The prevalence and antibiotic susceptibility of intestinal carriage of Gram-negative bacteria among preterm infants admitted to the neonatal intensive care unit (NICU) in a tertiary teaching hospital in Malaysia were determined. A total of 34 stool specimens were obtained from preterm infants upon admission and once weekly up to two weeks during hospitalization. The presumptive colonies of Escherichia coli and Klebsiella pneumoniae were selected for identification, antibiotic susceptibility testing, and subtyping by using pulsed-field gel electrophoresis (PFGE). Out of 76 Gram-negative isolates, highest resistance was detected for amoxicillin/clavulanate (30.8%, n = 16), ceftriaxone (42.3%, n = 22), ceftazidime (28.8%, n = 15), cefoxitin (28.8%, n = 15), aztreonam (36.5%, n = 19), and polymyxin B (23.1%, n = 12). Three colistin resistant K. pneumoniae have also been detected based on E-test analysis. Thirty-nine isolates of K. pneumoniae and 20 isolates of E. coli were resistant to more than three antimicrobial classes and were categorized as multidrug resistant (MDR). PFGE analysis revealed a higher diversity in pulsotypes for K. pneumoniae (18 pulsotypes) in comparison to E. coli (four pulsotypes). In addition, a total of fifteen pulsotypes was observed from 39 MDR K. pneumoniae. The risk factors for antibiotic resistance were assessed using random forest analysis. Gender was found to be the most important predictor for colistin resistant while length, OFC, and delivery mode were showing greater predictive power in the polymyxin B resistance. This study revealed worrying prevalence rates of intestinal carriage of multidrug-resistant K. pneumoniae and E. coli of hospitalized preterm infants in Malaysia, particularly high resistance to polymyxins.
The IgG avidity assay is an important tool in the management of suspected toxoplasmosis in pregnant women. This study aimed to produce new Toxoplasma gondii recombinant proteins and to assess their usefulness in an IgG avidity assay. Toxoplasma positive and negative serum samples were used, the former were categorized into low (LGA) and high (HGA) IgG avidity samples. Immunoblots were performed on 30 T. gondii cDNA clones to determine the reactivity and IgG avidity to the expressed proteins. Two of the clones were found to have diagnostic potential and were analyzed further; AG12b encoded T. gondii apical complex lysine methyltransferase (AKMT) protein and AG18 encoded T. gondii forkhead-associated (FHA) domain-containing protein. The His-tagged recombinant proteins, rAG12b and rAG18, were expressed and tested with LGA and HGA samples using an IgG avidity western blot and ELISA. With the IgG avidity western blot, rAG12b identified 86.4% of LGA and 90.9% of HGA samples, whereas rAG18 identified 81.8% of both LGA and HGA samples. With the IgG avidity ELISA, rAG12b identified 86.4% of both LGA and HGA samples, whereas rAG18 identified 77.3% of LGA and 86.4% of HGA serum samples. This study showed that the recombinant antigens were able to differentiate low avidity and high avidity serum samples, suggesting that they are potential candidates for use in the Toxoplasma IgG avidity assay.
Entamoeba histolytica infection remains a public health concern in developing countries. Early diagnosis of amoebiasis can avoid disease complications, thus this study was aimed at developing a test that can rapidly detect the parasite antigens in stool samples. Rabbits were individually immunized with recombinant pyruvate phosphate dikinase (rPPDK) and E. histolytica excretory-secretory antigens to produce polyclonal antibodies. A rapid dipstick test was produced using anti-rPPDK PAb lined on the dipstick as capture reagent and anti-EhESA PAb conjugated to colloidal gold as the detector reagent. Using E. histolytica-spiked in stool sample of a healthy individual, the detection limit of the dipstick test was found to be 1000 cells ml-1. Meanwhile when rPPDK was spiked in the stool sample, the minimum concentration detected by the dipstick test was 0.1 μg ml-1. The performances of the dipstick, commercial Techlab E. histolytica II enzyme-linked immunosorbent assays (ELISA) and real-time PCR were compared using 70 stool samples from patients infected with Entamoeba species (n = 45) and other intestinal pathogens (n = 25). When compared to real-time PCR, the diagnostic sensitivity of the dipstick for detection of E. histolytica was 65.4% (n = 17/26); while the diagnostic specificity when tested with stool samples containing other intestinal pathogens was 92% (23/25). In contrast, Techlab E. histolytica II ELISA detected 19.2% (5/26) of the E. histolytica-positive samples as compared to real-time PCR. The lateral flow dipstick test produced in this study enabled rapid detection of E. histolytica, thus it showed good potential to be further developed into a diagnostic tool for intestinal amoebiasis.
Toxoplasmosis, a parasitic disease in human and animals, is caused by Toxoplasma gondii. Our previous study has led to the discovery of a novel RAP domain binding protein antigen (TgRA15), an apparent in-vivo induced antigen recognised by antibodies in acutely infected individuals. This study is aimed to evaluate the humoral response and cytokine release elicited by recombinant TgRA15 protein in C57BL/6 mice, demonstrating its potential as a candidate vaccine for Toxoplasma gondii infection. In this study, the recombinant TgRA15 protein was expressed in Escherichia coli, purified and refolded into soluble form. C57BL/6 mice were immunised intradermally with the antigen and CASAC (Combined Adjuvant for Synergistic Activation of Cellular immunity). Antigen-specific humoral and cell-mediated responses were evaluated using Western blot and ELISA. The total IgG, IgG1 and IgG2a antibodies specific to the antigen were significantly increased in treatment group compare to control group. A higher level of interferon gamma (IFN-γ) secretion was demonstrated in the mice group receiving booster doses of rTgRA15 protein, suggesting a potential Th1-mediated response. In conclusion, the rTgRA15 protein has the potential to generate specific antibody response and elicit cellular response, thus potentially serve as a vaccine candidate against T. gondii infection.
This study aimed to evaluate vascular endothelial growth factor (VEGF) and pentraxin 3 (PTX-3) as predictive and diagnostic markers in differentiating severe dengue from non-severe dengue. The study was conducted in Ampang Health Clinic, Ampang Hospital and Serdang Hospital. The plasma levels of VEGF and PTX-3 were compared between severe dengue and non-severe dengue by ELISA from the day of presentation until discharged. Multiple logistic regression was used to develop predictive and diagnostic models by incorporating other clinical parameters. The receiver operating characteristics (ROC) analysis was used to assess the accuracy of the biomarkers and the developed models. Eighty-two patients were recruited, 29 with severe dengue and four died. The Area Under the Curve (AUC) was statistically significant in VEGF as diagnostic marker at Day 2 and 3 of illness with sensitivity of 80.00%-100.00% and specificity of 76.47%-80.00%. The predictive model with AUC of 0.84 (p
The increasing prevalence of antibiotic resistant pathogens poses a serious threat to global health. However, less emphasis has been placed to co-relate the gene expression and metabolism of antibiotic resistant pathogens. This study aims to elucidate gene expression and variations in metabolism of multidrug resistant Klebsiella pneumoniae after exposure to antibiotics. Phenotypic responses of three genotypically distinct carbapenem resistant Klebsiella pneumoniae (CRKP) strains untreated and treated with sub-lethal concentrations of imipenem were investigated via phenotype microarrays (PM). The gene expression and metabolism of the strain harboring blaNDM-1 before and after exposure to sub-lethal concentration of imipenem were further investigated by RNA-sequencing (RNA-Seq) and 1H NMR spectroscopy respectively. Most genes related to cell division, central carbon metabolism and nucleotide metabolism were downregulated after imipenem treatment. Similarly, 1H NMR spectra obtained from treated CRKP showed decrease in levels of bacterial end products (acetate, pyruvate, succinate, formate) and metabolites involved in nucleotide metabolism (uracil, xanthine, hypoxanthine) but elevated levels of glycerophosphocholine. The presence of anserine was also observed for the treated CRKP while FAPγ-adenine and methyladenine were only present in untreated bacterial cells. As a conclusion, the studied CRKP strain exhibited decrease in central carbon metabolism, cell division and nucleotide metabolism after exposure to sub-lethal concentrations of imipenem. The understanding of the complex biological system of this multidrug resistant bacterium may help in the development of novel strategies and potential targets for the management of the infections.
Lymphatic filariasis (LF) is a vector borne disease caused by parasitic worms such as Wuchereria bancrofti, Brugia malayi and B. timori, which are transmitted by mosquitoes. Current therapeutics to treat LF are mainly microfilarcidal, and lack activity against adult worms. This set back, poses a challenge for the control and elimination of filariasis. Thus, in this study the activities of caffeic acid phenethyl ester (CAPE) against the filarial worm B. pahangi and its bacterial endosymbiont, Wolbachia were evaluated. Different concentrations (2, 5, 10, 15, 20 μg/ml) of CAPE were used to assess its effects on motility, viability and microfilarial (mf) production of B. pahangi in vitro. Anti-Wolbachial activity of CAPE was measured in worms by quantification of Wolbachial wsp gene copy number using real-time polymerase chain reaction. Our findings show that CAPE was found to significantly reduce adult worm motility, viability, and mf release both in vitro and in vivo. 20 μg/ml of CAPE halts the release of mf in vitro by day 6 of post treatment. Also, the number of adult worms recovered in vivo were reduced significantly during and after treatment with 50 mg/kg of CAPE relative to control drugs, diethylcarbamazine and doxycycline. Real time PCR based on the Wolbachia ftsZ gene revealed a significant reduction in Wolbachia copy number upon treatment. Anti-Wolbachia and antifilarial properties of CAPE require further investigation as an alternative strategy to treat LF.
An. culicifacies is the major vector of malaria in tribal community and tribal dominated areas in India. Development of resistance to insecticides is the major challenge to curb the transmission. Gadchiroli (Maharashtra) is a tribal district in central India where incidence of malaria increased from 2012 to 2015 despite indoor space spray with synthetic pyrethroids. To determine the susceptibility status of An. culicifacies against commonly used insecticides in public health program in Gadchiroli. standard WHO method and test kit were used. The insecticide impregnated papers were procured from vector control unit Malaysia. An. culicifacies found resistance to three major groups of pesticides i.e. organochlorine (DDT 4%), organophosphorous (Malathion 5%) and pyrethroids (Cyfluthrin 0.15%, Deltametherin 0.05% and Lambdacyhalothrin 0.05%). The susceptibility status in Permethrin 0.75% needs further confirmation. Development of resistance to different insecticides of varied groups is an adverse finding for the elimination of malaria, explaining the recent increase in malaria incidence in Gadchiroli. The phenomenon further needs to be studied in different locations and the susceptibility needs to test against other insecticides. The findings may have significant implications to the choice of insecticides in the malaria control program in tribal areas.
Immunoinformatics plays a pivotal role in vaccine design, immunodiagnostic development, and antibody production. In the past, antibody design and vaccine development depended exclusively on immunological experiments which are relatively expensive and time-consuming. However, recent advances in the field of immunological bioinformatics have provided feasible tools which can be used to lessen the time and cost required for vaccine and antibody development. This approach allows the selection of immunogenic regions from the pathogen genomes. The ideal regions could be developed as potential vaccine candidates to trigger protective immune responses in the hosts. At present, epitope-based vaccines are attractive concepts which have been successfully trailed to develop vaccines which target rapidly mutating pathogens. In this article, we provide an overview of the current progress of immunoinformatics and their applications in the vaccine design, immune system modeling and therapeutics.
Streptococcus pneumoniae (S. pneumoniae) is one of the main causative agents of pneumococcal diseases. To date, more than 90 distinct serotypes have been identified. Implementation of vaccines has caused a drastic reduction in vaccine-serotype pneumococcal diseases but increase in cases due to non-vaccine serotype has been observed in Malaysia. However, further investigation on different serotype incidence in Malaysia is needed and the rate of pneumococcal vaccination for new-born babies in Malaysia remains low. The recent emergence of drug-resistant S. pneumoniae (DRSP) has also been a global concern, especially penicillin resistance. This study determined the serotypes of S. pneumoniae strains (n = 95) isolated from nasopharyngeal specimens from children admitted to UMMC from 2013 to 2015. In accordance with previous studies, PCR result showed 40% of NT isolates were successfully typed as 3 less common serotypes, namely 9N/L, 17A, and 23B. The repetitive-element PCR (REP-PCR) result revealed genetic variations among the strains whereby five major clusters were observed at the similarity of 80% by clustering analysis based on fingerprint data. Penicillin-binding proteins (pbps) of selected isolates were studied by PCR and sequencing. Three strains with ≤19-mm diameter zone for Oxacillin Disc Diffusion (ODD) test previously were recorded to have mutation on all pbp1a, pbp2b, and pbp2x with MIC of 4 µg/ml, which were penicillin-intermediate resistance according to the CLSI breakpoints.
Leptospirosis causes a wide range of clinical outcomes, including organ failure and death. Early treatment significantly increases the chances of cure. Interleukin-8 (IL-8) is a chemoattractant cytokine for neutrophil and is associated with multiple organ failure. Research has indicated IL-8 to be raised in severe and fatal cases of leptospirosis, but its suitability as a prognostic biomarker has yet to be confirmed. This study aimed to evaluate the significance of IL-8 with the clinical outcomes of leptospirosis patients. Plasma IL-8 was measured in fifty-two samples from hospitalized patients and nineteen healthy controls. The comparisons were made between mild, severe-survived and fatal groups identified by clinical or laboratory findings. IL-8 was significantly higher in fatal (p = 0.01) compared to mild cases. IL-8 was also significantly higher in fatal (p = 0.02) when compared to survived cases of leptospirosis. IL-8 levels in the plasma of fatal leptospirosis cases were significantly elevated compared to survived cases and may serve as a potential prognostic biomarker in determining the possible outcome of leptospirosis patients.
CURCUMA LONGA: (C. longa) rhizome extract has been traditionally used to treat many infections. Curcumin, a pure compound isolated from the plant, has been documented to possess a wide spectrum of pharmacological effects. The present study aimed to investigate the effects of Thai medicinal plant extracts including C. longa extract and Curcumin on Acanthamoeba triangularis, a causative agent of human Acanthamoeba keratitis. The parasite was isolated from the recreational reservoir at Walailak University, Thailand. The organism was identified as A. triangularis using morphology and 18S rDNA nucleotide sequences. The pathogen was tested for their susceptibility to ethanol extracts of Thai medicinal plants based on eye infection treatment. The ethanol C. longa extract showed the strongest anti-Acanthamoeba activity against both the trophozoites and cysts, followed by Coscinium fenestratum, Coccinia grandis, and Acmella oleracea extracts, respectively. After 24 h, 95% reduction of trophozoite viability was significantly decreased following the treatment with C. longa extract at 125 µg/mL, compared with the control (P
A cross-sectional survey was conducted among 1,142 Orang Ali schoolchildren in six states of Peninsular Malaysia to investigate the current prevalence and risk factors of STH infections. Faecal samples were examined using direct smear, formalin-ether sedimentation, Kato-Katz, and Harada-Mori methods. A pre-tested questionnaire was used to collect information on the demographic, socioeconomic, personal hygiene, and health status of the participants. Overall, 70.1% (95% CI = 67.4, 72.7) of the participants were infected with at least one of the STH species. The prevalence of Ascaris lumbricoides, Trichuris trichiura, and hookworm infections was 63.1%, 61.8% and 11.5%, respectively. Moderate-to-heavy STH infections accounted for 61.3% of the total infections. Univariate and logistic regression analyses revealed different sets of risk factors, with age (> 10 years) being the significant risk factor of all three STH species. Moreover, other species-specific risk factors were identified including being a member of the Senoi tribe, family size (≥ 7 members), school size (150-250 pupils), maternal unemployment, unimproved source of drinking water, lacking improved toilet in the house, inadequate WASH facilities at school, not washing hands before eating, and not washing fruits before eating; presence of domestic animals, and not wearing shoes when outside. The high prevalence of STH infections found in the study population exceeds the WHO policy intervention threshold (20% prevalence). Thus, an innovative holistic approach should be adopted to control STH infections among these children as part of the efforts to improve the quality of life of the entire Orang Asli population. .
Health-care-associated infections (HAIs) are considered a serious public health issues that contribute substantially to the global burden of mortality and morbidity with respect to infectious diseases. The aim is to assess the burden of health-care-associated infections by collation of available data from published point prevalence surveys (PPS) on HAIs to give future guidance. Study protocol and methodology were designed according to preferred reporting items for systematic reviews and meta-analysis (PRISMA) guidelines. Published research papers that conducted a point prevalence survey of HAIs in hospital settings by following the structured survey methodology employed by European Centre of Disease Prevention and Control (ECDC) were included. Of 1212 articles, 67 studies were included in the final analysis conducted across different countries. Overall, 35 studies were conducted in Europe, 21 in Asia, 9 in America, and 2 in Africa. The highest prevalence of HAIs was recorded in a study conducted in adult ICU settings of 75 regions of Europe (51.3%). The majority of the studies included HAI data on urinary tract infections, respiratory tract infections, and bloodstream infections. Klebsiella pneumonia, Pseudomonas aeruginosa and E. coli were the most frequent pathogens responsible for HAIs. PPS is an useful tool to quantify HAIs and provides a robust baseline data for policymakers. However, a standardize surveillance method is required. In order to minimize the burden of HAIs, infection prevention and control programs and antibiotic stewardship may be effective strategies to minimize the risk of HAIs.
Amoebiasis, caused by Entamoeba histolytica, is one of the leading parasitic infections in the world. This study was aimed at profiling antigenic membrane proteins of a virulent variant of E. histolytica HM-1:IMSS. The membrane proteins were extracted using ProteoExtract® kit (Merck, Darmstadt, Germany) or conventional method, separated using OFFGEL 3100 fractionator (Agilent Technologies, Santa Clara, California), followed by SDS-PAGE and Western blot analysis. Selected antigenic membrane proteins were identified using LC-ESI-MS/MS. Subsequently, the proteins were classified according to their biological processes and predictions were made on membrane and membrane-associated proteins. When the proteins were probed with pooled sera from amoebic liver abscess (ALA) patients, 10 and 15 antigenic proteins with molecular weights 25 to 200 kDa were identified using the ProteoExtract® kit and conventional method, respectively. LC-ESI-MS/MS identified 13 antigenic proteins, and both extraction methods predicted six of them as membrane and membrane-associated proteins. The topmost biological processes which comprised of six proteins were involved in cellular processes.. These antigenic membrane proteins merit further investigations as potential candidates for vaccine studies.