Shamala Devi Sekaran

Abstract: Water-soluble phenolics from the oil palm possess significant biological properties. In this study, we aimed to discover the role of oil palm phenolics (OPP) in influencing the gene expression changes caused by an atherogenic diet in mice. We fed mice with either a low-fat normal diet (14.6 % kcal/kcal fat) with distilled water, or a high-fat atherogenic diet (40.5 % kcal/kcal fat) containing cholesterol. The latter group was given either distilled water or OPP. We harvested major organs such as livers, spleens and hearts for microarray gene expression profiling analysis. We determined how OPP changed the gene expression profiles caused by the atherogenic diet. In addition to gene expression studies, we carried out physiological observations, blood hematology as well as clinical biochemistry, cytokine profiling and antioxidant assays on their blood sera. Using Illumina microarrays, we found that the atherogenic diet caused oxidative stress, inflammation and increased turnover of metabolites and cells in the liver, spleen and heart. In contrast, OPP showed signs of attenuating these effects. The extract increased unfolded protein response in the liver, attenuated antigen presentation and processing in the spleen and up-regulated antioxidant genes in the heart. Real-time quantitative reverse transcription-polymerase chain reaction validated the microarray gene expression fold changes observed. Serum cytokine profiling showed that OPP attenuated inflammation by modulating the Th1/Th2 axis toward the latter. OPP also increased serum antioxidant activity to normal levels. This study suggests that OPP may possibly attenuate atherosclerosis and other forms of cardiovascular disease.

Abstract: Water-soluble phenolics from the oil palm possess significant biological properties. In this study, we aimed to discover the role of oil palm phenolics (OPP) in influencing the gene expression changes caused by an atherogenic diet in mice. We fed mice with either a low-fat normal diet (14.6 % kcal/kcal fat) with distilled water, or a high-fat atherogenic diet (40.5 % kcal/kcal fat) containing cholesterol. The latter group was given either distilled water or OPP. We harvested major organs such as livers, spleens and hearts for microarray gene expression profiling analysis. We determined how OPP changed the gene expression profiles caused by the atherogenic diet. In addition to gene expression studies, we carried out physiological observations, blood hematology as well as clinical biochemistry, cytokine profiling and antioxidant assays on their blood sera. Using Illumina microarrays, we found that the atherogenic diet caused oxidative stress, inflammation and increased turnover of metabolites and cells in the liver, spleen and heart. In contrast, OPP showed signs of attenuating these effects. The extract increased unfolded protein response in the liver, attenuated antigen presentation and processing in the spleen and up-regulated antioxidant genes in the heart. Real-time quantitative reverse transcription-polymerase chain reaction validated the microarray gene expression fold changes observed. Serum cytokine profiling showed that OPP attenuated inflammation by modulating the Th1/Th2 axis toward the latter. OPP also increased serum antioxidant activity to normal levels. This study suggests that OPP may possibly attenuate atherosclerosis and other forms of cardiovascular disease.

Abstract: Plasma leakage in severe dengue has been postulated to be associated with skewed cytokine immune responses. In this study, the association of cytokines with vascular permeability in dengue patients was investigated. Human serum samples collected from 48 persons (13 with dengue fever, 29 with dengue hemorrhagic fever, and 6 healthy) were subjected to cytokines analysis by using Luminex Multiplex Technology. Selected serum samples from patients with dengue hemorrhagic fever sera and recombinant human cytokines were then tested for roles on inducing vascular permeability by treatment of human umbilical vein endothelial cells. Confocal immunofluorescence staining indicated morphologic alteration of human umbilical vein endothelial cells treated with serum samples from patients with dengue hemorrhagic fever compared with serum samples from healthy persons. The findings suggest that cytokines produced during dengue hemorrhagic infections could induce alterations in the vascular endothelium, which may play a fundamental role in the pathophysiology of dengue.

Abstract: Melanoma incidence and mortality have risen dramatically in recent years. No effective treatment for metastatic melanoma exists; hence currently, an intense effort for new drug evaluation is being carried out. In this study, we investigated the effects of a palm oil-derived nanopolymer called Bio-12 against human malignant melanoma. The nanopolymers of Bio-12 are lipid esters derived from a range of fatty acids of palm oil. Our study aims to identify the anti-proliferative properties of Bio-12 against human malignant melanoma cell line (MeWo) and to elucidate the mode of actions whereby Bio-12 brings about cell death. Bio-12 significantly inhibited the growth of MeWo cells in a concentration- and time- dependent manner with a median inhibitory concentration (IC50) value of 1/25 dilution after 72 h but was ineffective on human normal skin fibroblasts (CCD-1059sk). We further investigated the mode of actions of Bio-12 on MeWo cells. Cell cycle flow cytometry demonstrated that MeWo cells treated with increasing concentrations of Bio-12 resulted in S-phase arrest, accompanied by the detection of sub-G1 content, indicative of apoptotic cell death. Induction of apoptosis was further confirmed via caspase (substrate) cleavage assay which showed induction of early apoptosis in MeWo cells. In addition, DNA strand breaks which are terminal event in apoptosis were evident through increase of TUNEL positive cells and formation of a characteristic DNA ladder on agarose gel electrophoresis. Moreover, treatment of MeWo cells with Bio-12 induced significant increase in lactate dehydrogenase (LDH) activity. These results show that Bio-12 possesses the ability to suppress proliferation of human malignant melanoma MeWo cells and this suppression is at least partly attributed to the initiation of the S-phase arrest, apoptosis and necrosis, suggesting that it is indeed worth for further investigations.

Abstract: Background: Quorum sensing is a term that describes an environmental sensing system that allows bacteria to monitor their own population density which contributes significantly to the size and development of the biofilm. Many gram negative bacteria use N-acyl-homoserine lactones as quorum sensing signal molecules. In this study, we sought to find out if the biofilm formation among clinical isolates of Acinetobacter spp. is under the control of autoinducing quorum sensing molecules. Methodology/Principal Findings: Biofilm formation among clinical isolates of Acinetobacter spp. was assessed and the production of signal molecules were detected with Chromobacterium violaceum CV026 biosensor system. Characterisation of autoinducers was carried out by mass spectrometric analysis. We have also reported the identification of an autoinducer synthase gene, abaI among the isolates that produce quorum sensing signal molecules and have reported that the mutation in the abaI gene influences their biofilm forming capabilities. Using a microtitre-plate assay it was shown that 60% of the 50 Acinetobacter spp. isolates significantly formed biofilms. Further detection with the biosensor strain showed that some of these isolates produced long chain signal molecules. Mass spectrometric analysis revealed that five of these isolates produced N-decanoyl homoserine lactone and two isolates produced acyl-homoserine lactone with a chain length equal to C-12. The abaI gene was identified and a tetracycline mutant of the abaI gene was created and the inhibition in biofilm formation in the mutant was shown. Conclusions/Significance: These data are of great significance as the signal molecules aid in biofilm formation which in turn confer various properties of pathogenicity to the clinical isolates including drug resistance. The use of quorum sensing signal blockers to attenuate bacterial pathogenicity is therefore highly attractive, particularly with respect to the emergence of multi antibiotic resistant bacteria.

Abstract: Background: Dengue virus-host cell interaction initiates when the virus binds to the attachment receptors followed by endocytic internalization of the virus particle. Successful entry into the cell is necessary for infection initiation. Currently, there is no protective vaccine or antiviral treatment for dengue infection. Targeting the viral entry pathway has become an attractive therapeutic strategy to block infection. This study aimed to investigate the effect of silencing the GRP78 and clathrin-mediated endocytosis on dengue virus entry and multiplication into HepG2 cells. Methodology/Principal Findings: HepG2 cells were transfected using specific siRNAs to silence the cellular surface receptor ( GRP78) and clathrin-mediated endocytosis pathway. Gene expression analysis showed a marked down-regulation of the targeted genes (87.2%, 90.3%, and 87.8% for GRP78, CLTC, and DNM2 respectively) in transfected HepG2 cells when measured by RT-qPCR. Intracellular and extracellular viral RNA loads were quantified by RT-qPCR to investigate the effect of silencing the attachment receptor and clathrin-mediated endocytosis on dengue virus entry. Silenced cells showed a significant reduction of intracellular (92.4%) and extracellular viral RNA load (71.4%) compared to non-silenced cells. Flow cytometry analysis showed a marked reduction of infected cells (89.7%) in silenced HepG2 cells compared to non-silenced cells. Furthermore, the ability to generate infectious virions using the plaque assay was reduced 1.07 log in silenced HepG2 cells. Conclusions/Significance: Silencing the attachment receptor and clathrin-mediated endocytosis using siRNA could inhibit dengue virus entry and multiplication into HepG2 cells. This leads to reduction of infected cells as well as the viral load, which might function as a unique and promising therapeutic agent for attenuating dengue infection and prevent the development of dengue fever to the severe life-threatening DHF or DSS. Furthermore, a decrease of viremia in humans can result in the reduction of infected vectors and thus, halt of the transmission cycle.

Abstract: Background: Dengue is an important medical problem, with symptoms ranging from mild dengue fever to severe forms of the disease, where vascular leakage leads to hypovolemic shock. Cytokines have been implicated to play a role in the progression of severe dengue disease; however, their profile in dengue patients and the synergy that leads to continued plasma leakage is not clearly understood. Herein, we investigated the cytokine kinetics and profiles of dengue patients at different phases of illness to further understand the role of cytokines in dengue disease. Methods and Findings: Circulating levels of 29 different types of cytokines were assessed by bead-based ELISA method in dengue patients at the 3 different phases of illness. The association between significant changes in the levels of cytokines and clinical parameters were analyzed. At the febrile phase, IP-10 was significant in dengue patients with and without warning signs. However, MIP-1 beta was found to be significant in only patients with warning signs at this phase. IP-10 was also significant in both with and without warning signs patients during defervescence. At this phase, MIP-1b beta and G-CSF were significant in patients without warning signs, whereas MCP-1 was noted to be elevated significantly in patients with warning signs. Significant correlations between the levels of VEGF, RANTES, IL-7, IL-12, PDGF and IL-5 with platelets; VEGF with lymphocytes and neutrophils; G-CSF and IP-10 with atypical lymphocytes and various other cytokines with the liver enzymes were observed in this study. Conclusions: The cytokine profile patterns discovered between the different phases of illness indicate an essential role in dengue pathogenesis and with further studies may serve as predictive markers for progression to dengue with warning signs.

Abstract: Background: Dengue is the most prevalent mosquito borne infection worldwide. Vertical transmissions after maternal dengue infection to the fetus and pregnancy losses in relation to dengue illness have been reported. The relationship of dengue to miscarriage is not known. Method: We aimed to establish the relationship of recent dengue infection and miscarriage. Women who presented with miscarriage (up to 22 weeks gestation) to our hospital were approached to participate in the study. For each case of miscarriage, we recruited 3 controls with viable pregnancies at a similar gestation. A brief questionnaire on recent febrile illness and prior dengue infection was answered. Blood was drawn from participants, processed and the frozen serum was stored. Stored sera were thawed and then tested in batches with dengue specific IgM capture ELISA, dengue non-structural protein 1 (NS1) antigen and dengue specific IgG ELISA tests. Controls remained in the analysis if their pregnancies continued beyond 22 weeks gestation. Tests were run on 116 case and 341 control sera. One case (a misdiagnosed viable early pregnancy) plus 45 controls (39 lost to follow up and six subsequent late miscarriages) were excluded from analysis. Findings: Dengue specific IgM or dengue NS1 antigen (indicating recent dengue infection) was positive in 6/115 (5.2%) cases and 5/296 (1.7%) controls RR 3.1 (95% CI 1.0-10) P = 0.047. Maternal age, gestational age, parity and ethnicity were dissimilar between cases and controls. After adjustments for these factors, recent dengue infection remained significantly more frequently detected in cases than controls (AOR 4.2 95% CI 1.2-14 P = 0.023). Interpretation: Recent dengue infections were more frequently detected in women presenting with miscarriage than in controls whose pregnancies were viable. After adjustments for confounders, the positive association remained.

Abstract: Background: Dengue is an important medical problem, with symptoms ranging from mild dengue fever to severe forms of the disease, where vascular leakage leads to hypovolemic shock. Cytokines have been implicated to play a role in the progression of severe dengue disease; however, their profile in dengue patients and the synergy that leads to continued plasma leakage is not clearly understood. Herein, we investigated the cytokine kinetics and profiles of dengue patients at different phases of illness to further understand the role of cytokines in dengue disease. Methods and Findings: Circulating levels of 29 different types of cytokines were assessed by bead-based ELISA method in dengue patients at the 3 different phases of illness. The association between significant changes in the levels of cytokines and clinical parameters were analyzed. At the febrile phase, IP-10 was significant in dengue patients with and without warning signs. However, MIP-1 beta was found to be significant in only patients with warning signs at this phase. IP-10 was also significant in both with and without warning signs patients during defervescence. At this phase, MIP-1b beta and G-CSF were significant in patients without warning signs, whereas MCP-1 was noted to be elevated significantly in patients with warning signs. Significant correlations between the levels of VEGF, RANTES, IL-7, IL-12, PDGF and IL-5 with platelets; VEGF with lymphocytes and neutrophils; G-CSF and IP-10 with atypical lymphocytes and various other cytokines with the liver enzymes were observed in this study. Conclusions: The cytokine profile patterns discovered between the different phases of illness indicate an essential role in dengue pathogenesis and with further studies may serve as predictive markers for progression to dengue with warning signs.

Abstract: Background: Dengue is the most prevalent mosquito borne infection worldwide. Vertical transmissions after maternal dengue infection to the fetus and pregnancy losses in relation to dengue illness have been reported. The relationship of dengue to miscarriage is not known. Method: We aimed to establish the relationship of recent dengue infection and miscarriage. Women who presented with miscarriage (up to 22 weeks gestation) to our hospital were approached to participate in the study. For each case of miscarriage, we recruited 3 controls with viable pregnancies at a similar gestation. A brief questionnaire on recent febrile illness and prior dengue infection was answered. Blood was drawn from participants, processed and the frozen serum was stored. Stored sera were thawed and then tested in batches with dengue specific IgM capture ELISA, dengue non-structural protein 1 (NS1) antigen and dengue specific IgG ELISA tests. Controls remained in the analysis if their pregnancies continued beyond 22 weeks gestation. Tests were run on 116 case and 341 control sera. One case (a misdiagnosed viable early pregnancy) plus 45 controls (39 lost to follow up and six subsequent late miscarriages) were excluded from analysis. Findings: Dengue specific IgM or dengue NS1 antigen (indicating recent dengue infection) was positive in 6/115 (5.2%) cases and 5/296 (1.7%) controls RR 3.1 (95% CI 1.0-10) P = 0.047. Maternal age, gestational age, parity and ethnicity were dissimilar between cases and controls. After adjustments for these factors, recent dengue infection remained significantly more frequently detected in cases than controls (AOR 4.2 95% CI 1.2-14 P = 0.023). Interpretation: Recent dengue infections were more frequently detected in women presenting with miscarriage than in controls whose pregnancies were viable. After adjustments for confounders, the positive association remained.

Abstract: Melanoma incidence and mortality have risen dramatically in recent years. No effective treatment for metastatic melanoma exists; hence currently, an intense effort for new drug evaluation is being carried out. In this study, we investigated the effects of a palm oil-derived nanopolymer called Bio-12 against human malignant melanoma. The nanopolymers of Bio-12 are lipid esters derived from a range of fatty acids of palm oil. Our study aims to identify the anti-proliferative properties of Bio-12 against human malignant melanoma cell line (MeWo) and to elucidate the mode of actions whereby Bio-12 brings about cell death. Bio-12 significantly inhibited the growth of MeWo cells in a concentration- and time- dependent manner with a median inhibitory concentration (IC50) value of 1/25 dilution after 72 h but was ineffective on human normal skin fibroblasts (CCD-1059sk). We further investigated the mode of actions of Bio-12 on MeWo cells. Cell cycle flow cytometry demonstrated that MeWo cells treated with increasing concentrations of Bio-12 resulted in S-phase arrest, accompanied by the detection of sub-G1 content, indicative of apoptotic cell death. Induction of apoptosis was further confirmed via caspase (substrate) cleavage assay which showed induction of early apoptosis in MeWo cells. In addition, DNA strand breaks which are terminal event in apoptosis were evident through increase of TUNEL positive cells and formation of a characteristic DNA ladder on agarose gel electrophoresis. Moreover, treatment of MeWo cells with Bio-12 induced significant increase in lactate dehydrogenase (LDH) activity. These results show that Bio-12 possesses the ability to suppress proliferation of human malignant melanoma MeWo cells and this suppression is at least partly attributed to the initiation of the S-phase arrest, apoptosis and necrosis, suggesting that it is indeed worth for further investigations.

Abstract: Plasma leakage in severe dengue has been postulated to be associated with skewed cytokine immune responses. In this study, the association of cytokines with vascular permeability in dengue patients was investigated. Human serum samples collected from 48 persons (13 with dengue fever, 29 with dengue hemorrhagic fever, and 6 healthy) were subjected to cytokines analysis by using Luminex Multiplex Technology. Selected serum samples from patients with dengue hemorrhagic fever sera and recombinant human cytokines were then tested for roles on inducing vascular permeability by treatment of human umbilical vein endothelial cells. Confocal immunofluorescence staining indicated morphologic alteration of human umbilical vein endothelial cells treated with serum samples from patients with dengue hemorrhagic fever compared with serum samples from healthy persons. The findings suggest that cytokines produced during dengue hemorrhagic infections could induce alterations in the vascular endothelium, which may play a fundamental role in the pathophysiology of dengue.

Abstract: Background: Dengue virus-host cell interaction initiates when the virus binds to the attachment receptors followed by endocytic internalization of the virus particle. Successful entry into the cell is necessary for infection initiation. Currently, there is no protective vaccine or antiviral treatment for dengue infection. Targeting the viral entry pathway has become an attractive therapeutic strategy to block infection. This study aimed to investigate the effect of silencing the GRP78 and clathrin-mediated endocytosis on dengue virus entry and multiplication into HepG2 cells. Methodology/Principal Findings: HepG2 cells were transfected using specific siRNAs to silence the cellular surface receptor ( GRP78) and clathrin-mediated endocytosis pathway. Gene expression analysis showed a marked down-regulation of the targeted genes (87.2%, 90.3%, and 87.8% for GRP78, CLTC, and DNM2 respectively) in transfected HepG2 cells when measured by RT-qPCR. Intracellular and extracellular viral RNA loads were quantified by RT-qPCR to investigate the effect of silencing the attachment receptor and clathrin-mediated endocytosis on dengue virus entry. Silenced cells showed a significant reduction of intracellular (92.4%) and extracellular viral RNA load (71.4%) compared to non-silenced cells. Flow cytometry analysis showed a marked reduction of infected cells (89.7%) in silenced HepG2 cells compared to non-silenced cells. Furthermore, the ability to generate infectious virions using the plaque assay was reduced 1.07 log in silenced HepG2 cells. Conclusions/Significance: Silencing the attachment receptor and clathrin-mediated endocytosis using siRNA could inhibit dengue virus entry and multiplication into HepG2 cells. This leads to reduction of infected cells as well as the viral load, which might function as a unique and promising therapeutic agent for attenuating dengue infection and prevent the development of dengue fever to the severe life-threatening DHF or DSS. Furthermore, a decrease of viremia in humans can result in the reduction of infected vectors and thus, halt of the transmission cycle.

Abstract: Background: Quorum sensing is a term that describes an environmental sensing system that allows bacteria to monitor their own population density which contributes significantly to the size and development of the biofilm. Many gram negative bacteria use N-acyl-homoserine lactones as quorum sensing signal molecules. In this study, we sought to find out if the biofilm formation among clinical isolates of Acinetobacter spp. is under the control of autoinducing quorum sensing molecules. Methodology/Principal Findings: Biofilm formation among clinical isolates of Acinetobacter spp. was assessed and the production of signal molecules were detected with Chromobacterium violaceum CV026 biosensor system. Characterisation of autoinducers was carried out by mass spectrometric analysis. We have also reported the identification of an autoinducer synthase gene, abaI among the isolates that produce quorum sensing signal molecules and have reported that the mutation in the abaI gene influences their biofilm forming capabilities. Using a microtitre-plate assay it was shown that 60% of the 50 Acinetobacter spp. isolates significantly formed biofilms. Further detection with the biosensor strain showed that some of these isolates produced long chain signal molecules. Mass spectrometric analysis revealed that five of these isolates produced N-decanoyl homoserine lactone and two isolates produced acyl-homoserine lactone with a chain length equal to C-12. The abaI gene was identified and a tetracycline mutant of the abaI gene was created and the inhibition in biofilm formation in the mutant was shown. Conclusions/Significance: These data are of great significance as the signal molecules aid in biofilm formation which in turn confer various properties of pathogenicity to the clinical isolates including drug resistance. The use of quorum sensing signal blockers to attenuate bacterial pathogenicity is therefore highly attractive, particularly with respect to the emergence of multi antibiotic resistant bacteria.

Abstract: It is well established that plant phenolics elicit various biological activities, with positive effects on health. Palm oil production results in large volumes of aqueous by-products containing phenolics. In the present study, we describe the effects of oil palm phenolics (OPP) on several degenerative conditions using various animal models. OPP reduced blood pressure in a NO-deficient rat model, protected against ischaemia-induced cardiac arrhythmia in rats and reduced plaque formation in rabbits fed an atherogenic diet. In Nile rats, a spontaneous model of the metabolic syndrome and type 2 diabetes, OPP protected against multiple aspects of the syndrome and diabetes progression. In tumour-inoculated mice, OPP protected against cancer progression. Microarray studies on the tumours showed differential transcriptome profiles that suggest anti-tumour molecular mechanisms involved in OPP action. Thus, initial studies suggest that OPP may have potential against several chronic disease outcomes in mammals.

Abstract: Background: Serological tests for IgM and IgG are routinely used in clinical laboratories for the rapid diagnosis of dengue and can differentiate between primary and secondary infections. Dengue virus non-structural protein 1 (NS1) has been identified as an early marker for acute dengue, and is typically present between days 1-9 post-onset of illness but following seroconversion it can be difficult to detect in serum. Aims: To evaluate the performance of a newly developed Panbio (R) Dengue Early Rapid test for NS1 and determine if it can improve diagnostic sensitivity when used in combination with a commercial IgM/IgG rapid test. Methodology: The clinical performance of the Dengue Early Rapid was evaluated in a retrospective study in Vietnam with 198 acute laboratory-confirmed positive and 100 negative samples. The performance of the Dengue Early Rapid in combination with the IgM/IgG Rapid test was also evaluated in Malaysia with 263 laboratory-confirmed positive and 30 negative samples. Key Results: In Vietnam the sensitivity and specificity of the test was 69.2% (95% CI: 62.8% to 75.6%) and 96% (95% CI: 92.2% to 99.8) respectively. In Malaysia the performance was similar with 68.9% sensitivity (95% CI: 61.8% to 76.1%) and 96.7% specificity (95% CI: 82.8% to 99.9%) compared to RT-PCR. Importantly, when the Dengue Early Rapid test was used in combination with the IgM/IgG test the sensitivity increased to 93.0%. When the two tests were compared at each day post-onset of illness there was clear differentiation between the antigen and antibody markers. Conclusions: This study highlights that using dengue NS1 antigen detection in combination with anti-glycoprotein E IgM and IgG serology can significantly increase the sensitivity of acute dengue diagnosis and extends the possible window of detection to include very early acute samples and enhances the clinical utility of rapid immunochromatographic testing for dengue.

Abstract: Pseudomonas aeruginosa is considered as one of the leading causes of nosocomial infections. The start of antimicrobial therapy is often empirical and selective pressure on panel of antibiotics; therefore, it is important to know the susceptibility pattern of pathogens in order to select the most appropriate antibiotic. The aim of the current study is to update the rational empirical antimicrobial therapy recommendations. Antimicrobial resistance was done using the E-test method. Urine and wound swab samples were the highest encountered isolates; results were interpreted according to National Committee for Clinical Laboratory Standards guidelines. A total of 88 clinical isolates of P. aeruginosa were collected randomly from April 2009 to March 2010 from the University of Malaya Medical Center. P. aeruginosa isolated from various clinical samples has lost susceptibility and showed increasing resistance to Gentamicin with 94.3%, followed by (ciprofloxacin) 92%, (ceftazidime) 89.8%, (imipenem) 73.9%, Pipracilline/tazobactam 61.4%, (aztreonam) 52.3%, and (amikacin) 50% and only susceptible to colistin with 92%. In conclusion, most of the isolates showed high levels of resistance to examined antibiotics except colistin and this may indicate the importance of antibiotic susceptibility testing and optimal treatment by combination of drugs.

Abstract: Background: Dengue infection ranks as one of the most significant viral diseases of the globe. Currently, there is no specific vaccine or antiviral therapy for prevention or treatment. Monocytes/macrophages are the principal target cells for dengue virus and are responsible for disseminating the virus after its transmission. Dengue virus enters target cells via receptor-mediated endocytosis after the viral envelope protein E attaches to the cell surface receptor. This study aimed to investigate the effect of silencing the CD-14 associated molecule and clathrin-mediated endocytosis using siRNA on dengue virus entry into monocytes. Methodology/Principal Findings: Gene expression analysis showed a significant down-regulation of the target genes (82.7%, 84.9 and 76.3% for CD-14 associated molecule, CLTC and DNM2 respectively) in transfected monocytes. The effect of silencing of target genes on dengue virus entry into monocytes was investigated by infecting silenced and non-silenced monocytes with DENV-2. Results showed a significant reduction of infected cells (85.2%), intracellular viral RNA load (73.0%), and extracellular viral RNA load (63.0%) in silenced monocytes as compared to non-silenced monocytes. Conclusions/Significance: Silencing the cell surface receptor and clathrin mediated endocytosis using RNA interference resulted in inhibition of the dengue virus entry and subsequently multiplication of the virus in the monocytes. This might serve as a novel promising therapeutic target to attenuate dengue infection and thus reduce transmission as well as progression to severe dengue hemorrhagic fever.

Abstract: Nosocomial infections are major clinical threats to hospitalised patients and represent an important source of morbidity and mortality. It is necessary to develop rapid detection assays of nosocomial pathogens for better prognosis and initiation of antimicrobial therapy in patients. In this study, we present the development of molecular methods for the detection of six common nosocomial pathogens including Escherichia coli, Staphylococcus aureus, Streptococcus pneumoniae, Klebsiella pneumoniae, Pseudomonas aeruginosa and Acinetobacter spp. Conventional multiplex PCR and SYBR Green based real time PCR assays were performed using genus and species specific primers. Blind testing with 300 clinical samples was also carried out. The two assays were found to be sensitive and specific. Eubacterial PCR assay exhibited positive results for 46 clinical isolates from which 43 samples were detected by real time PCR assay. The sensitivity of the assay is about 93.7% in blind test isolates. The PCR results were reconfirmed using the conventional culture method. This assay has the potential to be a rapid, accurate and highly sensitive molecular diagnostic tool for simultaneous detection of Escherichia coli, Staphylococcus aureus, Streptococcus pneumoniae, Klebsiella pneumoniae, Pseudomonas aeruginosa and Acinetobacter spp. This assay has the potential to detect nosocomial pathogens within 5 to 6 hours, helping to initiate infection control measures and appropriate treatment in paediatric and elderly (old aged) patients, pre-and post surgery patients and organ transplant patients and thus reduces their hospitalization duration.

Abstract: Background: Dengue virus is transmitted by mosquitoes and has four serotypes. Cross-protection to other serotypes lasting for a few months is observed following infection with one serotype. There is evidence that low-affinity T and/or B cells from primary infections contribute to the severe syndromes often associated with secondary dengue infections. such pronounced immune-mediated enhancement suggests a dengue-specific pattern of immune cell activation. This study investigates the acute and early convalescent B cell response leading to the generation of cross-reactive and neutralizing antibodies following dengue infection. Methodology/Principal Findings: We assayed blood samples taken from dengue patients with primary or secondary infection during acute disease and convalescence and compared them to samples from patients presenting with non-dengue related fever. Dengue induced massive early plasmablast formation, which correlated with the appearance of polyclonal, cross-reactive IgG for both primary and secondary infection. Surprisingly, the contribution of IgG to the neutralizing titer 4-7 days after fever onset was more than 50% even after primary infection. Conclusions/Significance: Poly-reactive and virus serotype cross-reactive IgG are an important component of the innate response in humans during both primary and secondary dengue infection, and "innate specificities'' seem to constitute part of the adaptive response in dengue. While of potential importance for protection during secondary infection, cross-reactive B cells will also compete with highly neutralizing B cells and possibly interfere with their development.

Abstract: Background: Current chemotherapeutic drugs kill cancer cells mainly by inducing apoptosis. However, they become ineffective once cancer cell has the ability to metastasize, hence the poor prognosis and high mortality rate. Therefore, the purpose of this study was to evaluate the antimetastatic potential of Phyllanthus (P. niruri, P. urinaria, P. watsonii, and P. amarus) on lung and breast carcinoma cells. Methodology/Principal Findings: Cytotoxicity of Phyllanthus plant extracts were first screened using the MTS reduction assay. They were shown to inhibit MCF-7 (breast carcinoma) and A549 (lung carcinoma) cells growth with IC(50) values ranging from 50-180 mu g/ml and 65-470 mu g/ml for methanolic and aqueous extracts respectively. In comparison, they have lower toxicity on normal cells with the cell viability percentage remaining above 50% when treated up to 1000 mu g/ml for both extracts. After determining the non-toxic effective dose, several antimetastasis assays were carried out and Phyllanthus extracts were shown to effectively reduce invasion, migration, and adhesion of both MCF-7 and A549 cells in a dose-dependent manner, at concentrations ranging from 20-200 mu g/ml for methanolic extracts and 50-500 mu g/ml for aqueous extracts. This was followed by an evaluation of the possible modes of cell death that occurred along with the antimetastatic activity. Phyllanthus was shown to be capable of inducing apoptosis in conjunction with its antimetastastic action, with more than three fold increase of caspases-3 and -7, the presence of DNA-fragmentation and TUNEL-positive cells. The ability of Phyllanthus to exert antimetastatic activities is mostly associated to the presence of polyphenol compounds in its extracts. Conclusions/Significance: The presence of polyphenol compounds in the Phyllanthus plant is critically important in the inhibition of the invasion, migration, and adhesion of cancer cells, along with the involvement of apoptosis induction. Hence, Phyllanthus could be a valuable candidate in the treatment of metastatic cancers.

Abstract: Background: Streptococcus pneumoniae is a major causative agent of severe infections, including sepsis, pneumonia, meningitis, and otitis media, that has since become a major public health concern. In this study, the serotypes distribution of pneumococcal isolates was investigated to predict the efficacy of the 7-valent pneumococcal conjugate vaccine (PCV7) among the Malaysian populations. Methodology/Principal Findings: A total of 151 clinical isolates were serotyped using multiplex PCR assays. Out of them, there were 21.2% penicillin-resistant, 29.1% penicillin-intermediate, and 49.7% penicillin-susceptible S. pneumoniae strains. Serotypes detected among the Malaysian isolates were 1, 3, 10A, 11A/11D, 12F/12A, 14, 15A, 15B/15C, 16F, 18C/18B/18A/18F, 19A, 19F, 23F, 35B, 35F/47F, 6A/6B, 7C/7B/40, 7F/7A, 9V/9A, and 34. Serotype 19F and 23F were the two most prevalent serotypes detected. Serotypes are highly associated with invasiveness of isolates (p = 0.001) and penicillin susceptibility (p<0.001). Serotype 19F was observed to have increased resistance against penicillin while serotype 19A has high invasive tendency. Age of patients was an important factor underlying the pneumococcal serotypes (p = 0.03) and clinical sites of infections (p<0.001). High prevalence of pneumococcal isolates were detected among children <5 years old at nasopharyngeal sites while elderly adults >= 60 years old were at increased risk for pneumococcal bacteremia. Conclusion/Significance: Current study revealed that a number of serotypes, especially those associated with high penicillin resistance, have been formulated in the PCV7. Therefore, the protections expected from the routine use of PCV7 would be encouraging for the Malaysian. However, it is not possible to predict serotypes that might become predominant in the future and hence continued surveillance of circulating serotypes will be needed.

Abstract: Background: Plant phenolics are important nutritional antioxidants which could aid in overcoming chronic diseases such as cardiovascular disease and cancer, two leading causes of death in the world. The oil palm (Elaeis guineensis) is a rich source of water-soluble phenolics which have high antioxidant activities. This study aimed to identify the in vivo effects and molecular mechanisms involved in the biological activities of oil palm phenolics (OPP) during healthy states via microarray gene expression profiling, using mice supplemented with a normal diet as biological models. Results: Having confirmed via histology, haematology and clinical biochemistry analyses that OPP is not toxic to mice, we further explored the gene expression changes caused by OPP through statistical and functional analyses using Illumina microarrays. OPP showed numerous biological activities in three major organs of mice, the liver, spleen and heart. In livers of mice given OPP, four lipid catabolism genes were up-regulated while five cholesterol biosynthesis genes were down-regulated, suggesting that OPP may play a role in reducing cardiovascular disease. OPP also up-regulated eighteen blood coagulation genes in spleens of mice. OPP elicited gene expression changes similar to the effects of caloric restriction in the hearts of mice supplemented with OPP. Microarray gene expression fold changes for six target genes in the three major organs tested were validated with real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR), and the correlation of fold changes obtained with these two techniques was high (R-2 = 0.9653). Conclusions: OPP showed non-toxicity and various pleiotropic effects in mice. This study implies the potential application of OPP as a valuable source of wellness nutraceuticals, and further suggests the molecular mechanisms as to how dietary phenolics work in vivo.

Abstract: The major L-amino acid oxidase (LAAO, EC 1.4.3.2) of king cobra (Ophiophagus hannah) venom is known to be an unusual form of snake venom LAAO as it possesses unique structural features and unusual thermal stability. The antibacterial effects of king cobra venom LAAO were tested against several strains of clinical isolates including Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa, Klebsiella pneumoniae, and Escherichia coli using broth microdilution assay. For comparison, the antibacterial effects of several antibiotics (cefotaxime, kanamycin, tetracycline, vancomycin and penicillin) were also examined using the same conditions. King cobra venom LAAO was very effective in inhibiting the two Gram-positive bacteria (S. aureus and S. epidermidis) tested, with minimum inhibitory concentration (MIC) of 0.78 mu g/mL (0.006 mu M) and 1.56 mu g/mL (0.012 mu M) against S. aureus and S. epidermidis, respectively. The MICs are comparable to the MICs of the antibiotics tested, on a weight basis.. However, the LAAO was only moderately effective against three Gram-negative bacteria tested (P. aeruginosa, K. pneumoniae and E. coli), with MIC ranges from 25 to 50 mu g/mL (0.2-0.4 mu M). Catalase at the concentration of 1 mg/mL abolished the antibacterial effect of LAAO, indicating that the antibacterial effect of the enzyme involves generation of hydrogen peroxide. Binding studies indicated that king cobra venom LAAO binds strongly to the Gram-positive S. aureus and S. epidermidis, but less strongly to the Gram-negative E. coli and P. aeruginosa, indicating that specific binding to bacteria is important for the potent antibacterial activity of the enzyme. (C) 2010 Elsevier Inc. All rights reserved.

Abstract: The major L-amino acid oxidase (LAAO, EC 1.4.3.2) of king cobra (Ophiophagus hannah) venom is known to be an unusual form of snake venom LAAO as it possesses unique structural features and unusual thermal stability. The antibacterial effects of king cobra venom LAAO were tested against several strains of clinical isolates including Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa, Klebsiella pneumoniae, and Escherichia coli using broth microdilution assay. For comparison, the antibacterial effects of several antibiotics (cefotaxime, kanamycin, tetracycline, vancomycin and penicillin) were also examined using the same conditions. King cobra venom LAAO was very effective in inhibiting the two Gram-positive bacteria (S. aureus and S. epidermidis) tested, with minimum inhibitory concentration (MIC) of 0.78 mu g/mL (0.006 mu M) and 1.56 mu g/mL (0.012 mu M) against S. aureus and S. epidermidis, respectively. The MICs are comparable to the MICs of the antibiotics tested, on a weight basis.. However, the LAAO was only moderately effective against three Gram-negative bacteria tested (P. aeruginosa, K. pneumoniae and E. coli), with MIC ranges from 25 to 50 mu g/mL (0.2-0.4 mu M). Catalase at the concentration of 1 mg/mL abolished the antibacterial effect of LAAO, indicating that the antibacterial effect of the enzyme involves generation of hydrogen peroxide. Binding studies indicated that king cobra venom LAAO binds strongly to the Gram-positive S. aureus and S. epidermidis, but less strongly to the Gram-negative E. coli and P. aeruginosa, indicating that specific binding to bacteria is important for the potent antibacterial activity of the enzyme. (C) 2010 Elsevier Inc. All rights reserved.

Abstract: Background: Streptococcus pneumoniae is a major causative agent of severe infections, including sepsis, pneumonia, meningitis, and otitis media, that has since become a major public health concern. In this study, the serotypes distribution of pneumococcal isolates was investigated to predict the efficacy of the 7-valent pneumococcal conjugate vaccine (PCV7) among the Malaysian populations. Methodology/Principal Findings: A total of 151 clinical isolates were serotyped using multiplex PCR assays. Out of them, there were 21.2% penicillin-resistant, 29.1% penicillin-intermediate, and 49.7% penicillin-susceptible S. pneumoniae strains. Serotypes detected among the Malaysian isolates were 1, 3, 10A, 11A/11D, 12F/12A, 14, 15A, 15B/15C, 16F, 18C/18B/18A/18F, 19A, 19F, 23F, 35B, 35F/47F, 6A/6B, 7C/7B/40, 7F/7A, 9V/9A, and 34. Serotype 19F and 23F were the two most prevalent serotypes detected. Serotypes are highly associated with invasiveness of isolates (p = 0.001) and penicillin susceptibility (p<0.001). Serotype 19F was observed to have increased resistance against penicillin while serotype 19A has high invasive tendency. Age of patients was an important factor underlying the pneumococcal serotypes (p = 0.03) and clinical sites of infections (p<0.001). High prevalence of pneumococcal isolates were detected among children <5 years old at nasopharyngeal sites while elderly adults >= 60 years old were at increased risk for pneumococcal bacteremia. Conclusion/Significance: Current study revealed that a number of serotypes, especially those associated with high penicillin resistance, have been formulated in the PCV7. Therefore, the protections expected from the routine use of PCV7 would be encouraging for the Malaysian. However, it is not possible to predict serotypes that might become predominant in the future and hence continued surveillance of circulating serotypes will be needed.

Abstract: Background: Current chemotherapeutic drugs kill cancer cells mainly by inducing apoptosis. However, they become ineffective once cancer cell has the ability to metastasize, hence the poor prognosis and high mortality rate. Therefore, the purpose of this study was to evaluate the antimetastatic potential of Phyllanthus (P. niruri, P. urinaria, P. watsonii, and P. amarus) on lung and breast carcinoma cells. Methodology/Principal Findings: Cytotoxicity of Phyllanthus plant extracts were first screened using the MTS reduction assay. They were shown to inhibit MCF-7 (breast carcinoma) and A549 (lung carcinoma) cells growth with IC(50) values ranging from 50-180 mu g/ml and 65-470 mu g/ml for methanolic and aqueous extracts respectively. In comparison, they have lower toxicity on normal cells with the cell viability percentage remaining above 50% when treated up to 1000 mu g/ml for both extracts. After determining the non-toxic effective dose, several antimetastasis assays were carried out and Phyllanthus extracts were shown to effectively reduce invasion, migration, and adhesion of both MCF-7 and A549 cells in a dose-dependent manner, at concentrations ranging from 20-200 mu g/ml for methanolic extracts and 50-500 mu g/ml for aqueous extracts. This was followed by an evaluation of the possible modes of cell death that occurred along with the antimetastatic activity. Phyllanthus was shown to be capable of inducing apoptosis in conjunction with its antimetastastic action, with more than three fold increase of caspases-3 and -7, the presence of DNA-fragmentation and TUNEL-positive cells. The ability of Phyllanthus to exert antimetastatic activities is mostly associated to the presence of polyphenol compounds in its extracts. Conclusions/Significance: The presence of polyphenol compounds in the Phyllanthus plant is critically important in the inhibition of the invasion, migration, and adhesion of cancer cells, along with the involvement of apoptosis induction. Hence, Phyllanthus could be a valuable candidate in the treatment of metastatic cancers.

Abstract: Background: Plant phenolics are important nutritional antioxidants which could aid in overcoming chronic diseases such as cardiovascular disease and cancer, two leading causes of death in the world. The oil palm (Elaeis guineensis) is a rich source of water-soluble phenolics which have high antioxidant activities. This study aimed to identify the in vivo effects and molecular mechanisms involved in the biological activities of oil palm phenolics (OPP) during healthy states via microarray gene expression profiling, using mice supplemented with a normal diet as biological models. Results: Having confirmed via histology, haematology and clinical biochemistry analyses that OPP is not toxic to mice, we further explored the gene expression changes caused by OPP through statistical and functional analyses using Illumina microarrays. OPP showed numerous biological activities in three major organs of mice, the liver, spleen and heart. In livers of mice given OPP, four lipid catabolism genes were up-regulated while five cholesterol biosynthesis genes were down-regulated, suggesting that OPP may play a role in reducing cardiovascular disease. OPP also up-regulated eighteen blood coagulation genes in spleens of mice. OPP elicited gene expression changes similar to the effects of caloric restriction in the hearts of mice supplemented with OPP. Microarray gene expression fold changes for six target genes in the three major organs tested were validated with real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR), and the correlation of fold changes obtained with these two techniques was high (R-2 = 0.9653). Conclusions: OPP showed non-toxicity and various pleiotropic effects in mice. This study implies the potential application of OPP as a valuable source of wellness nutraceuticals, and further suggests the molecular mechanisms as to how dietary phenolics work in vivo.

Abstract: It is well established that plant phenolics elicit various biological activities, with positive effects on health. Palm oil production results in large volumes of aqueous by-products containing phenolics. In the present study, we describe the effects of oil palm phenolics (OPP) on several degenerative conditions using various animal models. OPP reduced blood pressure in a NO-deficient rat model, protected against ischaemia-induced cardiac arrhythmia in rats and reduced plaque formation in rabbits fed an atherogenic diet. In Nile rats, a spontaneous model of the metabolic syndrome and type 2 diabetes, OPP protected against multiple aspects of the syndrome and diabetes progression. In tumour-inoculated mice, OPP protected against cancer progression. Microarray studies on the tumours showed differential transcriptome profiles that suggest anti-tumour molecular mechanisms involved in OPP action. Thus, initial studies suggest that OPP may have potential against several chronic disease outcomes in mammals.

Abstract: Background: Serological tests for IgM and IgG are routinely used in clinical laboratories for the rapid diagnosis of dengue and can differentiate between primary and secondary infections. Dengue virus non-structural protein 1 (NS1) has been identified as an early marker for acute dengue, and is typically present between days 1-9 post-onset of illness but following seroconversion it can be difficult to detect in serum. Aims: To evaluate the performance of a newly developed Panbio (R) Dengue Early Rapid test for NS1 and determine if it can improve diagnostic sensitivity when used in combination with a commercial IgM/IgG rapid test. Methodology: The clinical performance of the Dengue Early Rapid was evaluated in a retrospective study in Vietnam with 198 acute laboratory-confirmed positive and 100 negative samples. The performance of the Dengue Early Rapid in combination with the IgM/IgG Rapid test was also evaluated in Malaysia with 263 laboratory-confirmed positive and 30 negative samples. Key Results: In Vietnam the sensitivity and specificity of the test was 69.2% (95% CI: 62.8% to 75.6%) and 96% (95% CI: 92.2% to 99.8) respectively. In Malaysia the performance was similar with 68.9% sensitivity (95% CI: 61.8% to 76.1%) and 96.7% specificity (95% CI: 82.8% to 99.9%) compared to RT-PCR. Importantly, when the Dengue Early Rapid test was used in combination with the IgM/IgG test the sensitivity increased to 93.0%. When the two tests were compared at each day post-onset of illness there was clear differentiation between the antigen and antibody markers. Conclusions: This study highlights that using dengue NS1 antigen detection in combination with anti-glycoprotein E IgM and IgG serology can significantly increase the sensitivity of acute dengue diagnosis and extends the possible window of detection to include very early acute samples and enhances the clinical utility of rapid immunochromatographic testing for dengue.

Abstract: Background: Dengue virus is transmitted by mosquitoes and has four serotypes. Cross-protection to other serotypes lasting for a few months is observed following infection with one serotype. There is evidence that low-affinity T and/or B cells from primary infections contribute to the severe syndromes often associated with secondary dengue infections. such pronounced immune-mediated enhancement suggests a dengue-specific pattern of immune cell activation. This study investigates the acute and early convalescent B cell response leading to the generation of cross-reactive and neutralizing antibodies following dengue infection. Methodology/Principal Findings: We assayed blood samples taken from dengue patients with primary or secondary infection during acute disease and convalescence and compared them to samples from patients presenting with non-dengue related fever. Dengue induced massive early plasmablast formation, which correlated with the appearance of polyclonal, cross-reactive IgG for both primary and secondary infection. Surprisingly, the contribution of IgG to the neutralizing titer 4-7 days after fever onset was more than 50% even after primary infection. Conclusions/Significance: Poly-reactive and virus serotype cross-reactive IgG are an important component of the innate response in humans during both primary and secondary dengue infection, and "innate specificities'' seem to constitute part of the adaptive response in dengue. While of potential importance for protection during secondary infection, cross-reactive B cells will also compete with highly neutralizing B cells and possibly interfere with their development.

Abstract: Background: Dengue infection ranks as one of the most significant viral diseases of the globe. Currently, there is no specific vaccine or antiviral therapy for prevention or treatment. Monocytes/macrophages are the principal target cells for dengue virus and are responsible for disseminating the virus after its transmission. Dengue virus enters target cells via receptor-mediated endocytosis after the viral envelope protein E attaches to the cell surface receptor. This study aimed to investigate the effect of silencing the CD-14 associated molecule and clathrin-mediated endocytosis using siRNA on dengue virus entry into monocytes. Methodology/Principal Findings: Gene expression analysis showed a significant down-regulation of the target genes (82.7%, 84.9 and 76.3% for CD-14 associated molecule, CLTC and DNM2 respectively) in transfected monocytes. The effect of silencing of target genes on dengue virus entry into monocytes was investigated by infecting silenced and non-silenced monocytes with DENV-2. Results showed a significant reduction of infected cells (85.2%), intracellular viral RNA load (73.0%), and extracellular viral RNA load (63.0%) in silenced monocytes as compared to non-silenced monocytes. Conclusions/Significance: Silencing the cell surface receptor and clathrin mediated endocytosis using RNA interference resulted in inhibition of the dengue virus entry and subsequently multiplication of the virus in the monocytes. This might serve as a novel promising therapeutic target to attenuate dengue infection and thus reduce transmission as well as progression to severe dengue hemorrhagic fever.

Abstract: Pseudomonas aeruginosa is considered as one of the leading causes of nosocomial infections. The start of antimicrobial therapy is often empirical and selective pressure on panel of antibiotics; therefore, it is important to know the susceptibility pattern of pathogens in order to select the most appropriate antibiotic. The aim of the current study is to update the rational empirical antimicrobial therapy recommendations. Antimicrobial resistance was done using the E-test method. Urine and wound swab samples were the highest encountered isolates; results were interpreted according to National Committee for Clinical Laboratory Standards guidelines. A total of 88 clinical isolates of P. aeruginosa were collected randomly from April 2009 to March 2010 from the University of Malaya Medical Center. P. aeruginosa isolated from various clinical samples has lost susceptibility and showed increasing resistance to Gentamicin with 94.3%, followed by (ciprofloxacin) 92%, (ceftazidime) 89.8%, (imipenem) 73.9%, Pipracilline/tazobactam 61.4%, (aztreonam) 52.3%, and (amikacin) 50% and only susceptible to colistin with 92%. In conclusion, most of the isolates showed high levels of resistance to examined antibiotics except colistin and this may indicate the importance of antibiotic susceptibility testing and optimal treatment by combination of drugs.

Abstract: Nosocomial infections are major clinical threats to hospitalised patients and represent an important source of morbidity and mortality. It is necessary to develop rapid detection assays of nosocomial pathogens for better prognosis and initiation of antimicrobial therapy in patients. In this study, we present the development of molecular methods for the detection of six common nosocomial pathogens including Escherichia coli, Staphylococcus aureus, Streptococcus pneumoniae, Klebsiella pneumoniae, Pseudomonas aeruginosa and Acinetobacter spp. Conventional multiplex PCR and SYBR Green based real time PCR assays were performed using genus and species specific primers. Blind testing with 300 clinical samples was also carried out. The two assays were found to be sensitive and specific. Eubacterial PCR assay exhibited positive results for 46 clinical isolates from which 43 samples were detected by real time PCR assay. The sensitivity of the assay is about 93.7% in blind test isolates. The PCR results were reconfirmed using the conventional culture method. This assay has the potential to be a rapid, accurate and highly sensitive molecular diagnostic tool for simultaneous detection of Escherichia coli, Staphylococcus aureus, Streptococcus pneumoniae, Klebsiella pneumoniae, Pseudomonas aeruginosa and Acinetobacter spp. This assay has the potential to detect nosocomial pathogens within 5 to 6 hours, helping to initiate infection control measures and appropriate treatment in paediatric and elderly (old aged) patients, pre-and post surgery patients and organ transplant patients and thus reduces their hospitalization duration.

Abstract: Early definitive diagnosis of dengue virus infection may help in the timely management of dengue virus infection. We evaluated the Standard Diagnostics (SD, South Korea) dengue virus nonstructural protein NS1 antigen enzyme-linked immunosorbent assay (SD dengue NS1 Ag ELISA) for the detection of dengue virus NS1 antigen in patients' sera, using a total of 399 serum samples in a comparison with real-time reverse transcription (RT)-PCR, an in-house IgM capture (MAC)-ELISA, and a hemagglutination inhibition (HI) assay. Of the 320 dengue sera, 205 (64%) tested positive for NS1 antigen compared to 300 (93.75%) by either MAC-ELISA or RT-PCR, 161 (50.31%) by RT-PCR, and 226 (70.36%) by MAC-ELISA only. The assay was able to detect NS1 antigen in convalescent-phase sera until day 14 of infection. The NS1 detection rate is inversely proportional while the IgM detection rate is directly proportional to the presence of IgG antibodies. The overall sensitivity and specificity of the SD dengue NS1 Ag ELISA in the detection of "confirmed dengue virus" sera are 76.76% and 98.31%, respectively. This suggests that the SD kit is highly specific and sensitive for the detection of NS1 antigen. However, caution is needed when the kit is used as a single assay, as detection in samples that contained the virus was only about 81.97%. Combining this assay with an IgM and/or IgG assay will increase the sensitivity of detection, especially in areas with a higher prevalence of secondary dengue virus infections.

Abstract: Early definitive diagnosis of dengue virus infection may help in the timely management of dengue virus infection. We evaluated the Standard Diagnostics (SD, South Korea) dengue virus nonstructural protein NS1 antigen enzyme-linked immunosorbent assay (SD dengue NS1 Ag ELISA) for the detection of dengue virus NS1 antigen in patients' sera, using a total of 399 serum samples in a comparison with real-time reverse transcription (RT)-PCR, an in-house IgM capture (MAC)-ELISA, and a hemagglutination inhibition (HI) assay. Of the 320 dengue sera, 205 (64%) tested positive for NS1 antigen compared to 300 (93.75%) by either MAC-ELISA or RT-PCR, 161 (50.31%) by RT-PCR, and 226 (70.36%) by MAC-ELISA only. The assay was able to detect NS1 antigen in convalescent-phase sera until day 14 of infection. The NS1 detection rate is inversely proportional while the IgM detection rate is directly proportional to the presence of IgG antibodies. The overall sensitivity and specificity of the SD dengue NS1 Ag ELISA in the detection of "confirmed dengue virus" sera are 76.76% and 98.31%, respectively. This suggests that the SD kit is highly specific and sensitive for the detection of NS1 antigen. However, caution is needed when the kit is used as a single assay, as detection in samples that contained the virus was only about 81.97%. Combining this assay with an IgM and/or IgG assay will increase the sensitivity of detection, especially in areas with a higher prevalence of secondary dengue virus infections.

Abstract: A commercial Dengue Duo rapid test kit was evaluated for early dengue diagnosis by detection of dengue virus NS1 antigen and immunoglobulin M (IgM)/IgG antibodies A total of 420 patient serum samples were subjected to real-time reverse transcription-polymerase chain reaction (RT-PCR). in-house IgM capture enzyme-linked immunosorbent assay (ELISA). hemagglutination inhibition assay. and the SD Dengue Duo rapid test Of the 320 dengue acute and convalescent sera. dengue infection was detected by either serology or RT-PCR in 300 samples (93 75%). as compared with 289 samples (90.31%) in the combined SD Duo NSI/IgM The NS1 detection rate is inversely proportional. whereas the IgM detection rate is directly proportional to the presence of IgG antibodies. The sensitivity and specificity in diagnosing acute dengue infection in the SD Duo NSI/IgM were 88 65% and 98 75%. respectively The assay is sensitive and highly specific Detection of both NS1 and IgM by SD Duo gave comparable detection rate by either serology or RT-PCR

Abstract: The human leukocyte antigen alleles have been implicated as probable genetic markers in predicting the susceptibility and/or protection to severe manifestations of dengue virus (DENV) infection. In this present study, we aimed to investigate for the first time, the genotype variants of HLA Class 1(-A and -B) of DENV infected patients against healthy individuals in Malaysia. Methodology/Principal Findings: This study was carried out with 92 dengue disease patients and 95 healthy controls from three different ethnic groups (Malay, Chinese and Indian) in Malaysia. All patients with clinical and laboratory confirmation of DENV infection were typed for the HLA-A and B loci, using polymerase chain reaction-sequence specific primer techniques. In our total population, a significant increase for HLA-B*53 (P=0.042, Pc=1.008) allele and a significant decrease for A*03 (P=0.015, Pc=0.18, OR=5.23, 95% CI=1.19-23.02) and B*18 (P=0.017, Pc=0.408) alleles were noted in DHF patients as compared to healthy donors. We also observed that in the Malay DHF patients, allele B*13 (P=0.049, Pc=1.176, OR=0.18, 95% CI=0.03-0.90) was present at a significantly higher frequency in this population while allele HLA-B*18 (P=0.024, Pc=0.576) was seen to be negatively associated with DHF. Conclusions/Significance: These are the first findings on genetic polymorphisms in our population and we conclude that: (1) In our total population, HLA-B*53 probably involve in disease susceptibility, while the HLA-A*03 and HLA-B*18 may confer protection from progression to severe disease; (2) In the Malay population, HLA-B*13 and B*18 are probably associated in disease susceptibility and protection, respectively. These results could furnish as a valuable predictive tool to identify ethnically different individuals at risk and/or protection from severe forms of DENV infection and would provide valuable informations for the design of future dengue vaccine.

Abstract: Background: Early diagnosis of dengue can assist patient triage and management and prevent unnecessary treatments and interventions. Commercially available assays that detect the dengue virus protein NS1 in the plasma/serum of patients offers the possibility of early and rapid diagnosis. Methodology/Principal Findings: The sensitivity and specificity of the Pan-E Dengue Early ELISA and the Platelia (TM) Dengue NS1 Ag assays were compared against a reference diagnosis in 1385 patients in 6 countries in Asia and the Americas. Platelia was more sensitive (66%) than Pan-E (52%) in confirmed dengue cases. Sensitivity varied by geographic region, with both assays generally being more sensitive in patients from SE Asia than the Americas. Both kits were more sensitive for specimens collected within the first few days of illness onset relative to later time points. Pan-E and Platelia were both 100% specific in febrile patients without evidence of acute dengue. In patients with other confirmed diagnoses and healthy blood donors, Platelia was more specific (100%) than Pan-E (90%). For Platelia, when either the NS1 test or the IgM test on the acute sample was positive, the sensitivity versus the reference result was 82% in samples collected in the first four days of fever. NS1 sensitivity was not associated to disease severity (DF or DHF) in the Platelia test, whereas a trend for higher sensitivity in DHF cases was seen in the Pan-E test (however combined with lower overall sensitivity). Conclusions/Significance: Collectively, this multi-country study suggests that the best performing NS1 assay (Platelia) had moderate sensitivity (median 64%, range 34-76%) and high specificity (100%) for the diagnosis of dengue. The poor sensitivity of the evaluated assays in some geographical regions suggests further assessments are needed. The combination of NS1 and IgM detection in samples collected in the first few days of fever increased the overall dengue diagnostic sensitivity.

Abstract: The accurate and rapid identification of bacteria in the enteric tract is necessary for early treatment. In this study, we describeD a novel system which consists of a multiplex polymerase chain reaction (PCR) to simultaneously identify a group of six Enterobacteriaceae members including Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Citrobacter spp., Enterobacter cloacae and Salmonella typhi. Genus and species specific primers were designed for this group of pathogens and conventional multiplex PCR and SYBR green based real time PCR assays were performed to detect these pathogens. All the samples were analysed with a eubacterial real-time PCR assay that enables detection of bacterial DNA and then detection of the organisms was determined using genus and species specific PCR assays. This assay was evaluated using clinical specimens and was found to be quite sensitive and specific. Their PCR results matched with the conventional culture identifications. The conventional and SYBR green real time multiplex PCR assays takes only 3 h to be performed and has the potential to replace the conventional culture technique and thus can speed up the treatment process. This technique has the potential to be a valuable diagnostic tool for simultaneous identification of E. coli, K. pneumoniae, P. mirabilis, Citrobacter spp., E. cloacae and S. typhi.

Abstract: This paper reports the development of a one-step SYBR-Green I-based real-time RT-PCR assay for the detection and quantification of Chikungunya virus (CHIKV) in human, monkey and mosquito samples by targeting the El structural gene. A preliminary evaluation of this assay has been successfully completed using 71 samples, consisting of a panel of negative control sera, sera from healthy individuals, sera from patients with acute disease from which CHIKV had been isolated, as well as monkey sera and adult mosquito samples obtained during the chikungunya fever outbreak in Malaysia in 2008. The assay was found to be 100-fold more sensitive than the conventional RT-PCR with a detection limit of 4.12x10(0) RNA copies/mu l. The specificity of the assay was tested against other related viruses such as Dengue (serotypes 1-4), Japanese encephalitis, Herpes Simplex, Parainfluenza, Sindbis, Ross River, Yellow fever and West Nile viruses. The sensitivity, specificity and efficiency of this assay were 100%, 100% and 96.8% respectively. This study on early diagnostics is of importance to all endemic countries, especially Malaysia, which has been facing increasingly frequent and bigger outbreaks due to this virus since 1999.

Abstract: ASSURE (R) Dengue IgA Rapid Test, an immunochromatographic anti-Dengue IgA Rapid Test based on reverse flow technology, was evaluated using archived sera The sera were obtained during hospital admission and discharge of 204 patients during 2000 to 2001 dengue outbreak in Bangladesh and 220 negative sera collected in 2009 Based on characterization by reference ELISA (nonstructural protein 1 [NS1] Ag ELISA, immunoglobulin M [IgM]-Cap ELISA, and immunoglobulin G [IgG]-Cap ELISA), 179 (87 7%) patients were positive for dengue infection, and the remaining 245 patients had nondengue febrile illness The performance of Dengue IgA Rapid Test was compared to reference ELISA Of 179 dengue-positive sera, 79 (44 1%) were positive by NS1 Ag ELISA, 174 (97 2%) were positive by IgM-Cap ELISA, and 142 (79 3%) were positive by IgG-Cap ELISA Among 142 IgG-positive cases, 121 (85 2%) patients had shown high level of IgG (PanBio units equivalent to hemagglutination inhibition (HI) titer >= 2560) during hospital admission, indicating secondary infections Dengue IgA Rapid Test demonstrated 994% (178 of 179) sensitivity in diagnosing dengue infection with the ability to detect 100% primary (58 of 58) and 99 2% (120 of 121) secondary infections, and the specificity was found 99 2%(2 of 245) The capability of Dengue IgA Rapid Test in detecting dengue infection in terms of day of Illness was comparable to reverse transcriptase polymerase chain reaction and was found better than in-house IgM ELISA Compared with in-house IgM ELISA, Dengue IgA Rapid Test also detected similar number of dengue virus (DENV) 1, DENV 2, and more DENV 3 and DENV 4 cases The overall performance thus suggested its usefulness as one of the dengue early diagnostic tools where diagnostic facility is limited Crown Copyright (C) 2010 Published by Elsevier Inc All rights reserved

Abstract: Background & objectives: Carbapenem-resistant Acinetobacter spp. have gained increasing significance as opportunistic pathogens in hospitalized patients. Carbapenem resistance is often associated with the loss and/or decrease in outer membrane proteins (OMP) and overexpression of multidrug efflux systems. However, carbapenem-hydrolysing beta-lactamases of Ambler Class B (metallo-enzymes) and Ambler Class D (oxacillinases) have also been detected in Acinetobacter spp. In this study we have investigated the role of the iron regulated outer membrane protein (IROMPs) and the loss of a 29-kDa OMP in carbapenem resistance of Acinetobacter calcoaceticus. Methods: Carbapenem resistant clinical isolates (n=39) of Acinetobacter baumannii/calcoaceticus were used. Identification of Acinetobacter spp. at species level was done by amplified ribosomal DNA restriction analysis (ARDRA). MIC was evaluated using agar dilution method according to CLSI standards. Presence of outer membrane proteins were determined by SDS-PAGE. A representative strain of A. calcoaceticus, S26 with the loss of 29-kDa OMP was selected for further analysis as strain S26 had unique resistance mechanism, that is, the presence of IMP-4 metallo-beta-lactamases. IROMPs were expressed under iron deficit conditions. Bands corresponding to IROMPs were excised from SDS-PAGE and used to immunize rabbits for the production of polyclonal antibodies. The antibodies raised against IROMPs were detected by an in-house ELISA and then used for bactericidal activity against carbapenem resistant A. baumannii/calcoaceticus. Results: All isolates were resistant to all antibiotics including imipenem and meropenem and had loss of a 29-kDa OMP. The polyclonal antibodies showed bactericidal effect against the organism tested and it specifically killed the bacteria grown in iron deficit medium. Interpretation & conclusions: In this study, a 29-kDa OMP has been identified to be the major outer membrane protein in A. baumannii/calcoaceticus and loss of this porin and overexpression of IROMPs have contributed to carbapenem resistance. Polyclonal antibodies raised against IROMPs may have a role in antimicrobial therapy in these isolates.

Abstract: Enterovirus 71 (EV71) causes childhood hand, foot, and mouth disease and neurological complications, and no vaccines or therapeutic drugs are currently available. Formaldehyde-inactivated whole-virus vaccines derived from EV71 clinical isolates and a mouse-adapted virus (MAV) were tested in a mouse model of EV71 encephalomyelitis. After only two immunizations, given to mice at 1 and 7 days of age, the MAV vaccine protected mice at 14 days of age from disease. Tissues from immunized mice were negative for virus by viral culture, reverse transcriptase PCR, immunohistochemistry analysis, and in situ hybridization. Cross-neutralizing EV71 antibodies to strains with genotypes B3, B4, and C1 to C5 generated in immunized adult mice were able to passively protect 14-day-old mice from disease.

Abstract: Background: Phyllanthus is a traditional medicinal plant that has been used in the treatment of many diseases including hepatitis and diabetes. The main aim of the present work was to investigate the potential cytotoxic effects of aqueous and methanolic extracts of four Phyllanthus species (P. amarus, P. niruri, P. urinaria and P. watsonii) against skin melanoma and prostate cancer cells. Methodology/Principal Findings: Phyllanthus plant appears to possess cytotoxic properties with half-maximal inhibitory concentration (IC(50)) values of 150-300 mu g/ml for aqueous extract and 50-150 mu g/ml for methanolic extract that were determined using the MTS reduction assay. In comparison, the plant extracts did not show any significant cytotoxicity on normal human skin (CCD-1127Sk) and prostate (RWPE-1) cells. The extracts appeared to act by causing the formation of a clear "ladder'' fragmentation of apoptotic DNA on agarose gel, displayed TUNEL-positive cells with an elevation of caspase-3 and -7 activities. The Lactate Dehydrogenase (LDH) level was lower than 15% in Phyllanthus treated-cancer cells. These indicate that Phyllanthus extracts have the ability to induce apoptosis with minimal necrotic effects. Furthermore, cell cycle analysis revealed that Phyllanthus induced a Go/G1-phase arrest on PC-3 cells and a S-phase arrest on MeWo cells and these were accompanied by accumulation of cells in the Sub-G1 (apoptosis) phase. The cytotoxic properties may be due to the presence of polyphenol compounds such as ellagitannins, gallotannins, flavonoids and phenolic acids found both in the water and methanol extract of the plants. Conclusions/Significance: Phyllanthus plant exerts its growth inhibition effect in a selective manner towards cancer cells through the modulation of cell cycle and induction of apoptosis via caspases activation in melanoma and prostate cancer cells. Hence, Phyllanthus may be sourced for the development of a potent apoptosis-inducing anticancer agent.

Abstract: The major outer membrane protein (OmpH) of 4 local Malaysian strains of Pasteurella multocida serotype B:2 were characterized in comparison to ATCC strains. Three major peptide bands of MW 26, 32 and 37 kDa were characterized using SDS-PAGE. Two of these fragments, the 32 kDa and 37 kDa were observed to be more reactive with a mouse polyclonal antiserum in all of the local isolates as well as the ATCC strains in a Western blot. However, the 32 kDa fragment was found to cross react with other Gram negative bacteria. Therefore, the 37 kDa OmpH was selected as vaccine candidate. The 37 kDa ompH gene of the isolated strain 1710 was cloned into an Escherichia coil expression vector to produce large amounts of recombinant OmpH (rOmpH). The 37 kDa ompH gene of strain 1710 was sequenced. In comparison to a reference strain X-73 of the ompH of R multocida, 39bp was found deleted in the 37 kDa ompH gene. However, the deletion did not shift the reading frame or change the amino acid sequence. The rOmpH was used in a mice protection study. Mice immunized and challenged intraperitoneally resulted 100% protection against P. multocida whilst mice immunized subcutaneously and challenged intraperitoneally only resulted 80% protection. The rOmpH is therefore a suitable candidate for vaccination field studies. The same rOmpH was also used to develop a potential diagnostic kit in an ELISA format.

Abstract: Purpose: The aim of this study is to evaluate the role of real-time polymerase chain reaction (PCR) and conventional bacterial culture methods in the detection of Pseudomonas aeruginosa in contact lens-induced severe, partially treated corneal ulcers referred to a tertiary center. Methods: The study duration was 6 months. All patients with contact lens-related corneal ulcer, requiring admission during the study period were recruited. Samples from corneal scrapings were simultaneously sent at the time of admission for PCR and culture testing. An in-house real-time PCR was developed to detect the P. aeruginosa lasA gene. The results of PCR and culture were compared using McNemar's chi(2) test. Results: Ten patients were recruited. The mean age was 33 years (20-45 years). All the patients had contact lens-related keratitis (> 4 mm) of which eight (80%) were found positive for P. aeruginosa by PCR or culture. There was no significant difference between PCR and culture in detecting P. aeruginosa (P < 0.05). Conclusions: PCR is, at least, as good as conventional cultures in detecting P. aeruginosa. It is a rapid assay as compared with culture, and early detection enables prompt treatment thus reducing the destructive effect of the organism on the cornea.

Abstract: Background & objectives: Carbapenem-resistant Acinetobacter spp. have gained increasing significance as opportunistic pathogens in hospitalized patients. Carbapenem resistance is often associated with the loss and/or decrease in outer membrane proteins (OMP) and overexpression of multidrug efflux systems. However, carbapenem-hydrolysing beta-lactamases of Ambler Class B (metallo-enzymes) and Ambler Class D (oxacillinases) have also been detected in Acinetobacter spp. In this study we have investigated the role of the iron regulated outer membrane protein (IROMPs) and the loss of a 29-kDa OMP in carbapenem resistance of Acinetobacter calcoaceticus. Methods: Carbapenem resistant clinical isolates (n=39) of Acinetobacter baumannii/calcoaceticus were used. Identification of Acinetobacter spp. at species level was done by amplified ribosomal DNA restriction analysis (ARDRA). MIC was evaluated using agar dilution method according to CLSI standards. Presence of outer membrane proteins were determined by SDS-PAGE. A representative strain of A. calcoaceticus, S26 with the loss of 29-kDa OMP was selected for further analysis as strain S26 had unique resistance mechanism, that is, the presence of IMP-4 metallo-beta-lactamases. IROMPs were expressed under iron deficit conditions. Bands corresponding to IROMPs were excised from SDS-PAGE and used to immunize rabbits for the production of polyclonal antibodies. The antibodies raised against IROMPs were detected by an in-house ELISA and then used for bactericidal activity against carbapenem resistant A. baumannii/calcoaceticus. Results: All isolates were resistant to all antibiotics including imipenem and meropenem and had loss of a 29-kDa OMP. The polyclonal antibodies showed bactericidal effect against the organism tested and it specifically killed the bacteria grown in iron deficit medium. Interpretation & conclusions: In this study, a 29-kDa OMP has been identified to be the major outer membrane protein in A. baumannii/calcoaceticus and loss of this porin and overexpression of IROMPs have contributed to carbapenem resistance. Polyclonal antibodies raised against IROMPs may have a role in antimicrobial therapy in these isolates.

Abstract: A commercial Dengue Duo rapid test kit was evaluated for early dengue diagnosis by detection of dengue virus NS1 antigen and immunoglobulin M (IgM)/IgG antibodies A total of 420 patient serum samples were subjected to real-time reverse transcription-polymerase chain reaction (RT-PCR). in-house IgM capture enzyme-linked immunosorbent assay (ELISA). hemagglutination inhibition assay. and the SD Dengue Duo rapid test Of the 320 dengue acute and convalescent sera. dengue infection was detected by either serology or RT-PCR in 300 samples (93 75%). as compared with 289 samples (90.31%) in the combined SD Duo NSI/IgM The NS1 detection rate is inversely proportional. whereas the IgM detection rate is directly proportional to the presence of IgG antibodies. The sensitivity and specificity in diagnosing acute dengue infection in the SD Duo NSI/IgM were 88 65% and 98 75%. respectively The assay is sensitive and highly specific Detection of both NS1 and IgM by SD Duo gave comparable detection rate by either serology or RT-PCR

Abstract: Dengue is an arthropod-borne flavivirus that comprises four distinct serotypes (DEN-1, DEN-2, DEN-3 and DEN-4) that constitute an antigenic complex of the genus flavivirus, family Flaviviridae. Infection by one serotype induces life-long immunity against reinfection by the same serotype, but only transient and partial protection against infection with the other serotypes

Abstract: This paper reports the development of a one-step SYBR-Green I-based real-time RT-PCR assay for the detection and quantification of Chikungunya virus (CHIKV) in human, monkey and mosquito samples by targeting the El structural gene. A preliminary evaluation of this assay has been successfully completed using 71 samples, consisting of a panel of negative control sera, sera from healthy individuals, sera from patients with acute disease from which CHIKV had been isolated, as well as monkey sera and adult mosquito samples obtained during the chikungunya fever outbreak in Malaysia in 2008. The assay was found to be 100-fold more sensitive than the conventional RT-PCR with a detection limit of 4.12x10(0) RNA copies/mu l. The specificity of the assay was tested against other related viruses such as Dengue (serotypes 1-4), Japanese encephalitis, Herpes Simplex, Parainfluenza, Sindbis, Ross River, Yellow fever and West Nile viruses. The sensitivity, specificity and efficiency of this assay were 100%, 100% and 96.8% respectively. This study on early diagnostics is of importance to all endemic countries, especially Malaysia, which has been facing increasingly frequent and bigger outbreaks due to this virus since 1999.

Abstract: The accurate and rapid identification of bacteria in the enteric tract is necessary for early treatment. In this study, we describeD a novel system which consists of a multiplex polymerase chain reaction (PCR) to simultaneously identify a group of six Enterobacteriaceae members including Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Citrobacter spp., Enterobacter cloacae and Salmonella typhi. Genus and species specific primers were designed for this group of pathogens and conventional multiplex PCR and SYBR green based real time PCR assays were performed to detect these pathogens. All the samples were analysed with a eubacterial real-time PCR assay that enables detection of bacterial DNA and then detection of the organisms was determined using genus and species specific PCR assays. This assay was evaluated using clinical specimens and was found to be quite sensitive and specific. Their PCR results matched with the conventional culture identifications. The conventional and SYBR green real time multiplex PCR assays takes only 3 h to be performed and has the potential to replace the conventional culture technique and thus can speed up the treatment process. This technique has the potential to be a valuable diagnostic tool for simultaneous identification of E. coli, K. pneumoniae, P. mirabilis, Citrobacter spp., E. cloacae and S. typhi.

Abstract: The human leukocyte antigen alleles have been implicated as probable genetic markers in predicting the susceptibility and/or protection to severe manifestations of dengue virus (DENV) infection. In this present study, we aimed to investigate for the first time, the genotype variants of HLA Class 1(-A and -B) of DENV infected patients against healthy individuals in Malaysia. Methodology/Principal Findings: This study was carried out with 92 dengue disease patients and 95 healthy controls from three different ethnic groups (Malay, Chinese and Indian) in Malaysia. All patients with clinical and laboratory confirmation of DENV infection were typed for the HLA-A and B loci, using polymerase chain reaction-sequence specific primer techniques. In our total population, a significant increase for HLA-B*53 (P=0.042, Pc=1.008) allele and a significant decrease for A*03 (P=0.015, Pc=0.18, OR=5.23, 95% CI=1.19-23.02) and B*18 (P=0.017, Pc=0.408) alleles were noted in DHF patients as compared to healthy donors. We also observed that in the Malay DHF patients, allele B*13 (P=0.049, Pc=1.176, OR=0.18, 95% CI=0.03-0.90) was present at a significantly higher frequency in this population while allele HLA-B*18 (P=0.024, Pc=0.576) was seen to be negatively associated with DHF. Conclusions/Significance: These are the first findings on genetic polymorphisms in our population and we conclude that: (1) In our total population, HLA-B*53 probably involve in disease susceptibility, while the HLA-A*03 and HLA-B*18 may confer protection from progression to severe disease; (2) In the Malay population, HLA-B*13 and B*18 are probably associated in disease susceptibility and protection, respectively. These results could furnish as a valuable predictive tool to identify ethnically different individuals at risk and/or protection from severe forms of DENV infection and would provide valuable informations for the design of future dengue vaccine.

Abstract: Background: Early diagnosis of dengue can assist patient triage and management and prevent unnecessary treatments and interventions. Commercially available assays that detect the dengue virus protein NS1 in the plasma/serum of patients offers the possibility of early and rapid diagnosis. Methodology/Principal Findings: The sensitivity and specificity of the Pan-E Dengue Early ELISA and the Platelia (TM) Dengue NS1 Ag assays were compared against a reference diagnosis in 1385 patients in 6 countries in Asia and the Americas. Platelia was more sensitive (66%) than Pan-E (52%) in confirmed dengue cases. Sensitivity varied by geographic region, with both assays generally being more sensitive in patients from SE Asia than the Americas. Both kits were more sensitive for specimens collected within the first few days of illness onset relative to later time points. Pan-E and Platelia were both 100% specific in febrile patients without evidence of acute dengue. In patients with other confirmed diagnoses and healthy blood donors, Platelia was more specific (100%) than Pan-E (90%). For Platelia, when either the NS1 test or the IgM test on the acute sample was positive, the sensitivity versus the reference result was 82% in samples collected in the first four days of fever. NS1 sensitivity was not associated to disease severity (DF or DHF) in the Platelia test, whereas a trend for higher sensitivity in DHF cases was seen in the Pan-E test (however combined with lower overall sensitivity). Conclusions/Significance: Collectively, this multi-country study suggests that the best performing NS1 assay (Platelia) had moderate sensitivity (median 64%, range 34-76%) and high specificity (100%) for the diagnosis of dengue. The poor sensitivity of the evaluated assays in some geographical regions suggests further assessments are needed. The combination of NS1 and IgM detection in samples collected in the first few days of fever increased the overall dengue diagnostic sensitivity.

Abstract: Background: Phyllanthus is a traditional medicinal plant that has been used in the treatment of many diseases including hepatitis and diabetes. The main aim of the present work was to investigate the potential cytotoxic effects of aqueous and methanolic extracts of four Phyllanthus species (P. amarus, P. niruri, P. urinaria and P. watsonii) against skin melanoma and prostate cancer cells. Methodology/Principal Findings: Phyllanthus plant appears to possess cytotoxic properties with half-maximal inhibitory concentration (IC(50)) values of 150-300 mu g/ml for aqueous extract and 50-150 mu g/ml for methanolic extract that were determined using the MTS reduction assay. In comparison, the plant extracts did not show any significant cytotoxicity on normal human skin (CCD-1127Sk) and prostate (RWPE-1) cells. The extracts appeared to act by causing the formation of a clear "ladder'' fragmentation of apoptotic DNA on agarose gel, displayed TUNEL-positive cells with an elevation of caspase-3 and -7 activities. The Lactate Dehydrogenase (LDH) level was lower than 15% in Phyllanthus treated-cancer cells. These indicate that Phyllanthus extracts have the ability to induce apoptosis with minimal necrotic effects. Furthermore, cell cycle analysis revealed that Phyllanthus induced a Go/G1-phase arrest on PC-3 cells and a S-phase arrest on MeWo cells and these were accompanied by accumulation of cells in the Sub-G1 (apoptosis) phase. The cytotoxic properties may be due to the presence of polyphenol compounds such as ellagitannins, gallotannins, flavonoids and phenolic acids found both in the water and methanol extract of the plants. Conclusions/Significance: Phyllanthus plant exerts its growth inhibition effect in a selective manner towards cancer cells through the modulation of cell cycle and induction of apoptosis via caspases activation in melanoma and prostate cancer cells. Hence, Phyllanthus may be sourced for the development of a potent apoptosis-inducing anticancer agent.

Abstract: ASSURE (R) Dengue IgA Rapid Test, an immunochromatographic anti-Dengue IgA Rapid Test based on reverse flow technology, was evaluated using archived sera The sera were obtained during hospital admission and discharge of 204 patients during 2000 to 2001 dengue outbreak in Bangladesh and 220 negative sera collected in 2009 Based on characterization by reference ELISA (nonstructural protein 1 [NS1] Ag ELISA, immunoglobulin M [IgM]-Cap ELISA, and immunoglobulin G [IgG]-Cap ELISA), 179 (87 7%) patients were positive for dengue infection, and the remaining 245 patients had nondengue febrile illness The performance of Dengue IgA Rapid Test was compared to reference ELISA Of 179 dengue-positive sera, 79 (44 1%) were positive by NS1 Ag ELISA, 174 (97 2%) were positive by IgM-Cap ELISA, and 142 (79 3%) were positive by IgG-Cap ELISA Among 142 IgG-positive cases, 121 (85 2%) patients had shown high level of IgG (PanBio units equivalent to hemagglutination inhibition (HI) titer >= 2560) during hospital admission, indicating secondary infections Dengue IgA Rapid Test demonstrated 994% (178 of 179) sensitivity in diagnosing dengue infection with the ability to detect 100% primary (58 of 58) and 99 2% (120 of 121) secondary infections, and the specificity was found 99 2%(2 of 245) The capability of Dengue IgA Rapid Test in detecting dengue infection in terms of day of Illness was comparable to reverse transcriptase polymerase chain reaction and was found better than in-house IgM ELISA Compared with in-house IgM ELISA, Dengue IgA Rapid Test also detected similar number of dengue virus (DENV) 1, DENV 2, and more DENV 3 and DENV 4 cases The overall performance thus suggested its usefulness as one of the dengue early diagnostic tools where diagnostic facility is limited Crown Copyright (C) 2010 Published by Elsevier Inc All rights reserved

Abstract: The major outer membrane protein (OmpH) of 4 local Malaysian strains of Pasteurella multocida serotype B:2 were characterized in comparison to ATCC strains. Three major peptide bands of MW 26, 32 and 37 kDa were characterized using SDS-PAGE. Two of these fragments, the 32 kDa and 37 kDa were observed to be more reactive with a mouse polyclonal antiserum in all of the local isolates as well as the ATCC strains in a Western blot. However, the 32 kDa fragment was found to cross react with other Gram negative bacteria. Therefore, the 37 kDa OmpH was selected as vaccine candidate. The 37 kDa ompH gene of the isolated strain 1710 was cloned into an Escherichia coil expression vector to produce large amounts of recombinant OmpH (rOmpH). The 37 kDa ompH gene of strain 1710 was sequenced. In comparison to a reference strain X-73 of the ompH of R multocida, 39bp was found deleted in the 37 kDa ompH gene. However, the deletion did not shift the reading frame or change the amino acid sequence. The rOmpH was used in a mice protection study. Mice immunized and challenged intraperitoneally resulted 100% protection against P. multocida whilst mice immunized subcutaneously and challenged intraperitoneally only resulted 80% protection. The rOmpH is therefore a suitable candidate for vaccination field studies. The same rOmpH was also used to develop a potential diagnostic kit in an ELISA format.

Abstract: Purpose: The aim of this study is to evaluate the role of real-time polymerase chain reaction (PCR) and conventional bacterial culture methods in the detection of Pseudomonas aeruginosa in contact lens-induced severe, partially treated corneal ulcers referred to a tertiary center. Methods: The study duration was 6 months. All patients with contact lens-related corneal ulcer, requiring admission during the study period were recruited. Samples from corneal scrapings were simultaneously sent at the time of admission for PCR and culture testing. An in-house real-time PCR was developed to detect the P. aeruginosa lasA gene. The results of PCR and culture were compared using McNemar's chi(2) test. Results: Ten patients were recruited. The mean age was 33 years (20-45 years). All the patients had contact lens-related keratitis (> 4 mm) of which eight (80%) were found positive for P. aeruginosa by PCR or culture. There was no significant difference between PCR and culture in detecting P. aeruginosa (P < 0.05). Conclusions: PCR is, at least, as good as conventional cultures in detecting P. aeruginosa. It is a rapid assay as compared with culture, and early detection enables prompt treatment thus reducing the destructive effect of the organism on the cornea.

Abstract: Dengue is an arthropod-borne flavivirus that comprises four distinct serotypes (DEN-1, DEN-2, DEN-3 and DEN-4) that constitute an antigenic complex of the genus flavivirus, family Flaviviridae. Infection by one serotype induces life-long immunity against reinfection by the same serotype, but only transient and partial protection against infection with the other serotypes

Abstract: Enterovirus 71 (EV71) causes childhood hand, foot, and mouth disease and neurological complications, and no vaccines or therapeutic drugs are currently available. Formaldehyde-inactivated whole-virus vaccines derived from EV71 clinical isolates and a mouse-adapted virus (MAV) were tested in a mouse model of EV71 encephalomyelitis. After only two immunizations, given to mice at 1 and 7 days of age, the MAV vaccine protected mice at 14 days of age from disease. Tissues from immunized mice were negative for virus by viral culture, reverse transcriptase PCR, immunohistochemistry analysis, and in situ hybridization. Cross-neutralizing EV71 antibodies to strains with genotypes B3, B4, and C1 to C5 generated in immunized adult mice were able to passively protect 14-day-old mice from disease.

Abstract: Introduction : The AdeABC pump of Acinetobacter spp. confers resistance to various antibiotic classes. This pump is composed of the AdeA, AdeB, and AdeC proteins where AdeB is a member of the resistance-nodulation-division efflux pump superfamily. The adeA, adeB, and adeC genes are contiguous and adjacent to adeS and adeR, which are transcribed in the opposite direction and which specify proteins homologous to sensors and regulators of two-component systems, respectively. In this study, an attempt is made to elucidate the role of the AdeABC efflux pump in carbapenem resistance in Acinetobacter spp. Methods : 39 carbapenem-resistant clinical isolates of Acinetobacter spp. were used. Minimum inhibitory concentrations were evaluated using the agar dilution method according to Clinical and Laboratory Standards Institute standards. The presence of carbapenem hydrolysing oxacillinases and AdeABC efflux pump genes were determined by PCR amplification. Subsequently, each gene was inactivated by plasmid insertion in order to study the contribution of these genes in developing antibiotic resistance and the resulting mutants were tested for their antimicrobial susceptibilities. Results : Among the multidrug-resistant strains, 36 strains had all the three (A, B, C) genes detected, while the remaining three strains had one or two of the genes detected. Inactivation of these individual genes showed decreased antimicrobial susceptibility indicating its contribution towards the development of antimicrobial resistance. Conclusion: The presence of AdeABC multidrug efflux pump plays a major role in the development of antimicrobial resistance in Acinetobacter spp. The presence of either one or an interplay between these genes may have an effect on antimicrobial resistance in Acinetobacter spp.

Abstract: We have evaluated a commercial enzyme immunoassay for the rapid detection of dengue NS1 antigen in human sera. The PLATELIA (TM) Dengue antigen assay was compared with the in-house IgM and TaqMan real-time RT-PCR using a panel of sera from primary acute and convalescent dengue infections, secondary acute and convalescent dengue infections, IgM-positive samples, tissue culture supernatant and other flaviviral infections. Of the 93 acute serum samples 82 were positive for NS1 antigen using the PLATELIA (TM) Dengue antigen assay. Overall, the NS1 detection rate was much higher in the acute primary dengue (100%) than in the acute secondary dengue (53.3%) serum samples. Both the PLATELIA (TM) Dengue antigen assay and the TaqMan real-time RT-PCR assay were highly specific (100%). The overall sensitivity of the PLATELIA (TM) Dengue antigen assay was 93.9% and 55% in the absence and presence of IgM, respectively. The results indicate that the PLATELIA (TM) Dengue antigen assay is a specific and sensitive assay for the detection of dengue virus infections during the primary acute phase when IgM is not detectable.

Abstract: Anti-dengue virus immunoglobulin M kits were evaluated. Test sensitivities were 21%-99% and specificities were 77%-98% compared with reference ELISAs. False-positive results were found for patients with malaria or past dengue infections. Three ELISAs showing strong agreement with reference ELISAs will be included in the World Health Organization Bulk Procurement Scheme.

Abstract: The full-length genomes of two DENV-1 viruses isolated during the 2005-2006 dengue incidents in Brunei were sequenced. Twenty five primer sets were designed to amplify contiguous overlapping fragments of approximately 500-600 base pairs spanning the entire sequence of the genome. The amplified PCR products were sent to a commercial laboratory for sequencing and the nucleotides and the deduced amino acids were determined. Sequence analysis of the envelope gene at the nucleotide and amino acid levels between the two isolates showed 92 and 96% identity, respectively. Comparison of the envelope gene sequences with 68 other DENV-1 viruses of known genotypes placed the two isolates into two different genotypic groups. Isolate DS06/210505 belongs to genotype V together with some of the recent isolates from India (2003) and older isolates from Singapore (11990) and Burma (11976), while isolate DS212/110306 was clustered in genotype IV with the prototype Nauru strain (11974) and with some of the recent isolates from Indonesia (2004) and the Philippines (2002, 2001). In the full-length genome analysis at the nucleotide level, isolate DS06/210505 showed 94% identity to the French Guyana strain (11989) in genotype V while isolate DS212/110306 had 96% identity to the Nauru Island strain (11974) in genotype IV. This work constitutes the first complete genetic characterization of not only Brunei DENV-1 virus isolates, but also the first strain from Borneo Island. This study was the first to report the isolation of dengue virus in the country.

Abstract: Introduction : The AdeABC pump of Acinetobacter spp. confers resistance to various antibiotic classes. This pump is composed of the AdeA, AdeB, and AdeC proteins where AdeB is a member of the resistance-nodulation-division efflux pump superfamily. The adeA, adeB, and adeC genes are contiguous and adjacent to adeS and adeR, which are transcribed in the opposite direction and which specify proteins homologous to sensors and regulators of two-component systems, respectively. In this study, an attempt is made to elucidate the role of the AdeABC efflux pump in carbapenem resistance in Acinetobacter spp. Methods : 39 carbapenem-resistant clinical isolates of Acinetobacter spp. were used. Minimum inhibitory concentrations were evaluated using the agar dilution method according to Clinical and Laboratory Standards Institute standards. The presence of carbapenem hydrolysing oxacillinases and AdeABC efflux pump genes were determined by PCR amplification. Subsequently, each gene was inactivated by plasmid insertion in order to study the contribution of these genes in developing antibiotic resistance and the resulting mutants were tested for their antimicrobial susceptibilities. Results : Among the multidrug-resistant strains, 36 strains had all the three (A, B, C) genes detected, while the remaining three strains had one or two of the genes detected. Inactivation of these individual genes showed decreased antimicrobial susceptibility indicating its contribution towards the development of antimicrobial resistance. Conclusion: The presence of AdeABC multidrug efflux pump plays a major role in the development of antimicrobial resistance in Acinetobacter spp. The presence of either one or an interplay between these genes may have an effect on antimicrobial resistance in Acinetobacter spp.

Abstract: We determined the differential expression levels of proteins in peripheral blood mononuclear cells of patients with dengue fever (DF) and dengue hemorrhagic fever (DHF). Proteins were subjected to two-dimensional electrophoresis, mass spectrometry and Western blot analysis. We identified 8 proteins that were 2-fold or more LIP-regulated in patients compared to healthy control, three of which, aldolase, thioredoxin peroxidase and alpha tubulin, were related to dengue infection. Both thioredoxin peroxidase and alpha tubulin were over-expressed 4.9 and 3.3 times respectively in DHF compared to DF patients while aldolase was up-regulated 2.2 times in DF compared to DHF patients. Alpha tubulin and thioredoxin peroxidase have the potential to be utilized as biomarkers for DHF.

Abstract: Dengue infection is a major public health problem affecting millions of people living in tropical countries. With no suitable vaccines and specific antiviral drugs, treatment for dengue is usually symptomatic and supportive. Early diagnosis and recognition of severe disease is therefore crucial for better management of the patient. Two-dimension electrophoresis was used to identify disease-associated proteins that can be used for diagnosis and as drug targets for treatment. Two markers, identified by mass spectrometry analysis as alpha 1-antitrypsin and NS1 proteins were found to be upregulated in dengue fever (DF, n=10) and dengue haemorrhagic fever (DHF; n=10) patients compared with healthy individuals (n=8). Both alpha 1-antitrypsin and NS1 proteins were overexpressed two-foid in DHF patients compared with DF patients. Our study suggests that alpha 1-antitrypsin and NS1 protein could be used as biomarkers as early indicators of DHF risk among patients with suspected dengue infection. (C) 2008 Royal Society of Tropical Medicine and Hygiene. Published by Elsevier Ltd. All rights reserved.

Abstract: Background and purpose: To detect and characterize class 1 integrons among carbapenem-resistant strains of Acinetobacter spp. at University Malaya Medical Centre (UMMC), Kuala Lumpur, Malaysia. Methods: Thirty nine carbapen ern-resistant Acinetobacter strains were obtained from UMMC from August 2003 to March 2004 and analyzed for the presence of b/a(IMP) genes and class 1 integrons. Results: Class 1 integrons were detected in 31 of 39 strains. Two Acinetobacter calcoaceticus strains harbored an integron-borne b/a(IMP-4) metallo-beta-lactamase, 1 of which was located on a 36-kb plasmid. Two different amplified products were found in the 31 isolates with 3 restriction pattern profiles 1, 2, and 3. Correlation was observed between carriage of class 1 integrons and genomic relatedness among these isolates, indicating that particular mechanisms of carbapenem resistance could have been acquired by genotypically distinct clinical isolates of Acinetobacter spp. Conclusion: Although class 1 integrons are widely disseminated among clinical isolates of Acinetobacter spp. they do not play a major role in the spread of carbapenem resistance.

Abstract: Anti-dengue virus immunoglobulin M kits were evaluated. Test sensitivities were 21%-99% and specificities were 77%-98% compared with reference ELISAs. False-positive results were found for patients with malaria or past dengue infections. Three ELISAs showing strong agreement with reference ELISAs will be included in the World Health Organization Bulk Procurement Scheme.

Abstract: Dengue infection is a major public health problem affecting millions of people living in tropical countries. With no suitable vaccines and specific antiviral drugs, treatment for dengue is usually symptomatic and supportive. Early diagnosis and recognition of severe disease is therefore crucial for better management of the patient. Two-dimension electrophoresis was used to identify disease-associated proteins that can be used for diagnosis and as drug targets for treatment. Two markers, identified by mass spectrometry analysis as alpha 1-antitrypsin and NS1 proteins were found to be upregulated in dengue fever (DF, n=10) and dengue haemorrhagic fever (DHF; n=10) patients compared with healthy individuals (n=8). Both alpha 1-antitrypsin and NS1 proteins were overexpressed two-foid in DHF patients compared with DF patients. Our study suggests that alpha 1-antitrypsin and NS1 protein could be used as biomarkers as early indicators of DHF risk among patients with suspected dengue infection. (C) 2008 Royal Society of Tropical Medicine and Hygiene. Published by Elsevier Ltd. All rights reserved.

Abstract: The full-length genomes of two DENV-1 viruses isolated during the 2005-2006 dengue incidents in Brunei were sequenced. Twenty five primer sets were designed to amplify contiguous overlapping fragments of approximately 500-600 base pairs spanning the entire sequence of the genome. The amplified PCR products were sent to a commercial laboratory for sequencing and the nucleotides and the deduced amino acids were determined. Sequence analysis of the envelope gene at the nucleotide and amino acid levels between the two isolates showed 92 and 96% identity, respectively. Comparison of the envelope gene sequences with 68 other DENV-1 viruses of known genotypes placed the two isolates into two different genotypic groups. Isolate DS06/210505 belongs to genotype V together with some of the recent isolates from India (2003) and older isolates from Singapore (11990) and Burma (11976), while isolate DS212/110306 was clustered in genotype IV with the prototype Nauru strain (11974) and with some of the recent isolates from Indonesia (2004) and the Philippines (2002, 2001). In the full-length genome analysis at the nucleotide level, isolate DS06/210505 showed 94% identity to the French Guyana strain (11989) in genotype V while isolate DS212/110306 had 96% identity to the Nauru Island strain (11974) in genotype IV. This work constitutes the first complete genetic characterization of not only Brunei DENV-1 virus isolates, but also the first strain from Borneo Island. This study was the first to report the isolation of dengue virus in the country.

Abstract: Background and purpose: To detect and characterize class 1 integrons among carbapenem-resistant strains of Acinetobacter spp. at University Malaya Medical Centre (UMMC), Kuala Lumpur, Malaysia. Methods: Thirty nine carbapen ern-resistant Acinetobacter strains were obtained from UMMC from August 2003 to March 2004 and analyzed for the presence of b/a(IMP) genes and class 1 integrons. Results: Class 1 integrons were detected in 31 of 39 strains. Two Acinetobacter calcoaceticus strains harbored an integron-borne b/a(IMP-4) metallo-beta-lactamase, 1 of which was located on a 36-kb plasmid. Two different amplified products were found in the 31 isolates with 3 restriction pattern profiles 1, 2, and 3. Correlation was observed between carriage of class 1 integrons and genomic relatedness among these isolates, indicating that particular mechanisms of carbapenem resistance could have been acquired by genotypically distinct clinical isolates of Acinetobacter spp. Conclusion: Although class 1 integrons are widely disseminated among clinical isolates of Acinetobacter spp. they do not play a major role in the spread of carbapenem resistance.

Abstract: We determined the differential expression levels of proteins in peripheral blood mononuclear cells of patients with dengue fever (DF) and dengue hemorrhagic fever (DHF). Proteins were subjected to two-dimensional electrophoresis, mass spectrometry and Western blot analysis. We identified 8 proteins that were 2-fold or more LIP-regulated in patients compared to healthy control, three of which, aldolase, thioredoxin peroxidase and alpha tubulin, were related to dengue infection. Both thioredoxin peroxidase and alpha tubulin were over-expressed 4.9 and 3.3 times respectively in DHF compared to DF patients while aldolase was up-regulated 2.2 times in DF compared to DHF patients. Alpha tubulin and thioredoxin peroxidase have the potential to be utilized as biomarkers for DHF.

Abstract: We have evaluated a commercial enzyme immunoassay for the rapid detection of dengue NS1 antigen in human sera. The PLATELIA (TM) Dengue antigen assay was compared with the in-house IgM and TaqMan real-time RT-PCR using a panel of sera from primary acute and convalescent dengue infections, secondary acute and convalescent dengue infections, IgM-positive samples, tissue culture supernatant and other flaviviral infections. Of the 93 acute serum samples 82 were positive for NS1 antigen using the PLATELIA (TM) Dengue antigen assay. Overall, the NS1 detection rate was much higher in the acute primary dengue (100%) than in the acute secondary dengue (53.3%) serum samples. Both the PLATELIA (TM) Dengue antigen assay and the TaqMan real-time RT-PCR assay were highly specific (100%). The overall sensitivity of the PLATELIA (TM) Dengue antigen assay was 93.9% and 55% in the absence and presence of IgM, respectively. The results indicate that the PLATELIA (TM) Dengue antigen assay is a specific and sensitive assay for the detection of dengue virus infections during the primary acute phase when IgM is not detectable.

Abstract: In a previous study, we have reported the detection and isolation of dengue virus in Brunei (Osman, O., Fong, M.Y., Devi, S., 2007. A preliminary study of dengue infection in Brunei. JJID 60 (4), 205-208). DEN-2 was the predominant serotype followed by DEN-1. The full genomic sequences of 3 DEN-2 viruses isolated during the 2005-2006 dengue incident in Brunei were determined. Twenty-five primer sets were designed to amplify contiguous overlapping fragments of approximately 500-600 base pairs spanning the entire sequence of the viral genome. The amplified PCR products were sent for sequencing and their nucleoticles and the deduced amino acids were determined. All three DEN-2 virus isolated were clustered in the Cosmopolitan genotype of the DEN-2 classification by Twiddy et al. This work constitutes the first complete genetic characterization of three Brunei DEN-2 virus strains. (c) 2008 Elsevier B.V. All rights reserved.

Abstract: OBJECTIVE: To estimate prevalence rate of recent dengue infection in parturients, as well as the vertical transmission rate, and to compare pregnancy outcomes among infected women. METHOD: A prospective cohort study was performed. Maternal and paired umbilical cord bloods were taken at delivery. A brief questionnaire on febrile illness and dengue in pregnancy was completed by participants. The samples were sent to a World Health Organization Collaborating Center for Arbovirus Reference and Research. Maternal sera were tested with a dengue-specific immunoglobulin M (IgM) capture test. The paired umbilical cord serum was tested to determine when the maternal sample was positive. Dengue reverse transcriptase polymerase chain reaction (PCR) was performed on all dengue IgM-positive sera. Pregnancy outcome was extracted from delivery records. RESULTS: A total of 2,958 parturients were enrolled, and 2,531 paired maternal-umbilical cord blood samples were available for testing. Dengue-specific IgM was positive in 63 of 2,531 (2.5%, 95% confidence interval [CI] 1.9-3.2%) maternal samples. Only 1 of 64 (1.6%, 95% CI 0.0-9.5%) of the paired umbilical cord sera was IgM-positive. All reverse transcriptase PCR tests were negative. Fifty-six of 63 (88.9%) of dengue IgM-positive women did not report a febrile illness in pregnancy. Apart from a higher mean age in dengue IgM-positive women of 30.6 +/- 5.2 compared with 29.2 +/- 4.9 years (P=.025) compared with dengue IgM-negative women, all other characteristics were similar. Rates of preterm birth, mode of delivery, postpartum hemorrhage, low birth weight, and neonatal outcomes were not different. CONCLUSION: Recent dengue infection was demonstrated in 2.5% of parturients, with a vertical transmission rate of 1.6%. Pregnancy outcome of recently infected women was not different.

Abstract: Background: Heat shock proteins (HSPs) are known to be involved in the pathogenesis of Salmonella enterica serovar Typhi (S. Typhi), the causative agent of typhoid fever. The objective of this study was to apply a phage display library to identify mimotopes of two HSPs, HSP90 and DnaK in S. Typhi. Methodology: A 12-mer random peptide library expressed on the surface of the filamentous phage, M13, was used to select the mimotopes of two S. Typhi heat shock proteins by biopanning with monoclonal antibodies (mAbs), DnaK and HSP90. The immunogenicity of the selected peptides was determined through binding affinity with polyclonal antibodies from pooled typhoid-confirmed patients' sera and purified HSPs mAb using Western blotting and ELISA. Results: Five rounds of biopanning resulted in enrichment of phage clones expressing the binding motifs TDxSTRP and FPSHYWLYPPPT, respectively. The selected peptides showed strong immunoreactivity with patients' sera. Thus, monoclonal antibodies against HSP and patient sera can select common mimotopes from the random peptide library. Conclusion: These findings may provide fundamental information for further studies on diagnostic application or vaccine design against this aetiologic agent of typhoid fever.

Abstract: Difficulties in the management of dengue infections include the lack of rapid diagnostic methods and symptoms of dengue are often confused with those of other diseases. Five commercial kits were evaluated for the detection of dengue-specific IgM and IgG antibodies using sera obtained from patients with primary and secondary dengue infections, as well as other febrile illnesses. All kits were compared to the in-house IgM ELISA and HI assays. The rapid test kits took either 30 to 45 min (PanBio Dengue Duo Cassette, Accusens Dengue Virus Rapid Strip Test, and Unitest Dengue IgM and IgG Combo Rapid Test) or between 2 to 4 h (PanBio Dengue Duo Capture ELISA, and Antivirus IgM Detecion Kit 96 [Pentax Corporation]). Most kits were able to detect IgM in more than 90% of the secondary convalescent sera, while IgG detection was generally high (80 to 100%). All five kits showed high specificity when tested against sera from other febrile patients, and have been shown to be extremely useful in the diagnosis of dengue infections.

Abstract: Pneumococcal virulence determinants have been extensively studied but molecular evidence on virulence gene expression pattern is still lacking. We undertook this study to analyze the regulation pattern of adherence-associated genes; psaA, pspC, cbpG, including ply of serotypes 1, 7F, 19F and 23F clinical isolates during the bacterial adherence to human lung epithelial cells (in vitro), by real-time PCR. We were able to harvest the bacterial RNA (0.5-1 mu g mu L(-1)) from the infected host cell and analysis showed a consistent upregulation of psaA. Differential expressions were observed for pspC, cbpG and ply genes but the former was mostly upregulated whereas the later two frequently showed either no significant change or a downregulation. Partial nucleotide sequences of psaA, cbpG and ply were highly homologous among the isolates as well as against GenBank sequences (99%) whereas those for pspC were similar (98%) to allelic variants pspC-3 and pspC-5.

Abstract: Choline-binding proteins (CBP) have been associated with the pathogenesis of Streptococcus pneumoniae. We screened, using PCR, for the presence of genes (cbpA, D, E, G) encoding these proteins in 34 isolates of pneumococci of known serotypes and penicillin susceptibility from invasive and non-invasive disease. All isolates harboured cbpD and cbpE whereas cbpA and cbpG were found in 47% and 59% respectively; the latter were more frequent in vaccine-associated types and together accounted for 77% of these isolates. No association was observed with penicillin susceptibility but 85% of non-invasive isolates were positive for these genes.

Abstract: Outbreaks of dengue disease are constant threats to tropical and subtropical populations but range widely in severity, from mild to haemorrhagic fevers, for reasons that are still elusive. We investigated the interferon (IFN) response in infected human cell lines A549 and HepG2, using two strains (NGC and TSV01) of dengue serotype 2 (DEN2) and found that the two viruses exhibited a marked difference in inducing type I IFN response. While TSV01 infection led to activation of type I antiviral genes such as EIF2AK2 (PKR), OAS, ADAR and MX, these responses were absent in NGC-infected cells. Biochemical analysis revealed that NGC but not TSV01 suppressed STAT-1 and STAT-2 activation in response to type I IFN (alpha and beta). However, these two strains did not differ in their response to type I IFN (gamma). Although unable to suppress IFN signalling, TSV01 infection caused a weaker IFN-beta induction compared with NGC, suggesting an alternative mechanism of innate immune escape. We extended our study to clinical isolates of various serotypes and found that while MY10245 (DEN2) and MY22713 (DEN4) could suppress the IFN response in a similar fashion to NGC, three other strains of dengue [EDEN167 (DEN1), MY02569 (DEN11) and MY10340 (DEN2)] were unable to suppress the IFN response, suggesting that this difference is strain-dependent but not serotype-specific. Our report indicates the existence of a strain-specific virulence factor that may impact on disease severity.

Abstract: Background: Streptococcus pneumoniae is a major human pathogen. The emergence of penicillin resistant strains since the 1970s has been life threatening and the evolution of the bacteria have enabled itself to develop resistance to many other antibiotics such as the macrolides and the fluoroquinolones. This study aims to characterize S. pneumoniae isolates for the presence of penicillin and macrolide resistance genes. Methodology: One hundred and twenty clinical isolates of S. pneumoniae were obtained from patients of University Malaya Medical Centre (UMMC). The strains were screened using a multiplex real-time PCR method for the presence of alterations in the genes encoding the penicillin binding proteins: pbp2b, macrolide resistance determinant ermB and the pneumolysin gene, ply. Dual-labelled Taqman probes were used in the real-time detection method comprising three different genes labeled with individual fluorophores at different wavelengths. One hundred and twenty isolates from bacterial cultures and isolates directly from blood cultures samples were analyzed using this assay. Results: A multiplex PCR comprising the antibiotic resistance genes, ermB and pbp2b and pneumolysin gene (ply), a S. pneumoniae species specific gene, was developed to characterize strains of S. pneumoniae. Out of the 120 pneumococcal isolates, 58 strains were categorized as Penicillin Sensitive Streptococcus pneumoniae (PSSP), 36 as Penicillin Intermediate Streptococcus pneumoniae (PISP) and 26 as Penicillin Resistant Streptococcus pneumoniae (PRSP). All the 58 PSSP strains harboured the pbp2b gene while the 36 PISP and 26 PRSP strains did not harbour this gene, thus suggesting reduced susceptibility to penicillin. Resistance to erythromycin was observed in 47 of the pneumococcal strains while 15 and 58 were intermediate and sensitive to this drug respectively. Susceptibility testing to other beta-lactams (CTX and CRO) also showed reduced susceptibility among the strains within the PISP and PRSP groups but most PSSP strains were sensitive to other antibiotics. Conclusion: The characterization of pneumococcal isolates for penicillin and erythromycin resistance genes could be useful to predict the susceptibility of these isolates to other antibiotics, especially beta-lactams drugs. We have developed an assay with a shorter turnaround time to determine the species and resistance profile of Streptococcus pneumoniae with respect to penicillin and macrolides using the Real Time PCR format with fluorescent labeled Taqman probes, hence facilitating earlier and more definitive antimicrobial therapy which may lead to better patient management.

Abstract: Genes encoding the quinolones resistance determining regions (QRDRs) in Streptococcus pneumoniae were detected by PCR and the sequence analysis was carried out to identify point mutations within these regions. The study was carried out to observe mutation patterns among S. pneumoniae strains in Malaysia. Antimicrobial susceptibility testing of 100 isolates was determined against various antibiotics, out of which 56 strains were categorised to have reduced susceptibility to ciprofloxacin (2 g/mL). These strains were subjected to PCR amplification for presence of the gyrA, parC , gyrB and parE genes. Eight representative strains with various susceptibilities to fluoroquinolones were sequenced. Two out of the eight isolates that were sequenced were shown to have a point mutation in the gyrA gene at position Ser81. The detection of mutation at codon Ser81 of the gyrA gene suggested the potential of developing fluoroquinolone resistance among S. pneumoniae isolates in Malaysia. However, further experimental work is required to confirm the involvement of this mutation in the development of fluoroquinolone resistance in Malaysia.

Abstract: We describe a model of Enterovirus 71 encephalomyelitis in 2-week-old mice that shares many features with the human central nervous system (CNS) disease. Mice were infected via oral and parenteral routes with a murine-adapted virus strain originally from a fatal human case. The mice succumbed to infection after 2 to 5 days. Vacuolated and normal-appearing CNS neurons showed viral RNA and antigens and virions by in situ hybridization, immunohistochemistry, and electron microscopy; inflammation was minimal. The most numerous infected neurons were in anterior horns, motor trigeminal nuclei, and brainstem reticular formation; fewer neurons in the red nucleus, lateral cerebellar nucleus, other cranial nerve nuclei, motor cortex, hypothalamus, and thalamus were infected. Other CNS regions, dorsal root, and autonomic ganglia were spared. Intramuscular-inoculated mice killed 24 to 36 hours postinfection had viral RNA and antigens in ipsilateral lumbar anterior horn cells and adjacent axons. Upper cord motor neurons, brainstem, and contralateral motor cortex neurons were infected from 48-72 hours. Viral RNA and antigens were abundant in skeletal muscle and adjacent tissues but not in other organs. The distinct, stereotypic viral distribution in this model suggests that the virus enters the CNS via peripheral motor nerves after skeletal muscle infection, and spread within the CNS involves motor and other neural pathways. This model may be useful for further studies on pathogenesis and for testing therapies.

Abstract: Disease manifestation, pathology, and tissue tropism following infection with Tioman virus (TioPV), a newly isolated, bat-derived paramyxovirus, was investigated in subcutaneously (n = 12) and oronasally (n = 4) inoculated pigs. Pigs were either asymptomatic or developed pyrexia, but all of the animals produced neutralizing antibodies. The virus (viral antigen and/or genome) was detected in lymphocytes of the thymus, tonsils, spleen, lymph nodes and Peyer's patches (ileum), tonsillar epithelium, and thymic epithelioreticular cells. Virus was isolated from oral swabs but not from urine. Our findings suggest that the pig could act as an intermediate or amplifying host for TioPV and that oral secretion is a possible means of viral transmission.

Abstract: Tioman virus (TioPV) and Menangle virus (MenPV) are two antigenically and genetically related paramyxoviruses (genus: Rubulavirus, family: Paramyxoviridae) isolated from Peninsular Malaysia (2001) and Australia (1997), respectively. Both viruses are potential zoonotic agents. In the present study, the infectivity, growth kinetics, morphology and morphogenesis of these two paramyxoviruses in a human neuronal cell (SK-N-SH) line were investigated. Sub-confluent SK-N-SH cells were infected with TioPV and MenPV at similar multiplicity of infection. These cells were examined by conventional and immunoelectron microscopy, and virus titres in the supernatants were assayed. Syncytia were observed for both infections in SK-N-SH cells and were more pronounced during the early stages of TioPV infection. The TioPV titre increased consistently (10(1)) every 12 h after infection. In MenPV-infected cells, cellular material was frequently observed within budding virions, and microfilaments and microtubules were abundant. Viral budding was common, and extracellular MenPVs tended to be more pleomorphic compared to TioPVs, which appeared to be more spherical in appearance. The MenPV cytoplasmic viral inclusion appeared to be comparatively smaller, loose and interspersed with randomly scattered circle-like particles, whereas huge tubule-like cytoplasmic inclusions were observed in TioPV-infected cells. Both viruses also displayed different cellular pathology in the SK-N-SH cells. The intracellular ultrastructural characteristics of these two viruses in infected neuronal cells may allow them to be differentiated by electron microscopy.

Abstract: Over 100 viruses are known to cause acute viral encephalitis in human. In order to diagnose a viral central nervous system infection, various laboratory diagnosis methods have been used. In this study, we examined 220 cerebrospinal fluid samples that were received at the Diagnostic Virology Laboratory of University Malaya Medical Centre between year 2004 to 2006, by viral isolation, pathogen specific antibody ELISA, polymerase chain reaction (PCR) and Real-Time PCR. Majority of the samples were from patients <10 years old. Out of 220 samples, 3 were positive for viral isolation, 27 for PCR (inclusive for the 3 positive for viral isolation) and 39 for pathogen specific ELISA. The total positive detection rate of this study was 30%. Herpes virus was the most important aetiologic agent, responsible for 58% of infection, followed by paramyxovirus (especially measles virus) in 26% of infection, and 14% by enterovirus. Parvovirus and flavivirus were the other common viruses. Among the herpes viruses, herpes simplex and cytomegalovirus were the most common.

Abstract: Disease manifestation, pathology, and tissue tropism following infection with Tioman virus (TioPV), a newly isolated, bat-derived paramyxovirus, was investigated in subcutaneously (n = 12) and oronasally (n = 4) inoculated pigs. Pigs were either asymptomatic or developed pyrexia, but all of the animals produced neutralizing antibodies. The virus (viral antigen and/or genome) was detected in lymphocytes of the thymus, tonsils, spleen, lymph nodes and Peyer's patches (ileum), tonsillar epithelium, and thymic epithelioreticular cells. Virus was isolated from oral swabs but not from urine. Our findings suggest that the pig could act as an intermediate or amplifying host for TioPV and that oral secretion is a possible means of viral transmission.

Abstract: OBJECTIVE: To estimate prevalence rate of recent dengue infection in parturients, as well as the vertical transmission rate, and to compare pregnancy outcomes among infected women. METHOD: A prospective cohort study was performed. Maternal and paired umbilical cord bloods were taken at delivery. A brief questionnaire on febrile illness and dengue in pregnancy was completed by participants. The samples were sent to a World Health Organization Collaborating Center for Arbovirus Reference and Research. Maternal sera were tested with a dengue-specific immunoglobulin M (IgM) capture test. The paired umbilical cord serum was tested to determine when the maternal sample was positive. Dengue reverse transcriptase polymerase chain reaction (PCR) was performed on all dengue IgM-positive sera. Pregnancy outcome was extracted from delivery records. RESULTS: A total of 2,958 parturients were enrolled, and 2,531 paired maternal-umbilical cord blood samples were available for testing. Dengue-specific IgM was positive in 63 of 2,531 (2.5%, 95% confidence interval [CI] 1.9-3.2%) maternal samples. Only 1 of 64 (1.6%, 95% CI 0.0-9.5%) of the paired umbilical cord sera was IgM-positive. All reverse transcriptase PCR tests were negative. Fifty-six of 63 (88.9%) of dengue IgM-positive women did not report a febrile illness in pregnancy. Apart from a higher mean age in dengue IgM-positive women of 30.6 +/- 5.2 compared with 29.2 +/- 4.9 years (P=.025) compared with dengue IgM-negative women, all other characteristics were similar. Rates of preterm birth, mode of delivery, postpartum hemorrhage, low birth weight, and neonatal outcomes were not different. CONCLUSION: Recent dengue infection was demonstrated in 2.5% of parturients, with a vertical transmission rate of 1.6%. Pregnancy outcome of recently infected women was not different.

Abstract: Outbreaks of dengue disease are constant threats to tropical and subtropical populations but range widely in severity, from mild to haemorrhagic fevers, for reasons that are still elusive. We investigated the interferon (IFN) response in infected human cell lines A549 and HepG2, using two strains (NGC and TSV01) of dengue serotype 2 (DEN2) and found that the two viruses exhibited a marked difference in inducing type I IFN response. While TSV01 infection led to activation of type I antiviral genes such as EIF2AK2 (PKR), OAS, ADAR and MX, these responses were absent in NGC-infected cells. Biochemical analysis revealed that NGC but not TSV01 suppressed STAT-1 and STAT-2 activation in response to type I IFN (alpha and beta). However, these two strains did not differ in their response to type I IFN (gamma). Although unable to suppress IFN signalling, TSV01 infection caused a weaker IFN-beta induction compared with NGC, suggesting an alternative mechanism of innate immune escape. We extended our study to clinical isolates of various serotypes and found that while MY10245 (DEN2) and MY22713 (DEN4) could suppress the IFN response in a similar fashion to NGC, three other strains of dengue [EDEN167 (DEN1), MY02569 (DEN11) and MY10340 (DEN2)] were unable to suppress the IFN response, suggesting that this difference is strain-dependent but not serotype-specific. Our report indicates the existence of a strain-specific virulence factor that may impact on disease severity.

Abstract: Tioman virus (TioPV) and Menangle virus (MenPV) are two antigenically and genetically related paramyxoviruses (genus: Rubulavirus, family: Paramyxoviridae) isolated from Peninsular Malaysia (2001) and Australia (1997), respectively. Both viruses are potential zoonotic agents. In the present study, the infectivity, growth kinetics, morphology and morphogenesis of these two paramyxoviruses in a human neuronal cell (SK-N-SH) line were investigated. Sub-confluent SK-N-SH cells were infected with TioPV and MenPV at similar multiplicity of infection. These cells were examined by conventional and immunoelectron microscopy, and virus titres in the supernatants were assayed. Syncytia were observed for both infections in SK-N-SH cells and were more pronounced during the early stages of TioPV infection. The TioPV titre increased consistently (10(1)) every 12 h after infection. In MenPV-infected cells, cellular material was frequently observed within budding virions, and microfilaments and microtubules were abundant. Viral budding was common, and extracellular MenPVs tended to be more pleomorphic compared to TioPVs, which appeared to be more spherical in appearance. The MenPV cytoplasmic viral inclusion appeared to be comparatively smaller, loose and interspersed with randomly scattered circle-like particles, whereas huge tubule-like cytoplasmic inclusions were observed in TioPV-infected cells. Both viruses also displayed different cellular pathology in the SK-N-SH cells. The intracellular ultrastructural characteristics of these two viruses in infected neuronal cells may allow them to be differentiated by electron microscopy.

Abstract: In a previous study, we have reported the detection and isolation of dengue virus in Brunei (Osman, O., Fong, M.Y., Devi, S., 2007. A preliminary study of dengue infection in Brunei. JJID 60 (4), 205-208). DEN-2 was the predominant serotype followed by DEN-1. The full genomic sequences of 3 DEN-2 viruses isolated during the 2005-2006 dengue incident in Brunei were determined. Twenty-five primer sets were designed to amplify contiguous overlapping fragments of approximately 500-600 base pairs spanning the entire sequence of the viral genome. The amplified PCR products were sent for sequencing and their nucleoticles and the deduced amino acids were determined. All three DEN-2 virus isolated were clustered in the Cosmopolitan genotype of the DEN-2 classification by Twiddy et al. This work constitutes the first complete genetic characterization of three Brunei DEN-2 virus strains. (c) 2008 Elsevier B.V. All rights reserved.

Abstract: Over 100 viruses are known to cause acute viral encephalitis in human. In order to diagnose a viral central nervous system infection, various laboratory diagnosis methods have been used. In this study, we examined 220 cerebrospinal fluid samples that were received at the Diagnostic Virology Laboratory of University Malaya Medical Centre between year 2004 to 2006, by viral isolation, pathogen specific antibody ELISA, polymerase chain reaction (PCR) and Real-Time PCR. Majority of the samples were from patients <10 years old. Out of 220 samples, 3 were positive for viral isolation, 27 for PCR (inclusive for the 3 positive for viral isolation) and 39 for pathogen specific ELISA. The total positive detection rate of this study was 30%. Herpes virus was the most important aetiologic agent, responsible for 58% of infection, followed by paramyxovirus (especially measles virus) in 26% of infection, and 14% by enterovirus. Parvovirus and flavivirus were the other common viruses. Among the herpes viruses, herpes simplex and cytomegalovirus were the most common.

Abstract: Difficulties in the management of dengue infections include the lack of rapid diagnostic methods and symptoms of dengue are often confused with those of other diseases. Five commercial kits were evaluated for the detection of dengue-specific IgM and IgG antibodies using sera obtained from patients with primary and secondary dengue infections, as well as other febrile illnesses. All kits were compared to the in-house IgM ELISA and HI assays. The rapid test kits took either 30 to 45 min (PanBio Dengue Duo Cassette, Accusens Dengue Virus Rapid Strip Test, and Unitest Dengue IgM and IgG Combo Rapid Test) or between 2 to 4 h (PanBio Dengue Duo Capture ELISA, and Antivirus IgM Detecion Kit 96 [Pentax Corporation]). Most kits were able to detect IgM in more than 90% of the secondary convalescent sera, while IgG detection was generally high (80 to 100%). All five kits showed high specificity when tested against sera from other febrile patients, and have been shown to be extremely useful in the diagnosis of dengue infections.

Abstract: The purpose of this study was to examine the extent of dengue infection in Brunei and to determine the predominant serotype circulating in the country. The study generated useful epidemiological data on dengue infection in Brunei. A total of 271 samples from patients suspected of having dengue infections were selected and analyzed. All patients were seen in clinics and hospitals in Brunei. The samples were collected from April 2005 to April 2006 and transported to the WHO Collaborating Centre for Arbovirus Reference and Research, University of Malaya, Malaysia. The following tests were used to achieve the objectives: in-house IgM-capture enzyme-linked immunosorbent assay, virus isolation in mosquito albopictus cell line (C6/36), and viral RNA detection and serotyping by reverse transcriptase-polymerase chain reaction (RT-PCR). The results show that 45 people were positive for dengue-specific IgM (27 males and 18 females), while RT-PCR detected dengue viral RNA in 12 patients, 3 identified as DEN-1 and 9 as DEN-2. Dengue virus was isolated from 6 patients using the C6/36 cell line; 3 were DEN-2 isolates and 3 were DEN-1 isolates. These data show that dengue virus is circulating in Brunei and the predominant infecting serotype for that period was DEN-2 followed by DEN-1. This study is the first to report the detection and isolation of dengue virus from Brunei using RT-PCR and culture in the C6/36 albopictus mosquito cell line.

Abstract: Dengue virus infections are a major cause of morbidity and mortality in tropical and subtropical areas in the world. Attempts to develop effective vaccines have been hampered by the lack of understanding of the pathogenesis of the disease and the absence of suitable experimental models for dengue viral infection. The magnitude of T-cell responses has been reported to correlate with dengue disease severity. Sixty Malaysian adults with dengue viral infections were investigated for their dengue virus-specific T-cell responses to 32 peptides antigens from the structural and nonstructural regions from a dengue virus isolate. Seventeen different peptides from the C, E, NS2B, NS3, NS4A, NS4B, and NS5 regions were found to evoke significant responses in a gamma interferon enzyme-linked immunospot (ELISPOT) assay of samples from 13 selected patients with dengue fever (DF) and dengue hemorrhagic fever (DHF). NS3 and predominantly NS3(422-431) were found to be important T-cell targets. The highest peaks of T-cell responses observed were in responses to NS3(422-431) and NS5(563-571). in DHF patients. We also found almost a sevenfold increase in T-cell response in three DHF patients compared to three DF patient responses to peptide NS3(422-431). A large number of patients' T cells also responded to the NS2B,,-106 region. The ELISPOT analyses also revealed high frequencies of T cells that recognize both serotype-specific and cross-reactive dengue virus antigens in patients with DHF.

Abstract: A triplex real-time polymerase chain reaction (PCR) assay was used for the simultaneous detection of mecA (methicillin resistance), ermA (erythromycin resistance) and femA (Staphylococcus aureus identification) genes in a single assay. Among 93 clinical S. aureus hospital isolates, there were 48 methicillin-resistant S. aureus (MRSA) and 45 methicillin-sensitive S. aureus (MSSA) isolates. Screening the isolates using the triplex real-time PCR assay, the mecA, ermA and femA genes were detected in all MRSA isolates. The triplex real-time PCR assay was completed within 3 h and is a useful genotypic method for detecting the resistance determinants as well as for the identification of S. aureus isolates. These findings will assist the clinical laboratory in identifying these resistance genes and S. aureus rapidly, thus benefiting patient therapy. This study represents a valuable source of information for researchers to study the local antibiotic resistance pattern, which can increase our knowledge of the antibiotic resistance profile, using real-time PCR technology. (C) 2007 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.

Abstract: In this study we describe a triplex real-time PCR assay that enables the identification of S. aureus and detection of two important antibiotic resistant genes simultaneously using real-time PCR technology in a single assay. In this triplex real-time PCR assay, the mecA (methicillin resistant), femA (species specific S. aureus) and aacA-aphD (aminoglycoside resistant) genes were detected in a single test using dual-labeled Taqman probes. The assay gives simultaneous information for the identification of S. aureus and detection of methicillin and aminoglycoside resistance in staphylococcal isolates. 152 clinical isolates were subjected to this triplex real-time PCR assay. The results of the triplex real-time PCR assay correlated with the results of the phenotypic antibiotic susceptibility testing. The results obtained from triplex real-time PCR assay shows that the primer and probe sets were specific for the identification of S. aureus and were able to detect methicillin- and aminoglycoside-resistant genes. The entire assay can be performed within 3 h which is a very rapid method that can give simultaneous information for the identification of S. aureus and antibiotic resistance pattern of a staphylococcal isolate. The application of this rapid method in microbiology laboratories would be a valuable tool for the rapid identification of the S. aureus isolates and determination of their antibiotic resistance pattern with regards to methicillin and aminoglycosides. (c) 2006 Elsevier B.V. All rights reserved.

Abstract: Polyclonal sera from typhoid patients and a monoclonal antibody, mAb ATVi, which recognizes the capsular polysaccharide Vi antigen (ViCPS), were used to select for peptides that mimic the ViCPS by using a phage-displayed random 12-mer peptide library. Two major common mimotopes selected from the library carried the amino acid sequences TSHHDSHGLHRV and ENHSPVNIAHKL. Enzyme-linked immunosorbent assays (ELISAs) showed that these peptides carry mimotopes to ViCPS. Phage clones that contained the 12-mer peptides were also tested against pooled/individual typhoid patients' sera and found to have 3 to 5 times higher binding compared to normal sera. By using Phage-ELISA assays, the derived synthetic peptides, TSHHDSHGLHRV and ENHSPVNIAHKL, were tested against a monoclonal antibody mAb ATVi and over 2-fold difference in binding was found between these peptides and a control unrelated peptide, CTLTTKLYC. Inhibition of the mAb's binding to ViCPS indicated that the synthetic peptides successfully competed with the capsular polysaccharide for antibody binding.

Abstract: Tioman virus is a newly described bat-urine derived paramyxovirus isolated in Tioman Island, Malaysia in 2001. Hitherto, neither human nor animal infection by this virus has been reported. Nonetheless, its close relationship to another paramyxovirus, the Menangle virus which had caused diseases in humans and pigs [Philbey, A.W., Kirkland, P.D., Ross, A.D., Davis, R.J., Gleeson, A.B., Love, R.J., Daniels, P.W., Gould, A.R., Hyatt, A.D., 1998. An apparently new virus (family Paramyxoviridae) infectious for pigs, humans, and fruit bats. Emerg. Infect. Dis. 4, 269-271], raises the possibility that it may be potentially pathogenic. In this study, mice were experimentally infected with Tioman virus by intraperitoneal and intracerebral routes, and the cellular targets and topographical distribution of viral genome and antigens were examined using in situ hybridization and immunohistochemistry, respectively. The possible association between viral infection and apoptosis was also investigated using the TUNEL assay and immunohistochemistry to FasL, Caspase-3, Caspase-8, Caspase-9 and bcl-2. The results showed that Tioman virus inoculated intracerebrally was neurotropic causing plaque-like necrotic areas, and appeared to preferentially replicate in the neocortex and limbic system. Viral i infection of inflammatory cells was also demonstrated. TUNEL and Caspase-3 positivity was found in inflammatory cells but not in neurons, while FasL, Caspase-8 and Caspase-9 were consistently negative. This suggests that neuronal infection was associated with necrosis rather than apoptosis. Moreover, the data suggest that there may be an association between viral infection and apoptosis in inflammatory cells, and that it could, at least in part, involve Caspase-independent pathways. Bcl-2 was expressed in some neurons and inflammatory cells indicating its possible role in anti-apoptosis. There was no evidence of central nervous system infection via the intraperitoneal route. (C) 2007 Elsevier B.V. All rights reserved.

Abstract: The detection of leptospires in patient blood in the first week of the disease using PCR provides an early diagnostic tool. PCR using two sets of primers (G1/G2 and B64-1/B64-II) tested with samples seeded with 23 leptospiral strains from pathogenic and non-pathogenic strains was able to amplify leptospiral DNA from pathogenic strains only. Of the 39 antibody negative samples collected from patients suspected for leptospirosis, only 1 sample (2.6%) was PCR positive. Using LSSP-PCR, the G2 primers allowed the characterization of Leptopira species to 10 different genetic signatures which may have epidemiological value in determining species involved in outbreaks. Leptospiral outer membrane proteins from three strains were purified and reacted against patients sera and gave rise to different profiles for pathogenic and non-pathogenic strains. Lymphocytes of mice injected with OMPs proliferated and released IFN-(3) when stimulated in vitro using Leptospira OMP as antigens. This suggests that an immune response could be established using leptospiral OMPs as a putative vaccine. OMPs were also used in a Dot-ELISA to detect antibodies against Leptospira pathogens in humans.

Abstract: For rapid identification of methicillin-resistant Stapkylococcus aureus, molecular methods are generally targeting mecA and species-specific genes. Sa442 DNA fragment is a popular species-specific target. However, recently, there have been few reports on S. aureus isolates that are negative for Sa442 fragment; therefore, use of single gene or DNA-fragment-specific polymerase chain reaction (PCR) for identification of microbial isolate may result in misidentification. This study includes CoA gene in parallel with Sa442 marker for identification of S. aureus. This further improves the specificity of the assay by checking for 2 determinants simultaneously for the identification of S. aureus and can prevent misidentification of S. aureus isolates lacking Sa442 DNA fragment. In this study, the newly developed triplex real-time PCR assay was compared with a quadruplex conventional gel-based PCR assay using the same primer sets in both assays. The dual-labeled TaqMan probes (ProOligo, France) for these primers were specifically designed and used in a real-time PCR assay. The clinical isolates (n = 152) were subjected to both PCR assays. The results obtained from both assays proved that the primer and probe sets were 100% sensitive and 100% specific for identification of S. aureus and detection of methicillin resistance. This triplex real-time PCR assay represents a rapid and powerful method for S. aureus identification and detection of methicillin resistance. (c) 2006 Elsevier Inc. All rights reserved.

Abstract: The use of the polymerase chain reaction (PCR) in molecular diagnosis is now accepted worldwide and has become an essential toot in the research laboratory. In the laboratory, a rapid detection, serotyping and quantitation, one-step real-time RT-PCR assay was developed for dengue virus using TaqMan probes. In this assay, a set of forward and reverse primers were designed targeting the serotype conserved region at the NS5 gene. at the same time flanking a variable region for all four serotypes which were used to design the serotype-specific TaqMan probes. This multiplex one-step RT-PCR assay was evaluated using 376 samples collected during the year 2003. These groups included RNA from prototype dengue virus (1-4), RNA from acute serum from which dengue virus was isolated, RNA from tissue culture supernatants of dengue virus isolated, RNA from seronegative acute samples (which were culture and IgM negative) and RNA from samples of dengue IgM positive sera. The specificity of this assay was also evaluated using a panel of sera which were positive for other common tropical disease agents including herpes simplex virus, cytomegalovirus. measles virus, varicella-zoster virus, rubella virus, mumps virus, WWF, West Nile virus, Japanese encephalitis virus, S. typhi, Legionella, Leptospira, Chlamydia, and Mycoplasina. The sensitivity, specificity and real-time PCR efficiency of this assay were 89.54%, 100% and 91.5%. respectively. (c) 2006 Elsevier B.V. All rights reserved.

Abstract: The DNA sequences encompassing two hypervariable regions, VD II and III of the 56 kDa immunodominant protein gene of 21 Malaysian strains of Orientia tsutsugamushi were determined. Two strains demonstrated a 100% DNA homology with the Gilliam prototype strain, and one with TH1817 strain and TA678 strain respectively. High percentages of DNA similarity (95-99%) were observed with Karp (4 strains), Gilliam (2 strains), TH1817 (4 strains), TC586 (3 strains) and TA763 (1 strain). The remaining strains demonstrated the highest DNA similarity with TA763 (1 strain, 89%), TA078 (1 strain, 86%) and TA686 (1 strain, 87%). Our study provides additional evidence on the existence and the genetic heterogeneity of TA strains of the Southeast Asia and their closely related strains in Malaysia.

Abstract: Dengue viral antigens have been demonstrated in several types of naturally infected human tissues, but little is known of whether these same tissues have detectable viral RNA. We studied tissue specimens from patients with serologically or virologically confirmed dengue infections by immunohistochemistry (IHC) and in situ hybridization ( ISH), to localize viral antigen and RNA, respectively. IHC was performed on specimens obtained from 5 autopsies and 24 biopsies and on 20 blood-clot samples. For ISH, antisense riboprobes to the dengue E gene were applied to tissue specimens in which IHC was positive. Viral antigens were demonstrated in Kupffer and sinusoidal endothelial cells of the liver; macrophages, multinucleated cells, and reactive lymphoid cells in the spleen; macrophages and vascular endothelium in the lung; kidney tubules; and monocytes and lymphocytes in blood-clot samples. Positive-strand viral RNA was detected in the same IHC-positive cells found in the spleen and blood-clot samples. The strong, positive ISH signal in these cells indicated a high copy number of viral RNA, suggesting replication.

Abstract: The capsular polysaccharide Vi antigen (ViCPS) is an essential virulence factor and also a protective antigen of Salmonella enterica serovar Typhi. A random 12-mer phage-displayed peptide library was used to identify mimotopes (epitope analogues) of this antigen by panning against a ViCPS-specific monoclonal antibody (MAb) ATVi. Approximately 75% of the phage clones selected in the fourth round carried the peptide sequence TSHHDSHGLHRV, and the rest of the clones harbored ENHSPVNLA-HKL and other related sequences. These two sequences were also obtained in a similar panning process by using pooled sera from patients with a confirmed diagnosis of typhoid fever, suggesting they mimic immunodominant epitopes of ViCPS antigens. Binding of MAb ATVi to the mimotopes was specifically blocked by ViCPS, indicating that they interact with the same binding site (paratope) of the MAb. Data and reagents generated in this study have important implications for the development of peptide-base diagnostic tests and peptide vaccines and may also provide a better understanding of the pathogenesis of typhoid fever.

Abstract: In this study, recombinant proteins that encompassed the AD I-AD III regions of 56 kDa immunodominant gene of 2 Orientia tsutsugamushi (OT) serotypes; Gilliam and TA763 were expressed in Escherichia coli. Both recombinant proteins exhibited serologic cross-reactivity with the rabbit antisera against various OT serotypes, as evaluated by enzyme-linked immunosorbent assay (ELISA), but not against other rickettsial species, including Rickettsia typhi, R. prowazekii and TT118 SFG rickettsiae. The feasibility of using the recombinant proteins as a diagnostic reagent was further evaluated by ELISA using sera from blood donors and scrub typhus patients. The results suggested a higher affinity of the antihuman IgM than IgG with both recombinant proteins. The IgM ELISA findings were agreeable with the results of indirect immunoperoxidase (IIP) assay especially with sera of high antibody (1:1,600). However, more than one antigen are probably needed for development of an effective assay for serodiagnosis of scrub typhus in endemic areas. (C) 2002 Elsevier Science Inc. All rights reserved.

Abstract: Objective. To carry out a comparative study of the safety and immunogenicity of Vi polysaccharide vaccine against whole-cell killed (WCK) typhoid vaccine. Methods. The study was carried out on young adult recruits (aged 18-25 years) of the Malaysian Air Force. A total of 125 subjects received the Vi polyssacharide vaccine and 114 received the WCK vaccine. Findings. The Vi vaccine was significantly less reactogenic than the WCK vaccine with regard to systemic and local reactions. Following administration of the Vi vaccine, seroconversion rates (defined as the percentage of subjects with a 4-fold rise of baseline antibody level) of 75.5% and 67% were observed at 2 weeks and 6 weeks, respectively, after immunization, compared with 25% and 31.3% among recipients of the WCK vaccine. Of the 110 Vi vaccinees with serological data, 21 (19%) had high, seroprotective, pre-immunization levels of anti-Vi antibodies (greater than or equal to 1 mug/ml). The majority of subjects in this group came from a region in Malaysia which is known to have high typhoid endemicity. Interestingly, these antibody levels were boosted considerably following administration of vaccine at a level that was 5-fold higher than in subjects with low pre-immunization levels, In contrast, the seroconversion rates in those receiving the Vi vaccine were higher in subjects with low pre-immunization levels of anti-Vi antibodies (76-84%), compared to those with protective levels of greater than or equal to 1 mug/ml prior to immunization (48-57%). Conclusions. The study reaffirms the safety and efficacy of the Vi polysaccharide vaccine and identifies a hitherto unrecognized advantage in its use, Le. it is a potent immunogen that boosted considerably the protective antibody levels among a significant number of immunologically sensitized individuals living in typhoid-endemic regions.

Abstract: A serosurvey was conducted in 1995-97 among 1596 febrile patients from 8 health centres in Malaysia for antibodies against Orientia tsutsugamushi (OT), Rickettsia typhi (RT) and TT118 spotted fever group rickettsiae (SFGR) by using an indirect immunoperoxidase assay. A total of 51.4% patients had antibody against at least 1 of those rickettsiae. Antibody to SFGR was most prevalent (42.5%), followed by RT (28.1%) and OT (24.9%). The seroprevalences of antibodies to SFGR, RT or OT alone were 12.4, 3.6 and 4.3%, respectively. Antibodies against more than 1 species of rickettsiae were presence in 31.1% of the patients, suggesting the possibility of co-infection, previous exposures or serological cross-reactivities. Seroprevalence of the various rickettsiae varied according to locality, with SFGR antibodies being the most prevalent in most areas. There was no significant association of prevalence of rickettsial antibody with gender. The seroprevalence of OT, SFGR and RT increased with patient age but an increase of antibody titre with age was not significant. Those working in the agricultural sectors had significantly higher seroprevalence of OT, SFGR and RT than those not related with agricultural activities. Scrub typhus remains a public health problem with an estimated annual attack rate of 18.5%. Tick typhus and murine typhus as shown in this serosurvey appear much more widespread than scrub typhus in this country.