Molecular and Immunological Identifications of Giardia Sp. Isolated From Humans, Dogs and Rodents

Concentrations and staining methods for identification of Giardia parasites in faecal materials from humans and animals are still the routine methods of diagnosis of giardiasis. Introduction of new and sensitive immunological and molecular methods will definitely facilitate diagnosis and the identification of various Giardia species that will ultimately improve clinical management and control of disease transmission. The identification of specific proteins of Giardia parasites, which are genus and species specific, may further improve the specificity and sensitivity of diagnostic methods. In this study, identification and confirmation of the species of Giardia parasite found in Malaysia, particularly in humans, dogs and rodents were done by Polymerase Chain Reaction (PCR) using species-specific primers. Specific Heat-Shock Protein (HSP) as markers for species identification were done using Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) and Western Immunoblotting (WI). These markers were used as antigens for the production of species-specific monoclonal antibodies (MAb). The objectives of this study were: i) to identify the common Giardia sp. infecting humans and other mammals (dogs and rodents) in Malaysia using specific Giardia primers by PCR; ii) to produce in vitro and detect immunogenic HSP using polyclonal sera from rabbit immunised with antigens from known Giardia species on WI, and iii) to produce species-specific MAb from the identified HSP markers. Microscopically, this study has observed that G. intestinalis (GI) and G. duodenalis (GD) were indistinguishable but G. muris (GM) can be distinguished from GI or GD. In addition, this study has also confirmed that PCR using species-specific primers was more reliable and accurate in detecting the variant of GI found in humans and dogs. GD isolates recovered from dogs was found to be the actual variant of GI of humans. Clear morphological differentiations and identifications of GM and GI based on microscopical examination were observed and similar results were obtained by PCR using species-specific primers of respective species of Giardia. However, the SDSPAGE and WI failed to identify species-specific HSP markers, but WI using immunised rabbit sera detected four immunogenic HSP, with the molecular weight of 30 kDa, 34 kDa, 58 kDa and 66 kDa. These four immunogenic HSP were detected at 25°C, 37 °c and 50°C in both GI and GM. Three species-specific MAbs were produced using the combinations of the four immunogenic HSPs as antigens. These MAbs were designated as (i) [32 kDa HSPMAbGi(IgG3)], (ii) [29 kDa HSPMAbGm(IgM)], and (iii) [20 kDa HSPMAbGi(IgGl)]. [32 kDa HSPMAbGi(IgG3)] MAb was specific for GI variant found in humans, [29 kDa HSPMAbGm(IgM)] MAb was specific for GM and [20 kDa HSPMAbGi(IgGl)] was specific for GI variant in both humans and dogs. These findings suggest that GI is the main causative agent of giardiasis in both humans and urban dogs in Malaysia GM is the main Giardia parasite infecting rodents in both rural and urban areas in Malaysia

Molecular Identification of Giardia duodenalis Isolates from Fars Province, Iran

Background: Giardia duodenalis is one of the most common human intestinal protozoan parasites worldwide and is endemic throughout the world with a vast range of mammalian hosts. The present study aimed to identify the prevalence of G. duodenalis isolates and determine the most common of its assemblages in the patients referring to health centers and hospitals in Fars province, Iran that will be subjected to further molecular investigation. Methods: We collected 1000 human fecal samples from health centers and hospitals in Shiraz, Iran in a one year period from September 2009 to August 2010. Microscopic examination for the presence of G. duodenalis cysts and trophozoites was performed by direct wet mount before and after the concentration techniques. Extraction of DNA was performed by Phenol-Chloroform-Isoamylalcohol (PCI). G. duodenalis-positive specimens were analyzed by PCR. A fragment of the SSU-rDNA (292 bp) gene was amplified by PCR using the forward primer RH11 and the reverse primer RH4. Genotyping was performed using sequence analysis of G. duodenalis glutamate dehydrogenase gene using primers GDHeF, GDHiF, and GDHiR. Results: The prevalence of Giardia infection was 10.7% (107/1000) examined based on microscopic examination. PCR identified 80% (40/50) of the samples as positive for G. duodenalis based on SSU-rDNA amplification on sucrose gradient samples. Besides, genotyping results indicated 32 isolates (80%) as assemblage AII and 8 isolates (20%) as assemblage BIII and BIV based on the DNA sequence analysis of the glutamate dehydrogenase locus of G. duodenalis. Conclusion: The findings of this study emphasize that Iran (Fars Province) is a favorable area for giardiasis with an anthroponotic infection route

In vitro activity of Piper sarmentosum ethanol leaf extract against Toxoplasma gondii tachyzoites

Purpose: To evaluate the activity of the ethanol leaf extract of Piper sarmentosum against toxoplasmosis. Methods: An in vitro anti-Toxoplasma study was conducted using Vero cells as a host for T. gondii. Clindamycin used as the reference drug. Light microscopy technique was used to study the in situ antiparasitic activity of T. gondii. Non-toxic concentrations of the ethanol extract for Vero cells were determined by methyl thiazolyl tetrazolium (MTT) cell proliferation. The presence of Toxoplasma gondii was observed by Giemsa staining. Results: The results showed that significant (p < 0.05) anti-toxoplasma activity of the ethanol extract, though lower than that of clindamycin (control drug), was achieved, with half-maximal inhibitory concentration (IC50) of 12.4 and 7.2 μg/mL for the extract and reference drug, respectively. After 24 hours of exposure to the extract, the inoculated Vero cells showed lower parasitemia and no remarkable morphological changes. Conclusion: The findings demonstrate that the ethanol extract of P. sarmentosum leaves are active against toxoplasmosis in vitro. However, further studies are required to determine the therapeutic significance of these findings in viv

The impact of preventive fogging on entomological parameters in a university campus in Malaysia

Preventive fogging is defined as space spraying of insecticide against mosquitoes in order to prevent outbreak of mosquito borne infection. Despite provision of various preventive andcontrol activities against dengue and chikungunya infection by Ministry of Health Guideline, the detail on preventive fogging has not yet specified. However, this has been adopted by certain institutions as part of the routine strategies against dengue outbreak. A study on preventive fogging was conducted in one of the hostels in Universiti Putra Malaysia. The research was done for 16 weeks in which one routine fogging activity was done at the mid period of study. The main objectives of this study were to determine the effectiveness of preventive fogging activities against Aedes mosquitoes and to identify the distribution and abundance of Aedes mosquitoes in the area. Method: The fogging activity was carried out by the management staff as part of their preventive measures in the student hostels. Ovitrap was used as an indicator to monitor the impact of fogging activity and its continuous surveillance was monitored weekly. The ovitraps were placed indoors and outdoors. Species identification was carried out in the laboratory. The SPSS program was used to analyse the statistical data on the effectiveness of fogging activity. Larval count (indoors and outdoors) and ovitrap index (OI) readings were identified as ovitrap surveillance data for statistical analysis. Results: The results showed that Aedes albopictus was the only species of the genus Aedes found in this hostel. The area had been highly infested by Ae. Albopictus as indicated by high Ovitrap Index ranging between 48.33% to 90.00%. The mean (SD) of Ovitrap Index was reduced from 71.67% (12.73%) (before the preventive fogging), to 69.42% (14.40%) (after the fogging). Overall reduction in mosquito and larval density was also observed between pre and post fogging activity in this study. Conclusion: The implementation of preventive fogging has favourably reduced the dengue vector population up to 5 weeks after the introduction of preventive fogging. However, sole dependency on preventive fogging may lead to insecticide resistance. Revisiting the policy on preventive fogging; and identifying it as an additional tool for preventing dengue infection in higher learning institutions are recommended

Determination of diagnostic value of Toxoplasma gondii recombinant surface antigen (SAG1, P30) in mouse experimental model

The aim of this study was to test the potential diagnostic usefulness of recombinant Toxoplasma gondii SAG1 antigen and excretory-secretory antigen (ESA), with respect to toxoplasmosis detection and infection phase distinction in laboratory mouse by determining specifi c serum IgM and IgG antibodies with the use of indirect ELISA technique. The highest titre at the beginning of infection was demonstrated by immunoglobulin M while the highest titre at the end of the infection was displayed by immunoglobulin G. In contrast, sera of chronically infected mice, positive IgG titre was detected on day 14 p.i. for ESA or day seven p.i. with rSAG1 and increased thereafter until day 70 p.i. after which the titre stabilized. IgA antibody also showed similar kinetics as IgG. Potentially rSAG1 may be a suitable diagnostic antigen than ESA in the diagnosis of acute and chronic toxoplasmosis

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

Cutaneous larva migrans : a neglected disease and possible association with the use of long socks

Cutaneous larva migrans is a common parasitic skin disease that can be easily prevented by wearing 'protective' footwear. However, this has been under-emphasized in terms of what constitutes the protective footwear. Even though the disease resolves spontaneously, the significant duration of the disease along with severity of pruritus make treatment unavoidable. Here, we present a very long-standing creeping eruption, which puzzled many attending clinicians handling the case, and the possibility of long socks as a causal effect on the development of cutaneous larva migrans infection

Molecular characterization of a cDNA encoding an excretory–secretory antigen from Toxocara canis second stage larvae and its application to the immunodiagnosis of human toxocariasis

The cDNA encoding an excretory–secretory antigen from the second stage larvae of Toxocara canis has been characterized. Sequence analysis revealed an open reading frame encoding a protein of 226 amino acid residues (Mr=24 398). Sequence database searches showed similarities to regions corresponding to epidermal growth factor-like and lectin-like domains of the core proteins of vertebrate chondroitin sulfate proteoglycans, which are major components of the extracellular matrix. The T. canis core protein was expressed as a fusion protein with thioredoxin A using an Escherichia coli expression system, and then affinity purified on a metal affinity resin in the presence of 8 M urea. When the purified recombinant T. canis protein was used as an antigen, immunoblot analysis revealed the protein specifically reacted with sera from toxocariasis patients. The antigenic protein did not react with sera from patients with Brugia malayi infection, dirofilariasis, or ascariasis. In some cases of anisakiasis, cross-reactions were observed; however, the cross-reacting bands disappeared when anisakiasis sera preabsorbed with Anisakis antigen were used, indicating that the recombinant T. canis protein is very promising for use as an immunodiagnostic antigen for human toxocariasis

Current trend on the economic and public health significance of Salmonellosis in Iraq

Salmonellosis is reported as one of the main cause of diarrhoeal diseases globally. The disease is also associated with enteric fever, including typhoid which is a potentially fatal systemic illness bedeviling many developing countries. The disease is estimated to affect nearly 17 million people with over 150,000 deaths occurring annually. Salmonellosis is also beginning to emerge as a foodborne infection characterized by significant economic and public health hazard with global ramifications. High prevalence of the disease is directly related to poor sanitation and hygiene, consumption and use of unsafe water, overcrowding and social unrest. A significant number of Iraqis are affected annually with a death rate of 10-20 %, mainly resulting from limited access to fresh water and improper sewage disposal into the river bodies. This review provides an overview of Salmonella infection in human and animals, with emphasis on the economic and public health burden of the disease in Iraq

Molecular detection of Strongyloides ratti in faecal samples from wild rats in Serdang, Malaysia

Purpose: To detect Strongyloides ratti in faecal samples using conventional methods and to confirm the identification using a sensitive and specific method, namely, polymerase chain reaction (PCR). Methods: A PCR method targeting the small subunit of the rRNA gene was performed in this study for the detection of DNA from Strongyloides ratti (an animal model of S. stercoralis) in faecal samples of wild Brown rats, Rattus norvegicus. Results: Strongyloides ratti was detected in 34.2 % of collected rats by different conventional techniques and confirmed by PCR. The essay presented 100 % sensitivity with Strongyloides universal primer. Conclusion: The findings of this study suggest that the application of PCR with universal primer is a very sensitive methodology to detect S. ratti in faecal material of wild rats infected even with very low parasite burden