In Vitro Expression of Filarial SXPI Gene for the Development of a Nucleic Acid Based Vaccine

The objectives of this study were to clone gene that encode filarial SXPI protein followed by in vitm expression of the protein. The Special Programme for Research and Training in Tropical Diseases (TDR) WHO has advocated SXPl as one of the vaccine candidate to curb filarial infection. SXPl antigen has been reported to confer protective immunity, causing reduction of microfilaraemia levels in jirds (Meriones unguiculatus) blocking subsequent Brugia malayi infection. In this study, the gene that encode SXPl antigen was 517 bp in length and was extracted and amplified from the infective stage (L3) of subperiodic Brugia malayi. The gene was successfully cloned into replication vector p ~ ~ ~ 2(Inv. it1rog en) followed by subcloning into mammalian expression vector pVAXl (Invitrogen). The presence of SXPl gene in - both vectors were validated by polymerase chain reaction (PCR), restriction enzymes analysis (RE) and finally by automated sequencing. The cloned SXPl in pVAX was designated as pVAXISXP1. The plasmid bearing SXPI gene was transfected into two types of animal cell lines (COS-7 and CHO) using Polyfect Transfection Reagent (Qiagen). The successful expression of targeted gene in the mammalian cell lines were determined by RT-PCR and Western Blotting. The PCR product of the transfected cells was 517 bp on the agarose gel. In addition, the -20 kDa of expressed SXPl protein was detected on nitrocellulose membrane by rabbit polyclonal antibody against the SXPI protein. This study has successfully established the ground work for future deliberations towards the development of antibrugia transmission blocking genetic vaccine

Aedestech Mosquito Home System prevents the hatch of Aedes mosquito eggs and reduces its population

Dengue fever (DF) is a global health problem and considered to be endemic in Malaysia. Conventional mosquito traps currently applied as vector control do not effectively reduce Aedes mosquito population. AedesTech Mosquito Home System (AMHS) is an autocidal ovitraps for Aedes mosquitoes that uses the ‘lure and kill’ concept and is expected to be able to reduce Aedes mosquito population. The effectiveness of AMHS in reducing Aedes mosquito population was investigated in Block A, B and D (control) of the 17th College, Universiti Putra Malaysia (UPM). For the first two weeks (pre-intervention), the conventional ovitraps were used to obtain the initial abundance of mosquito population in Block A, B and D. Subsequently, AMHS was used for the next three months and again followed by the conventional ovitrap for the final two weeks (post-intervention). Ovitrap Index, Hatching Index and percentage of emergence of adult mosquitoes were calculated once every two weeks. Data were analysed using Paired Sample T-test. Values were considered significant at p≤0.05. The Ovitrap Index that indicates the mosquito population at Block A and B was significantly higher (p≤0.05) than of Block D. Hatching Index of AMHS was significantly lower (p≤0.05) then conventional ovitraps. All mosquito eggs collected in AMHS did not develop into adult mosquitoes. There was a significant reduction (p≤0.05) in the mosquito population between the pre- and post-intervention. In conclusion, AMHS was effective in reducing the mosquito population in 17th College, UPM. Therefore, it is believed to be a very promising vector management option to control the incidence of DF

Antiplasmodial and chloroquine chemosensitizing and resistance reversal effects of coumarin derivatives against Plasmodium falciparum 3D7 and K1

Background Emergence of chloroquine (CQ) resistance among different strains of Plasmodium falciparum is the worst incident that has ever faced the dedicated efforts to eradicate malaria. The main cause of CQ resistance is over-activity of the pumping mechanism that ousts CQ outside the DV. This urged the scientists to look for other alternatives or adjuvants that augment its action. CQ The study aimed to test the potential of five coumarin derivatives, namely; umbeliferon, esculetin, scopoletine, herniarin and 3-aminocoumarine to inhibit plasmodium growth and reverse CQ resistance in Plasmodium falciparum K1 and 3D7. They are highly ubiquitous in nature and are famous by their diverse pharmacological effects. SYBRE green-1 based drug sensitivity assay was used to screen the effect of CQ and each coumarin on the parasite growth and isobologram technique was to assess the interaction of the coumarins with CQ. Effect of each coumarin on both RBCs and Vero cells stability as well as on RBCs fragility were screened to exclude any toxic impact on normal cells. On the other hand, their effect on hemozoin formation was screened to investigate about their molecular mechanism. For molecular characterization, Their antioxidant properties were determined using the conventional in vitro tests and their characters were obtained from Molinspiration Simulation Software. Results showed that all of them were safe to human cells, have weak to moderate plasmodial growth inhibitory effect and only umbeliferon, 3- aminocoumarin and esculetin has interacted effectively with CQ. These actions are neither correlated with hemozoin formation inhibition nor to the antioxidant mechanisms. Further studies recommended to investigate the mechanism of their action. Overall, all the tested coumarins are not ideal to be used in the conventional malaria therapy and only umbeliferon, 3-aminocoumarin and esculetin can be suggested to potentiate CQ action

Investigations for the possible use of a monoclonal antibody produced against strongyloides ratti antigen as an immunodiagnostic reagent for active strongyloidiasis

Background: Currently, most of the available serological diagnostic kits for strongyloidiasis are based on the use of the crude antigens of Strongyloides ratti, which are good, but with less sensitivity towards the infection. Hence, this study aimed to produce and evaluate monoclonal antibody for detecting soluble parasite antigen in animal sera. Methods: The study was conducted in the Department of Medical Microbiology and Parasitology, University Putra Malaysia in 2014-2017. Saline extract protein from the infective larvae of S. ratti was used to immunize BALB/c mice and subsequent fusion of the B-cells with myeloma cells (SP2/0) using 50% PEG. The hybridomas were cultured in HAT medium and cloned by limiting dilutions. Positive hybrids were screened by indirect ELISA. The ascites fluid from the antibody-secreting hybridoma was purified and the MAb was characterized by western-blots and evaluated in sandwich ELISA for reactivity against the homologous and heterologous antigens. Results: An IgG1 that recognizes a 30 and 34 kDa protein bands was obtained. The MAb was recognized by all S. ratti-related antigens and cross-reacted with only Toxocara canis antigens in both assays. The minimum antigen detection limit was found to be 5 ng/ml. All antibody-positive rat and dog sera evaluated have shown antigen-positive reactions in Sandwich-ELISA. Conclusion: The MAb produced, was able to detect antigens in strongyloidiasis and toxocariasis in animal models and may also be useful for the serological detection of active strongyloidiasis and visceral toxocariasis in human sera

Anti-plasmodial and chloroquine resistance suppressive effects of embelin

Background: Emergence of chloroquine (CQ) resistance among different strains of Plasmodium falciparum is the worst catastrophe that has ever perplexed the dedicated efforts to eradicate malaria. This urged the scientists to search for new alternatives or sensitizers to augment its antiplasmodium effect. Materials and Method: In this experiment, the potential of embelin, isolated from Embelia ribes, to inhibit the growth and sensitize CQ action was screened using SYBRE-green-I based drug sensitivity and isobologram assays, respectively. Its effect on red blood cells stability was screened to assess its safety. To explore its molecular mechanism, its effect on plasmodial Hemozoin and the in vitro β-hematin formation was screened as well. Furthermore, its anti-oxidant activity was measured using the conventional in vitro tests and its molecular characters were obtained using Molispiration program. Results: The results showed that its anti-plasmodial effect was weaker than CQ but synergism was obtained when they were combined at ratios lower than 5:5 CQ/embelin. Furthermore, β-hematin formation was inhibited by embelin without showing any synergism after mixing with CQ. Conclusion: Overall, embelin is not ideal to be suggested as a conventional antiplasmodium but it has a potential to ameliorate CQ resistance. Furthermore, its action is not related to its impact on hemozoin formation. Further, investigations are recommended to illustrate its detailed mechanism of action

Glycerol: An unexpected major metabolite of energy metabolism by the human malaria parasite

<p>Abstract</p> <p>Background</p> <p>Malaria is a global health emergency, and yet our understanding of the energy metabolism of the principle causative agent of this devastating disease, <it>Plasmodium falciparum</it>, remains rather basic. Glucose was shown to be an essential nutritional requirement nearly 100 years ago and since this original observation, much of the current knowledge of <it>Plasmodium </it>energy metabolism is based on early biochemical work, performed using basic analytical techniques (e.g. paper chromatography), carried out almost exclusively on avian and rodent malaria. Data derived from malaria parasite genome and transcriptome studies suggest that the energy metabolism of the parasite may be more complex than hitherto anticipated. This study was undertaken in order to further characterize the fate of glucose catabolism in the human malaria parasite, <it>P. falciparum</it>.</p> <p>Methods</p> <p>Products of glucose catabolism were determined by incubating erythrocyte-freed parasites with D-[1-¹³C] glucose under controlled conditions and metabolites were identified using ¹³C-NMR spectroscopy.</p> <p>Results</p> <p>Following a 2 h incubation of freed-<it>P. falciparum </it>parasites with 25 mM D-[1-¹³C] glucose (<it>n </it>= 4), the major metabolites identified included; [3-¹³C] lactate, [1,3-¹³C] glycerol, [3-¹³C] pyruvate, [3-¹³C] alanine and [3-¹³C] glycerol-3-phosphate. Control experiments performed with uninfected erythrocytes incubated under identical conditions did not show any metabolism of D-[1-¹³C] glucose to glycerol or glycerol-3-phosphate.</p> <p>Discussion</p> <p>The identification of glycerol as a major glucose metabolite confirms the view that energy metabolism in this parasite is more complex than previously proposed. It is hypothesized here that glycerol production by the malaria parasite is the result of a metabolic adaptation to growth in O₂-limited (and CO₂elevated) conditions by the operation of a glycerol-3-phosphate shuttle for the re-oxidation of assimilatory NADH. Similar metabolic adaptations have been reported previously for other microaerobic/anaerobic organisms, such as yeast, rumen protozoa and human parasitic protozoa.</p> <p>Conclusion</p> <p>These data highlight the need to re-evaluate the carbon and redox balance of this important human pathogen, ultimately leading to a better understanding of how the parasite is able to adapt to the variable environments encountered during parasite development and disease progression.</p

Andrographolide effect on both Plasmodium falciparum infected and non infected RBCs membranes

Objective: To explore whether its antiplasmodium effect of andrographolide is attributed to its plausible effect on the plasma membrane of both Plasmodium falciparum infected and non-infected RBCs. Methods: Anti-plasmodium effect of andrographolide against Plasmodium falciparum strains was screened using the conventional malaria drug sensitivity assay. The drug was incubated with uninfected RBCs to monitor its effect on their morphology, integrity and osmotic fragility. It was incubated with the plasmodium infected RBCs to monitor its effect on the parasite induced permeation pathways. Its effect on the potential of merozoites to invade new RBCs was tested using merozoite invasion assay. Results: It showed that at andrographolide was innocuous to RBCs at concentrations approach its therapeutic level against plasmodia. Nevertheless, this inertness was dwindled at higher concentrations. Conclusions: In spite of its success to inhibit plasmodium induced permeation pathway and the potential of merozoites to invade new RBCs, its anti-plasmodium effect can't be attributed to these functions as they were attained at concentrations higher than what is required to eradicate the parasite. Consequently, other mechanisms may be associated with its claimed actions

An overview of the prevalence and distribution of gastrointestinal parasitic infections in post-war Iraq

Many modern-day diagnostic tests for parasitic diseases rely on conventional labour-intensive technologies such as serology and microscopy. Although major advances have been recorded in the diagnosis of infectious diseases in humans, parasitic diseases continue to present challenges, particularly in resource-poor countries, and this is mainly attributable to war and famine. Factors such as poverty, deteriorated health facilities and destruction of infrastructure are the consequence of the lack of suitable sanitary practices and proper hygiene, especially in refugee camps, that adversely promote infectious diseases to migrants, particularly among vulnerable children. Generally, the gastrointestinal tract is the predilection site for most helminths and protozoa. They are therefore regarded as a serious public-health problem, as they cause malabsorption, malnutrition and blood loss, leading to anaemia or even death. In addition to their health effects, parasitic infections cause physical and mental impairment in children, retard their educational achievements and hinder economic development.Keywords: Prevalence, Parasitic diseases, Intestinal parasite, Food-borne, Water-borne, Pathogens, Post-war, Ira

Intestinal microsporidiosis: a new entity in Malaysia?

Objective: Intestinal microsporidia is an emerging human disease caused by microsporidia. A study was conducted to determine the prevalence of microsporidia in patients with gastro-intestinal symptoms and to examine the clinical manifestations associated with intestinal microsporidiosis. Methods: A descriptive cross-sectional study using a well-structured questionnaire; a review of medical records was also undertaken. Positive stool samples were defined as presence of one or more pinkish-violet ovoid structures with a belt-like stripe under high power field (100x) using modified gram-chromotrope stain (MGC). Results: A total of 353 faecal specimens of patients was examined and 100 patients were found to have positive stool samples for microsporidia. The overall prevalence of microsporidia was 28.3%. Acute and chronic diarrhoea were seen in 49.0% and 36.0% patients, respectively. The commonest clinical presentations were diarrhoea (85.0%) with 83.0 % of patients having loose or watery stools, vomiting (75.0%), foul-smelling stools (60.0%), nausea (59.0%) and cramping abdominal pain (39.0%). The least common symptoms were fever (15.0%), mucous in stool (5.0%) and blood in stool (4.0%). Conclusion: This study concludes that the prevalence of microsporidia is still high (28.3%) and the majority of patients (93.0%) are symptomatic; the most common gastro-intestinal symptom is diarrhoea with loose or watery stools. Hence, it is recommended that a stool screening for microsporidia be done in selected patients presented with gastrointestinal symptoms