clinical and environmental isolates of Acinetobacter baumannii to tigecycline and other antibiotics was determined by disk diffusion method. The E-test was used to determine the minimum inhibitory concentration (MIC). The growth curves of tigecycline treated environmental and clinical strains were established. Fifty-seven percent and 75% of the clinical and environmental isolates were MDR strains, respectively. Ninety-five percent of the clinical isolates were susceptible to tigecycline and 5% showed intermediate resistance with MIC ranging between 0�·032 and 3 mg/l. Tigecycline susceptible and intermediate resistance among the environmental isolates were 40% and 60%, respectively, with a significantly lower MIC range of 0�·5â��4 mg/l. The bacterial growth curves demonstrated the higher ability of the environmental strains to tolerate the antibiotic effects than the clinical strains. The relatively high resistance profile among the environmental isolate suggests an insidious emergence of tigecycline resistance amongst A. baumannii. Strict infection control procedures are imperative to prevent the dissemination of tigecycline-resistant A. baumannii strains in the hospital environment

Abstract: Objective: We report a rare case of cervical necrotising fasciitis arising from poorly managed acute tonsillitis. Case report: A 23-year-old woman presented with a two-week history of fever and an eight-day history of painful neck swelling. Nine days before presentation, she had received digital manipulation of her throat by a neighbour, which had worsened her throat pain. There was associated progressive generalised neck swelling, odynophagia, dysphagia and dyspnoea. An X-ray of the neck soft tissue showed multiple gas collections. Conclusion: Cervical necrotising fasciitis is rare and usually odontogenic in origin. It is associated with a high mortality rate. Our patient responded to aggressive daily bedside wound debridements and dressings, appropriate intravenous antibiotics and high-protein nutritional support. In this way, exploration under general anaesthesia was avoided, in a developing country with limited facilities.

Abstract: Tsukamurella spp. are a rare but important cause of intravascular catheter-related bacteremia in immunocompromised patients. The organism is an aerobic, Gram-positive, weakly acid-fast bacillus that is difficult to differentiate using standard laboratory methods from other aerobic actinomycetales such as Nocardia spp., Rhododoccus spp., Gordonia spp., and the rapid growing Mycobacterium spp. We report a case of Tsukamurella tyrosinosolvens catheter-related bacteremia in a 51-year-old haematology patient who responded to treatment with imipenem and subsequent line removal. 16srRNA sequencing allowed for the prompt identification of this organism.

Abstract: Objective: To describe the outcomes of dorsal augmentation rhinoplasty with expanded polytetrafluoroethylene (ePTFE) implants in Southeast Asian patients from the Philippines. Methods: Retrospective review of 1054 patients. Results: Of the 1054 patients, 90.61% were women and 9.39% were men. One thousand eight patients (95.64%) underwent primary rhinoplasty; 46 (4.36%), secondary or revision rhinoplasty. One thousand thirty (97.72%) had desirable and 24 (2.28%) had undesirable outcomes. The most common undesirable outcome was implant deviation (1.04%), followed by a visible ePTFE implant (0.47%). Implant infection occurred in 0.38% of the patients, and 0.38% of the patients were not satisfied with their aesthetic outcome because of the presence of a high nasal bridge. Conclusions: We find ePTFE to be an excellent synthetic material with proven outcomes for augmenting the dorsum in rhinoplasty of the Southeast Asian patient. However, prudent use of this material is warranted to avoid undesirable outcomes.

Abstract: BACKGROUND: Dengue is a major mosquito-borne disease currently with no effective antiviral or vaccine available. Effort to find antivirals for it has focused on bioflavonoids, a plant-derived polyphenolic compounds with many potential health benefits. In the present study, antiviral activity of four types of bioflavonoid against dengue virus type -2 (DENV-2) in Vero cell was evaluated. Anti-dengue activity of these compounds was determined at different stages of DENV-2 infection and replication cycle. DENV replication was measured by Foci Forming Unit Reduction Assay (FFURA) and quantitative RT-PCR. Selectivity Index value (SI) was determined as the ratio of cytotoxic concentration 50 (CC50) to inhibitory concentration 50 (IC50) for each compound. RESULTS: The half maximal inhibitory concentration (IC50) of quercetin against dengue virus was 35.7 ��g mL-1 when it was used after virus adsorption to the cells. The IC50 decreased to 28.9 ��g mL-1 when the cells were treated continuously for 5 h before virus infection and up to 4 days post-infection. The SI values for quercetin were 7.07 and 8.74 ��g mL-1, respectively, the highest compared to all bioflavonoids studied. Naringin only exhibited anti-adsorption effects against DENV-2 with IC50 = 168.2 ��g mL-1 and its related SI was 1.3. Daidzein showed a weak anti-dengue activity with IC50 = 142.6 ��g mL-1 when the DENV-2 infected cells were treated after virus adsorption. The SI value for this compound was 1.03. Hesperetin did not exhibit any antiviral activity against DENV-2. The findings obtained from Foci Forming Unit Reduction Assay (FFURA) were corroborated by findings of the qRT-PCR assays. Quercetin and daidzein (50 ��g mL-1) reduced DENV-2 RNA levels by 67% and 25%, respectively. There was no significant inhibition of DENV-2 RNA levels with naringin and hesperetin. CONCLUSION: Results from the study suggest that only quercetin demonstrated significant anti-DENV-2 inhibitory activities. Other bioflavonoids, including daidzein, naringin and hesperetin showed minimal to no significant inhibition of DENV-2 virus replication. These findings, together with those previously reported suggest that select group of bioflavonoids including quercetin and fisetin, exhibited significant inhibitory activities against dengue virus. This group of flavonoids, flavonol, could be investigated further to discover the common mechanisms of inhibition of dengue virus replication.

Abstract: Conjoined twins are rare and are classified as symmetrical or asymmetrical, in which a member, the host (autosite), is near normal and bears the parasite, which is incomplete, smaller, and fully dependent for growth on it. This form of conjoined twins is referred to as heteropagus and when attached to the epigastrium of the autosite is called epigastric heteropagus. Only 44 cases of epigastric heteropagus twins have been previously reported in the world literature. We hereby report the successful separation of a pair of heteropagus twins. (C) 2011 Elsevier Inc. All rights reserved.

Abstract: Whitefly infestation and the begomoviruses that they transmit have been shown to affect the activities of plant defence proteins, but with no relation to heterophylly, a process of great importance underlying the overall biology of plants. Here, we have assessed the effects of Tomato yellow leaf curl virus (TYLCV) infection on Solanum lycopersicum peroxidase (POD) activity and have examined whether leaves of different ages exhibit differential POD activity in response to infection and infestation with Bemisia tabaci B biotype. We used leaf discs of two ages (juvenile and mature) with two different infection statuses (infected and healthy) to examine the activity of the tomato plant peroxidase using guaiacol as a substrate and taking exposure time into account. S. lycopersicum showed increased POD activity in the presence of TYLCV. The activity of the enzyme was higher in mature than in juvenile leaves. In general, both infected and healthy leaves exhibited greater POD activity during whitefly infestation. In the infested juvenile leaves, POD activity was much lower in the healthy leaves and increased gradually with period of exposure to B. tabaci B infestation. In contrast, the activity of the enzyme remained low in infested mature leaves in both the presence and absence of the virus even with increased exposure time. Determination of the distribution of an insect pest is critical for sampling and management. Leaf age is presumed to be associated with the within-host distribution of the geminivirus vector B. tabaci. Juvenile leaves will usually attract more insects due to increased nutritional value and weaker defences. Our results highlight the importance of leaf age/position on the whitefly - host plant - geminivirus interactions and have important implications for sampling and control strategies.

Abstract: In vitro antiviral activities of three flavonoids; fisetin, naringenin and rutin against DENV-2 (NGC strain) were evaluated. Inhibitory effects of each compound at the different stages of DENV-2 infection were examined using foci forming unit reduction assay (FFURA) and quantitative real-time polymerase chain amplification (qRT-PCR). Fisetin, rutin and naringenin showed cytotoxic effects against Vero cells with 50% cytotoxicity (CC(50)) values of 247, > 1000, and 87 mu g/mL, respectively. Fisetin when added to Vero cells after virus adsorption inhibited DENV replication with a half maximal inhibition concentration (IC(50)) value of 55 mu g/mL and selectivity index (SI) of 4.49. The IC(50) value of fisetin was 43.12 mu g/mL with SI=5.72 when Vero cells were treated for 5 h before virus infection and continuously up to 4 days post-infection. There was no direct virucidal activity or prophylactic activity of fisetin against DENV-2. Rutin and naringenin did not inhibit DENV-2 replication in Vero cells. Naringenin however, exhibited direct virucidal activity against DENV-2 with IC(50) = 52.64 mu g/mL but the SI was <1. The present study suggests that among the flavonoids examined, only fisetin showed significant in vitro anti dengue virus replication activity.

Abstract: Objectives: Acute appendicitis is a common surgical emergency. The etiology and pathophysiology of appendicitis have been well investigated. Aggregatibacter aphrophilus is a fastidious gram-negative coccobacilli. Detection of this organism in clinical samples and its differentiation from Haemophilus aphrophilus or from Aggregatibacter actinomycetemcomitans in routine microbiology settings could be difficult. Methods: In this rare case, we report the isolation of Aggregatibacter aphrophilus from the appendix of a 14-year old boy presented with acute appendicitis. The genotypic method using 16S rRNA sequencing was used for identification of the organism at species level. Conclusion: This case highlights the importance of detecting fastidious and rare microorganisms such as Aggregatibacter aphrophilus that could be associated with acute appendicitis.

Abstract: Discarded cigarette butts (DCB) waste occurs worldwide, pollutes landscapes, is unsightly, and results in added debris removal costs. There is, therefore, a great deal of current interest in making use of DCBs in beneficial ways. Despite evidence that DCBs are harmful to water fleas (Daphnia magna), which breed in aquatic environments as do mosquito larvae, their impact on dengue vectors is unknown. We examined whether Aedes albopictus alters its ovipositional responses, larval eclosion, and development in response to presence of DCBs in its habitats. We found oviposition activity in DCB-treated water similar to that of control water and that ovipositional activity in DCB solutions steadily increased over time as those solutions aged to 10 days. Larval eclosion was initially suppressed on day 1 in DCB solution, but increased thereafter to levels similar to control larval eclosion rates. The DCB-water solutions produced significantly higher mortality in both 1st and 2nd instars over control larvae for several days after initial exposure. Mortality rates decreased sharply 3 to 5 days postexposure as DCBs continued to decompose. We found increased survival rates during late development, but daily input of fresh DCBs prevented most young larvae from completing development. Taken together, these observations suggest that decomposing did not deter gravid Ae. albopictus females from ovipositing in treated containers and that DCB solutions had larvicidal effects on early instars. Our results are discussed in the context of DCB use to control container-breeding Ae. albopictus, a competent dengue vector in Asia and other parts of the world.

Abstract: 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.

Abstract: BACKGROUND: The mosquito Ae. albopictus is usually adapted to the peri-domestic environment and typically breeds outdoors. However, we observed its larvae in most containers within homes in northern peninsular Malaysia. To anticipate the epidemiological implications of this indoor-breeding, we assessed some fitness traits affecting vectorial capacity during colonization process. Specifically, we examined whether Ae. albopictus exhibits increased survival, gonotrophic activity and fecundity due to the potential increase in blood feeding opportunities. METHODOLOGY/PRINCIPAL FINDINGS: In a series of experiments involving outdoors and indoors breeding populations, we found that Ae. albopictus lives longer in the indoor environment. We also observed increased nighttime biting activity and lifetime fecundity in indoor/domestic adapted females, although they were similar to recently colonized females in body size. CONCLUSION/SIGNIFICANCE: Taken together these data suggest that accommodation of Ae. albopictus to indoor/domestic environment may increase its lifespan, blood feeding success, nuisance and thus vectorial capacity (both in terms of increased vector-host contacts and vector population density). These changes in the breeding behavior of Ae. albopictus, a potential vector of several human pathogens including dengue viruses, require special attention.

Abstract: Ancestral sylvatic dengue virus type 1, which was isolated from a monkey in 1972, was isolated from a patient with dengue fever in Malaysia. The virus is neutralized by serum of patients with endemic DENV-1 infection. Rare isolation of this virus suggests a limited spillover infection from an otherwise restricted sylvatic cycle.

Abstract: The normally high concentration of zinc in normal prostate gland is significantly reduced in malignant prostate tissues, but its precise role in prostate tumorigenesis remains unclear. The present study investigates the growth and transcriptional responses of LNCaP prostate cancer cells to prolonged high Zn2+ treatment. Restoration of high intracellular Zn2+ to LNCaP cells significantly reduced the cell proliferation rate by 42.2+/-7.4% at the exponential growth phase and the efficiency of colony formation on soft agar by 87.2+/-2.5% at week 5 post-treatment. At least 161 LNCaP cell genes responded to the high intracellular Zn2+, including approximately 10.6% genes that negatively regulate cell growth and approximately 16.1% genes that promote cancer cell proliferation. Inhibition of cell growth was transient as normal proliferation rate and colony formation efficiency were restored later even in the continuous presence of high intracellular Zn2+. RT-qPCR showed constitutively higher expression levels of FBL, CD164 and STEAP1 in LNCaP cells. FBL and CD164 were responsive to the treatment with Zn2+ in PNT2 prostate normal cells and were further overexpressed in the prolonged Zn2+-treated LNCaP cells. These observations suggest that in general high Zn2+ has suppressive effects on prostate cancer cell growth but continuous exposure to an environment of high Zn2+ can lead to the overexpression of cancer promoting genes such as FBL and CD164. This could be the antagonistic mechanism used to overcome the initial cell growth inhibitory effects of high Zn2+. These findings support a potential detrimental role of Zn2+ in prostate cancer.

Abstract: Moisture plays a major role in the dynamics of mosquito populations, especially those breeding in container habitats. Despite this importance, the role of moisture conditions as they affect oviposition and egg development in Aedes vectors remains largely unexplored. We investigated the effect of exposing gravid female Aedes albopictus mosquitoes and their eggs to different moisture levels (MLs) for various periods on oviposition and hatching. Overall, high-moisture substrates (HMSs; 66% and 72%) provided better environments for egg laying. The timing of initial egg laying was far longer at the lowest substrate moisture level (LSML, 25% and 41.2%) than at HMSs. The numbers of eggs laid were much lower in the drier environments. At LSMLs, gravid females retained increasing numbers of mature eggs until death, and egg retention decreased gradually with increasing ML. The HMSs also provided better environments for larval eclosion. The numbers of eggs hatched were lower at the LSML than the HSML environment. No egg hatching occurred after 1 h exposure to moisture. However, egg hatching occurred by installment, with spontaneous hatching (SH) increasing gradually with increasing ML. High-moisture conditions combined with long exposure (30 h and 48 h) favored SH. These results suggest that Ae. albopictus females can respond to better moisture conditions for increased success of embryonation and larval eclosion. This information may be useful in the colonization of floodwater Aedes species.

Abstract: Background: There are at least 51 adenovirus serotypes (AdV) known to cause human infections. The prevalence of the different human AdV (HAdV) serotypes varies among different regions. Presently, there are no reports of the prevalent HAdV types found in Malaysia. The present study was undertaken to identify the HAdV types associated primarily with respiratory tract infections (RTI) of young children in Malaysia. Methods Archived HAdV isolates from pediatric patients with RTI seen at the University of Malaya Medical Center (UMMC), Kuala Lumpur, Malaysia from 1999 to 2005 were used. Virus isolates were inoculated into cell culture and DNA was extracted when cells showed significant cytopathic effects. AdV partial hexon gene was amplified and the sequences together with other known HAdV hexon gene sequences were used to build phylogenetic trees. Identification of HAdV types found among young children in Malaysia was inferred from the phylograms. Results At least 2,583 pediatric patients with RTI sought consultation and treatment at the UMMC from 1999 to 2005. Among these patients, 48 (< 2%) were positive for HAdV infections. Twenty-seven isolates were recovered and used for the present study. Nineteen of the 27 ( 70%) isolates belonged to HAdV species C (HAdV-C) and six ( 22%) were of HAdV species B (HAdV-B). Among the HAdV-C species, 14 ( 74%) of them were identified as HAdV type 1 (HAdV-1) and HAdV type 2 (HAdV-2), and among the HAdV-B species, HAdV type 3 (HAdV-3) was the most common serotype identified. HAdV-C species also was isolated from throat and rectal swabs of children with hand, foot, and mouth disease (HFMD). Two isolates were identified as corresponding to HAdV-F species from a child with HFMD and a patient with intestinal obstruction. Conclusions HAdV-1 and HAdV-2 were the most common HAdV isolated from pediatric patients who sought treatment for RTI at the UMMC from 1999 to 2005. HAdV-B, mainly HAdV-3, was recovered from 22% of the patients. These findings provide a benchmark for future studies on the prevalence and epidemiology of HAdV types in Malaysia and in the region.

Abstract: Enterovirus 71 (EV71) outbreaks occur periodically in the Asia-Pacific region. In 2006, Brunei reported its first major outbreak of EV71 infections, associated with fatalities from neurologic complications. Isolated EV71 strains formed a distinct lineage with low diversity within subgeno-group B5, suggesting recent introduction and rapid spread within Brunei.

Abstract: Prostate cancer is an age-related disease that is linked to the inability of prostate cells to accumulate zinc following transformation. It is shown in the present study the the basal percentage of normal prostate cells expressing senescence-associated beta-galactosidase (SA-beta-gal) is higher than that of the cancer cells. In the presence of high zinc in the cell culture medium, the percentage of normal prostate cells expressing the SA-beta-gal increased but not that of the cancer cells. Increased intracellular zinc occurs in the prostate cancer cells treated with supraphysiologic concentration of zinc but it does not induce senescence or decrease the telomerase activities in these cells. Senescence, however, occurred when the prostate cancer cells DNA is damaged by irradiation. These findings suggest that prostate cancer cells are insensitive to the senescence-inducing effects of zinc but the cancer cells retain the capacity to undergo senescence through other pathways. (c) 2008 Elsevier GmbH. All rights reserved.

Abstract: A method to map the specific site on dengue virus envelope protein (E) that interacts with cells and a neutralizing antibody is developed using serially truncated dengue virus type 2 (DENV-2) E displayed on M13 phages as recombinant E-g3p fusion proteins. Recombinant phages displaying the truncated E consisting of amino acids 297-423 (EB2) and amino acids 379-423 (EB4) were neutralized by DENV-2 patient sera and the DENV-2 E-specific 3H5-1 monoclonal antibodies suggesting that the phages retained the DENV-2 E antigenic properties. The EB4 followed by EB2 recombinant phages bound the most to human monocytes (THP-1), African green monkey kidney (Vero) cells, mosquito (C6/36) cells, ScFv specific against E and C6/36 cell proteins. Two potential cell attachment sites were mapped to loop I (amino acids 297 to 312) and loop II (amino acids 379-385) of the DENV-2 E using the phage-displayed truncated DENV-2 E fragments and by the analysis of the E structure. Loop II was present only in EB4 recombinant phages. There was no competition for binding to C6/36 cell proteins between EB2 and EB4 phages. Loop I and loop II are similar to the sub-complex specific and type-specific neutralizing monoclonal antibody binding sites, respectively. Hence, it is proposed that binding and entry of DENV involves the interaction of loop I to cell surface glycosaminoglycan-motif and a subsequent highly specific interaction involving loop II with other cell proteins. The phage displayed truncated DENV-2 E is a powerful and useful method for the direct determination of DENV-2 E cell binding sites.

Abstract: Malignant prostate tissues have markedly reduced zinc (Zn(2+)) contents in comparison to non-malignant tissues. In this study, we restored a high intracellular Zn(2+) level to LNCaP prostate cancer cells by culturing the cells in a growth medium supplemented with a supraphysiological concentration of Zn(2+) (10 mu g/ml) over 5 weeks. The intracellular Zn(2+) level increased in the Zn(2+)-treated cells, and there was a marked increase in the presence of zincosomes, a Zn(2+)-specific intracellular organelle. The proliferation rate of the Zn(2+)-treated cells was markedly reduced. There was also a significant increase (36.6% +/- 6.4%) in the total tyrosine phosphorylated proteins. Vaccinia H1-related (VHR) phosphatase, zeta chain-associated protein-70 (ZAP-70) kinase and phosphorylated extracellular signal-regulated protein kinase 1 and 2 (p-ERK 1 and 2) were also present in higher abundance. Treatment with TPEN, which chelates Zn(2+), reduced the abundance of VHR phosphatase and ZAP-70 kinase, but increased the abundance of p-ERK 1. However, the TPEN treatment restored the Zn(2+)-treated LNCaP cell proliferation to a rate comparable to that of the non Zn(2+)-treated cells. These results highlight the importance of a high intracellular Zn(2+) content and the VHR/ZAP-70-associated pathways in the modulation of LNCaP prostate cancer cell growth.

Abstract: The application of proteomics in alga research is still quite limited. The present report describes the establishment of the proteome of a red alga of economic importance, Gracilaria changii (Xia et Abbott) Abbott, Zhang et Xia. Initially, four protein extraction methods including direct precipitation by trichloroacetic acid/acetone, direct lysis using urea buffer, Tris buffer, and phenol/chloroform extraction were compared for their suitability to generate G. changii proteins for two-dimensional gel electrophoresis (2-DE). The phenol/chloroform protein extraction method gave the best 2-DE resolution of the proteins. Using these 2-DE gels and mass spectrometry, several proteins including pigment proteins, metabolic enzymes, and ion transporters were identified. These findings highlight the potential of using proteomic approaches for the investigation of G. changii protein function.

Abstract: Background: Human enterovirus 71 (EV-71) is a common causative agent of hand, foot and mouth disease (HFMD). In recent years, the virus has caused several outbreaks with high numbers of deaths and severe neurological complications. Several new EV-71 subgenotypes were identified from these outbreaks. The mechanisms that contributed to the emergence of these subgenotypes are unknown. Results: Six EV-71 isolates from an outbreak in Malaysia, in 1997, were sequenced completely. These isolates were identified as EV-71 subgenotypes, B3, B4 and C2. A phylogenetic tree that correlated well with the present enterovirus classification scheme was established using these full genome sequences and all other available full genome sequences of EV-71 and human enterovirus A (HEV-A). Using the 5� UTR, P2 and P3 genomic regions, however, isolates of EV-71 subgenotypes B3 and C4 segregated away from other EV-71 subgenotypes into a cluster together with coxsackievirus A16 (CV-A16/G10) and EV-71 subgenotype C2 clustered with CV-A8. Results from the similarity plot analyses supported the clustering of these isolates with other HEV- A. In contrast, at the same genomic regions, a CV-A16 isolate, Tainan5079, clustered with EV-71. This suggests that amongst EV- 71 and CV-A16, only the structural genes were conserved. The 3� end of the virus genome varied and consisted of sequences highly similar to various HEV-A viruses. Numerous recombination crossover breakpoints were identified within the non-structural genes of some of these newer EV-71 subgenotypes. Conclusion: Phylogenetic evidence obtained from analyses of the full genome sequence supports the possible occurrence of inter-typic recombination involving EV-71 and various HEV-A, including CV-A16, the most common causal agent of HFMD. It is suggested that these recombination events played important roles in the emergence of the various EV-71 subgenotypes.

Abstract: Nipah virus infection of porcine stable kidney cells (PS), human neuronal cells (SK-N-MC), human lung fibroblasts cells (MRC-5), and human monocytes (THP-1) were examined. Rapid progression of cytopathic effects (CPE) and cell death were noted in PS cell cultures treated with Nipah virus, followed by MRC-5, SK-N-MC, and THP-1 cell cultures, in descending order of rapidity. Significant increase in the intracellular Nipah virus RNA occurred beginning at 24 hr Pl in all the infected cells. Whereas, the extracellular release of Nipah virus RNA increased significantly beginning at 48 and 72 hr Pl for the infected MRC-5 cells and PS cells, respectively. No significant release of extracellular Nipah virus RNA was detected from the Nipah virus-infected SK-N-MC and THP-1 cells. At its peak, approximately 6.6 log PFU/mu l of extracellular Nipah virus RNA was released from the Nipah virus-infected PS cells, with at least a 100-fold less virus RNA was recorded in the Nipah virus-infected SK-N-MC and THP-1. Approximately 15.2% (+/- 0.1%) of the released virus from the infected PS cell cultures was infectious in contrast to approximately 5.5% (+/- 0.7%) from the infected SK-N-MC cells. The findings suggest that there are no differences in the capacity to support Nipah virus replication between pigs and humans in fully susceptible PS and MRC-5 cells. However, there are differences between these cells and human neuronal cells and monocytes in the ability to support Nipah virus replication and virus release.

Abstract: Potential occurrence of dengue 2 virus (DENV-2)-induced apoptosis in mosquito larvae was investigated. Heads of Toxohrynchites splendens mosquito larvae were inoculated with DENV-2 inoculum at multiplicity of infection of 0.0005-0.001 plaque forming unit per cell. After 7 days of incubation, the larvae heads were severed and squashed onto slides. Following immunofluorescence staining with mouse polyclonal antibody against DENV-2, antigens were detected in 17.5% of the total cells counted. Counterstaining of the cells using colorimetric apoptotic staining method revealed that 21.7% of the antigen-positive cells were in apoptotic state. Statistical analyses of the data showed that the induction of apoptosis was directly correlated to the DENV-2 infection and not merely a metamorphic programmed cell death response.

Abstract: Dengue virus Type 2 C, prM, truncated E (NB-E and B-E), NS1, NS2A, NS2B/3 and NS3 genes were cloned and expressed using the pEGFP-N1 eukaryotic expression vector. Protein expression was visualized in transfected Vero cell cultures. Immunogenicity of the recombinant DNA plasmids was determined by inoculating 8 weeks old BALB/cJ mice with two doses of plasmid cocktail followed by a booster inoculation at two weeks apart. Mice were also injected with empty vector plasmids or vaccinated with virus inoculum as controls. Initial inoculation with the plasmid cocktail resulted in IgG response comparable to inoculation with virus inoculum alone which peaked after the booster dose. Rapid rise in IgG response was also observed when the mice were subsequently challenged with partially purified dengue virus Type 2, ten weeks after the first inoculation. These results suggest for the first time, that dengue virus DNA genes expressing antigens as fusion proteins with EGFP could stimulate immune responses in vivo.

Abstract: Dendritic cells (DC) are the main producers of the cytokine IL-12p70, through which they play a direct role in the development of IFN-gamma-secreting Th1 cells, costimulation of CTL differentiation and NK-cell activation. In contrast, IL-10, which is also produced by DC, negatively regulates IL-12 production. IL-12p70 production varies widely between individuals, and several polymorphisms in the gene encoding IL-12p40 (IL12B) have been identified that influence susceptibility and severity of infectious, autoimmune and neoplastic disease. Here we show that polymorphisms not only of IL12B, but also in the IL10 promoter, influence IL-12p70 secretion by monocyte-derived DC in response to LPS. Although IL12B promoter homozygotes were prone to making more IL-12p70, presence of the IL10 high genotype restricted IL-12p70 production in these individuals. These observations provide a further genetic control of IL-12p70 regulation and emphasize the complexity of production of this cytokine. They also suggest genotypes that might influence the outcome of DC immunotherapy.

Abstract: Background: Nipah virus is a zoonotic virus isolated from an outbreak in Malaysia in 1998. The virus causes infections in humans, pigs, and several other domestic animals. It has also been isolated from fruit bats. The pathogenesis of Nipah virus infection is still not well described. In the present study, Nipah virus replication kinetics were estimated from infection of African green monkey kidney cells (Vero) using the one-step SYBR (R) Green I-based quantitative real-time reverse transcriptase-polymerase chain reaction (qRT-PCR) assay. Results: The qRT-PCR had a dynamic range of at least seven orders of magnitude and can detect Nipah virus from as low as one PFU/mu L. Following initiation of infection, it was estimated that Nipah virus RNA doubles at every similar to 40 minutes and attained peak intracellular virus RNA level of similar to 8.4 log PFU/mu L at about 32 hours post-infection (PI). Significant extracellular Nipah virus RNA release occurred only after 8 hours PI and the level peaked at similar to 7.9 log PFU/mu L at 64 hours PI. The estimated rate of Nipah virus RNA released into the cell culture medium was similar to 0.07 log PFU/mu L per hour and less than 10% of the released Nipah virus RNA was infectious. Conclusion: The SYBR (R) Green I-based qRT-PCR assay enabled quantitative assessment of Nipah virus RNA synthesis in Vero cells. A low rate of Nipah virus extracellular RNA release and low infectious virus yield together with extensive syncytial formation during the infection support a cell-to-cell spread mechanism for Nipah virus infection.

Abstract: Background: 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. Results: Analysis of human enterovirus 71 (EV-71) subgenotype B3 genome sequences revealed that the 3D RNA polymerase and domain Z of the 3�-untranslating region RNA secondary structure had high similarity to CV-A16. Intracerebral inoculation of one-day old mice with the virus resulted in 16% of the mice showing swollen hind limbs and significantly lower weight gain in comparison to EV-71 B4-infected mice. None of the mice presented with hind leg paralysis typical in all the CV-A16 infected mice. CV-A16 genome sequences were amplified from the CV-A16- infected mice brain but no amplification was obtained from all the EV-71-inoculated mice suggesting that no replication had taken place in the suckling mice brain. The findings presented here suggest that EV-71 B3 viruses had CV-A16-liked non- structural gene features at the 3�-end of the genome. Their presence could have affected virulence by affecting the mice general health but was insufficient to confer the EV-71 B3 virus a CV-A16- liked neurovirulence in mice model infection.

Abstract: Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and mass spectrometry methods were established and utilized to examine the changes in protein expressions associated with post-germination of Hevea brasiliensis seed. No significant differences in the total number of proteins were observed but characteristic protein spots were present in both proteomes. The mature dry seed proteome contained clusters of proteins of about 36.5 and 23 kDa at pH 4-7 and a group of basic proteins at similar to pH 10. The presence of the 23 kDa proteins was markedly reduced in the post-germinated seed proteome. Approximately 60% of the proteins noted in the germinated seed proteome matched those of the mature dry seeds, with the remaining 40% protein spots as either unmatched or unique to the germinated seed proteome. Of the proteins detected, a putative beta-glucosidase, starch branching enzyme IIb and a MutT/nudix family of protein that were decreased in abundance in the germinated seed proteome and two proteins, acidic lectin and gibberellin 20-oxidase found unique to the mature dry seed proteome were identified by mass spectrometry. This report highlights the potential of using 2D-PAGE and mass spectrometry as means for identification of H. brasiliensis proteins and studying its seeds germination processes. (c) 2005 Elsevier Ireland Ltd. All rights reserved.

Abstract: Introduction: This study aims to identify Acinetobacter of clinical isolates from the University of Malaya Medical Centre (UMMC),Kuala Lumpur, to the species level by 16S rDNA sequencing. Methods: 12 representative Acinetobacter isolates of the UMMC inpatients were randomly picked and used for the study. The 16S rDNA sequences were determined and phylogenetic relationships to all known Acinetobacter species were established. Results: Based on the 16S rDNA sequences, all the UMMC isolates were identified as Acinetobacter baumannii. The isolates shared a common ancestral lineage with the prototypes Acinetobacter baumannii DSM30007 and DSM30008 with 99-100 percent sequence similarities. The isolates could be differentiated into two groups by a single nucleotide difference(thymine-cytosine) within the 16S rRNA sequence.Three different genotypes, 1, 3 and 4, were recognised using REP-PCR. The previously uncharacterised Acinetobacter isolates from the UMMC were identified by their 16S rDNA sequences as Acinetobacter baumannii. The isolates were distinguished into at least three different genotypes by REP-PCR genotyping. These findings confirmed for the first time the presence of Acinetobacter baumannii of different genotypes among patients at UMMC.

Abstract: 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.

Abstract: Binding of dengue virus 2 (DENV-2) to C6/36 mosquito cells protein was investigated. A 48 kDa DENV-2-binding C6/36 cells protein (D2BP) was detected in a virus overlay protein-binding assay. The binding occurred only to the C6/36 cells cytosolic protein fraction and it was inhibited by free D2BP. D2BP was shown to bind to DENV-2 E in the far-Western-binding studies and using mass spectrometry (MS) and MS/MS, peptide masses of the D2BP that matched to beta-tubulin and alpha-tubulin chains were identified. These findings suggest that DENV-2 through DENV-2 E binds directly to a 48 kDa tubulin or tubulin-like protein of C6/36 mosquito cells. (C) 2004 Elsevier Inc. All rights reserved.

Abstract: Two human enterovirus 71 (HEV71) isolates were identified from hand, foot and mouth disease patients with genome sequences that had high similarity to HEV71 (greater than or equal to93%) at 5�UTR, P1, and P2 and coxsackievirus A16 (CV-A16, greater than or equal to85%) at P3 and 3�UTR. Intertypic recombination is likely to have occurred between HEV71 and CV-A16 or an as-yet to be described CV-A16-like virus.

Abstract: The antibiotic susceptibility profiles and the repetitive extragenic palindromic sequence-based polymerase chain reaction (REP-PCR)-determined genotypes of 109 Acinetobacter strains collected from the University Malaya Medical Center (UMMC), Kuala Lumpur, Malaysia, in 1987 (N=21) and 1996-1998 (N=88) were established. Twelve antibiotic susceptibility profiles of antibiotics used at the UMMC were obtained. In descending order of effectiveness, imipenem, amikacin and ciprofloxacin were the most effective against the Acinetobacter strains. Compared with 1987 isolates, the isolates obtained in 1996-1998 had decreased susceptibility to these antibiotics and were tolerant to the antibiotics up to an MIC90 of greater than or equal to256 mg/L. REP-PCR DNA fingerprints of all the isolates revealed the presence of four Acinetobacter spp. lineages; 92% of all the isolates belonged to two dominant lineages (genotypes 1 and 4). Genotype 4 isolates predominant in 1987 showed increased resistance and antibiotic tolerance to imipenem, amikacin and ciprofloxacin compared with the 1996-1998 isolates. In contrast, genotype 1 isolates from 1996-1998 were mainly sensitive to these antibiotics. These findings demonstrate the presence of at least two independent Acinetobacter spp. lineages in the same hospital, and suggest the possibility that genotype 4 Acinetobacter spp. acquired the resistance phenotype in situ, whereas most of the genotype 1 isolates were probably introduced to the hospital in recent years.

Abstract: Nipah viruses from pigs from a Malaysian 1998 outbreak were isolated and sequenced. At least two different Nipah virus strains, including a previously unreported strain, were identified. The findings highlight the possibility that the Malaysia outbreaks had two origins of Nipah virus infections.

Abstract: Apoptosis was detected in Vero cell cultures expressing transfected dengue virus type 2 (DENV-2) genes. Approximately 17.5 and 51.5 % of cells expressing NS3 serine protease and NS2B-NS3(185) serine protease precursor protein [NS2B-NS3(185)(pro)] genes, respectively, were apoptotic. The percentage of apoptotic cells was significantly higher in cell cultures expressing NS2B-NS3(185)(pro). NS2B-NS3(185)(pro) was detected as NS2B-NS3(185)(pro)-EGFP fusion protein in cytoplasmic vesicular structures in the apoptotic cells. Site-directed mutagenesis which replaced His(51) with Ala within the protease catalytic triad significantly reduced the ability of the expressed NS3 and NS2B-NS3(185)(pro) to induce apoptosis. Results from the present study showed that DENV-2-encoded NS3 serine protease induces apoptosis, which is enhanced in cells expressing its precursor, NS2B-NS3(185)(pro). These findings suggest the importance of NS2B as a cofactor to NS3 protease-induced apoptosis.

Abstract: Dengue virus type-2 (DEN-2) has been isolated in Malaysia for more than three decades. The virus caused two major outbreaks in the early 1990s and late 1990s. Phylogenetic analyses performed using available E/NS1 junction sequences identified two DEN-2 genotypes: DEN-2 Asian 1 and DEN-2 Cosmopolitan. DEN-2 Cosmopolitan/Malaysia is the predominant genotype comprising more than 80% of the total isolates. Two different clades of DEN-2 Cosmopolitan/Malaysia genotype were identified. Clade I consisted of mainly the older isolates, whereas Clade II consisted of the more recent isolates, including that responsible for both the major DEN-2 outbreaks. Two different strains of DEN- 2 Cosmopolitan/Malaysia genotype were involved in the outbreaks, yet both strains appeared to share a common ancestral lineage with isolates from the early 1970s. Isolates from the late 1990s showed higher sequence similarities to the late-1960s isolates than the early-1990s isolates. These findings raised the possibility that the different DEN-2 strains noted over the last three decades in Malaysia might have evolved from a pre existing DEN-2 gene pool.

Abstract: 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.

Abstract: At least three major antigenic dengue 2 virus proteins were recognized by pooled dengue fever patients� sera in infected Aedes albopictus (C6/36) mosquito cells. Dengue virus envelope (E), premembrane (PrM) and non-structural protein 1 (NS 1) dimer were detected beginning on day 3 postinfection in both the cell membrane and cytosolic fractions. Using the patients� sera, the presence of antigenic intermediate core protein (C)-PrM and NS1-non-structural protein 2a (NS2a) in the cytoplasmic fraction of dengue 2 virus infected cells was revealed. The presence of a approximately 92 and approximately 84 kDa NS 1 dimer in the membrane (NS 1m) and cytosolic (NS 1c) fractions of C6/36 cells, respectively, was also recognized. Using individual patient�s serum, it was further confirmed that all patients� sera contained antibodies that specifically recognized E, NS 1 and PrM present in the dengue 2 virus-infected cell membrane fractions, suggesting that these glycosylated virus proteins were the main antigenic proteins recognized in vivo. Detection of dengue 2 virus C antibody in some patients further suggested that C could be antigenic if presented in vivo.

Abstract: 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.

Abstract: Dengue continues to be a major health threat to Malaysia a century after its first reported outbreak in 1902. Examination of the available outbreak data suggested that a major DF/DHF outbreak occurred in Malaysia in a cyclical pattern of approximately every 8 years. All four dengue virus serotypes are found co-circulating in Malaysia, but after the first and only major outbreak involving DEN-4 in 1960�s, only DEN-1, DEN-2 and DEN-3 were associated with DF/DHF outbreaks. It is argued that perhaps the spread of the later dengue virus serotypes followed the pattern of spread of the mosquito vector Aedes aegypti, whereas the former was associated with Aedes albopictus, the outdoor and rural area dwelling mosquito. Estimating from the trend and pattern of dengue and the associated dengue virus serotypes, unless there is a major breakthrough in dengue vaccine development, it is likely that dengue outbreaks will continue to occur in Malaysia throughout the 21st century.

Abstract: Dengue virus type 2 (DENV-2) infection induced apoptotic cellular DNA fragmentation in Vero cells within 8 days of infection. The addition of high concentrations of extracellular Zn2+ but not Ca2+, Mg2+ or Mn2+ to the cell culture medium hastened the detection of apoptosis to within 4 h after infection. No apoptotic cellular DNA fragmentation was detected in the cell culture treated with Zn2+ alone or infected with heat- or ultraviolet light-inactivated DENV-2 in the presence of Zn2+. These results suggest that (i) apoptosis is induced in African green monkey kidney cells infected with live DENV-2 and (ii) the addition of high extracellular Zn2+ accelerates detection of apoptosis in the DENV-2-infected cells. (C) 2002 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.

Abstract: The outbreak of enterovirus 71 infection in Taiwan, reported by Ho et al. (Sept. 23 issue), 1 occurred almost a year after the outbreak in Malaysia. Though both outbreaks occurred in Asia and both involved large numbers of deaths, it was not known whether the two outbreaks were related. We studied the nucleotide sequence and secondary RNA structure of some of the isolates, using the sequence of the 5� untranslated region (UTR). Enterovirus 71 isolates from patients in Singapore (seven isolates), Taiwan (two isolates), and Japan (one isolate) were examined and compared with those previously reported in Malaysia 2,3 or with sequences deposited in GenBank. A phylogenetic tree that we constructed using the aligned 5� UTR sequences revealed at least two major clusters of enterovirus 71 isolates. Cluster 1 included the isolate from Japan and six of the seven isolates from Singapore, together with isolates found predominantly in Malaysia during the 1997 outbreak. The isolates in this cluster had at least 89 percent sequence homology with enterovirus 71 MS isolates. The isolates in cluster 1 formed two subclusters. The isolate from Japan and five of the six isolates from Singapore were clustered with isolates from the Malaysian peninsula, forming one subcluster, and the remaining isolates, which included others from the Malaysian peninsula, Sarawak, and Singapore, formed the other subcluster. Three other isolates examined, one from Singapore and two from Taiwan, were in cluster 2, which consisted mostly of isolates from the 1998 outbreak in Taiwan. Cluster 2 also had two subclusters, with all the isolates from Taiwan, including the two sequenced in this study, in one subcluster and the remaining isolates from the Malaysian peninsula and Singapore in the other. The isolates in these two subclusters had at least 97 percent homology with each other and approximately 85 percent homology with the coxsackievirus A9 5� UTR sequence rather than with the enterovirus 71 MS group, which contained the other enterovirus 71 strains found predominantly in Malaysia and Singapore. A comparison of the 5� UTR secondary RNA structure, which has been associated with the degree of virulence, in the cluster 2 isolates revealed no significant differences in the structure of the three domains within the 5� UTR sequence. These findings suggest that the 5� UTR features of the enterovirus 71 strains from the Taiwanese outbreak were almost identical to those of the coxsackievirus A9�like strains isolated previously in the Malaysian peninsula. 3 Because of these similarities and the high frequency of travel between Malaysia and Taiwan, it is tempting to speculate that the predominant enterovirus 71 strains in the Taiwanese outbreak may have been accidental imports.

Abstract: Jane Cardosa and colleagues (Sept 18, p 987)1 report the discovery of a new fastidious adenovirus among patients with suspected hand, foot, and mouth disease (HFMD) in Sarawak. Subsequent to that outbreak, at least five deaths among young children with almost similar clinical presentations were reported in the Malaysian Peninsula. Enterovirus 71 (EV71) was isolated and identified in all five cases,2, 3 which suggested that perhaps the two viral outbreaks were unrelated. We detected enteroviruses including EV71 in about 51% (26 of 51) of samples from patients with suspected HFMD.4 We detected and confirmed adenovirus infection by cell culture and immunofluorescence staining from throat and rectal swab samples in only one patient from Sarawak. However, the adenovirus genome was detected by PCR amplification of the hexon gene in at least six other patients with suspected HFMD, including a patient who succumbed to brainstem encephalomyelitis caused by EV71. From this patient, the amplification product was detected only in Vero cells inoculated with pericardial and cerebrospinal fluid but not in cells inoculated with other tissue materials. Nonetheless, the presence of adenovirus in suspected HFMD patients was confirmed by amplification of Vero cells inoculated with blood from a 10-year-old boy. Amino acid sequence from nucleotide sequencing of amplification products showed that the patient�s adenovirus shared at least 95% identity with the hexon gene of adenovirus 7 and the new subgenus B adenovirus.1 This finding suggests that perhaps a similar adenovirus was circulating in Sarawak and the Malaysian Peninsula during the HFMD outbreak. Our attempts to propagate the virus or clone the sequence from the initial amplification product of other patients were not successful. However, EV71 was isolated from two of the six suspected adenovirus-positive patients. This includes the fatal infection attributed to EV71 and the HFMD case involving the 10-year-old boy from which EV71 and adenovirus were isolated from rectal swab and blood, respectively. In the latter case, the patient had fever, oral ulcer, and rashes on palms consistent with HFMD. Except for tachycardia and slight pleural effusion, no other overt neurological or cardiopulmonary symptoms were noted and the patient was discharged. Although there is no specific evidence to associate an adenovirus with the fatal HFMD-associated cases in the Malaysian Peninsula, EV71 was identified in almost all cases, not directly from patients� tissues but after inoculation of cell cultures mainly suitable for enterovirus isolations. Perhaps the presence of a fastidious adenovirus in the suspected HFMD cases was missed because of use of an unsuitable cell culture system.5 However, Cardosa and colleagues� report1 and our own findings suggest that the potential role of a fastidious adenovirus in EV71-associated HFMD needs to be examined further

Abstract: Background: The clinical sign of uvulo-palatoglossal junctional (UPJ) ulcers was first noted in 1983 in a 5.5-month-old baby with exanthem subitum (ES). An earlier prospective clinical study showed that there was a strong association of UPJ ulcers and occurrence of ES with a positive predictive value of 95.3% and negative predictive value of 100%. Objective: To determine the value of uvulo-palatoglossal junctional (UPJ) ulcers as an early clinical sign of exanthem subitum (ES) due to human herpesvirus 6 (HHV 6) infection. Study? design: A case-control study of 20 febrile children with UPJ ulcers versus 26 febrile children without UPJ ulcers. These children were followed up for any development of ES and investigated for human herpesvirus 6 (HHV 6) as the causative agents of the febrile episodes. Results: In this study, 20 out of 46 febrile children aged 3 months to 3 years with UPJ ulcers were virologically and/or serologically confirmed to be due to primary HHV 6 infection. The rest of the 26 children without ulcers did not have HHV 6 infection. Of the 20 children with UPJ ulcers, only 17 of the 19 children with adequate follow-up till subsidence of fever developed ES. None of the 26 children without UPJ ulcers developed ES. Conclusion: Statistically, then was a significant association of UPJ ulcers as an early sign of ES with a positive predictive value of 89.5% and negative predictive value of 100%. This finding also suggests that the presence of UPJ ulcers is a useful pathognomic clinical sign of symptomatic primary HHV 6 infection. (C) 2000 Elsevier Science B.V. All rights reserved.