Enteric Protozoan Parasite Research Articles

In Vitro Anti-Protozoal Activity of Propolis Extract and Cysteine Proteases Inhibitor ( Phenyl Vinyl Sulfone ) on Blastocystis Species

Blastocystis is one of the commonest enteric protozoan parasites worldwide. Despite its controversial clinical significance, frequent association with symptoms has necessitated treatment of cases with persistent symptoms. For long time, metronidzole (MTZ) was considered as a basic drug for blastocystosis treatment, however reports of treatment failure as well as its well-known side effects has promoted the search for more safe and effective alternatives. In vitro antiprotozoal activity of ethanolic extract of Egyptian propolis and a cysteine protease inhibitor, phenyl vinyl sulfone (PVS) on Blastocystis spp. was assessed through challenging with graded concentrations of propolis extract (125, 250, 500 & 1000pg/ml) and PVS (100, 200 and 300 ptg/ml) compared to MTZ (10, 50 and 100 pg/ml) and viable parasites were counted after 24, 48 and 72 hr. of incubation. Molecular subtyling of Blastocystis spp. was done using subtype specific sequence-tagged site (STS) primers. Propolis extract inhibited the growth of Blastocystis spp. in both of the detected subtypes (STI and ST3), which was especially observed in cultures exposed to 500 & 1000 μg/ml through all incuba- tion periods with the later concentration producing comparable results to MTZ. While PVS showed significant parasite count reduction on ST3 isolates, especially with the highest concentration, however the effect on STl isolate was nonsignificant. These findings highlight the potential antiprotozoal activity of propolis extract as a potent natural alternative for MTZ in treatment of blastocystosis.

Role of cysteine residues in the redox-regulated oligomerization and nucleotide binding to EhRabX3

The enteric protozoan parasite, Entamoeba histolytica, an etiological agent of amebiasis, is involved in the adhesion and destruction of human tissues. Worldwide, the parasite causes about 50 million cases of amebiasis and 100,000 deaths annually. EhRabX3, a unique amoebic Rab GTPase with tandem G-domains, possesses an unusually large number of cysteine residues in its N-terminal domain. Crystal structure of EhRabX3 revealed an intra-molecular disulfide bond between C39 and C163 which is critical for maintaining the 3-dimensional architecture and biochemical function of this protein. The remaining six cysteine residues were found to be surface exposed and predicted to be involved in inter-molecular disulfide bonds. In the current study, using biophysical and mutational approaches, we have investigated the role of the cysteine residues in the assembly of EhRabX3 oligomer. The self-association of EhRabX3 is found to be redox sensitive, in vitro. Furthermore, the oligomeric conformation of EhRabX3 failed to bind and exchange the guanine nucleotide, indicating structural re-organization of the active site. Altogether, our results provide valuable insights into the redox-dependent oligomerization of EhRabX3 and its implication on nucleotide binding.

Evaluation of five commercial methods for the extraction and purification of DNA from human faecal samples for downstream molecular detection of the enteric protozoan parasites Cryptosporidium spp., Giardia duodenalis, and Entamoeba spp

High quality, pure DNA is required for ensuring reliable and reproducible results in molecular diagnosis applications. A number of in-house and commercial methods are available for the extraction and purification of genomic DNA from faecal material, each one offering a specific combination of performance, cost-effectiveness, and easiness of use that should be conveniently evaluated in function of the pathogen of interest. In this comparative study the marketed kits QIAamp DNA stool mini (Qiagen), SpeedTools DNA extraction (Biotools), DNAExtract-VK (Vacunek), PowerFecal DNA isolation (MoBio), and Wizard magnetic DNA purification system (Promega Corporation) were assessed for their efficacy in obtaining DNA of the most relevant enteric protozoan parasites associated to gastrointestinal disease globally. A panel of 113 stool specimens of clinically confirmed patients with cryptosporidiosis (n=29), giardiasis (n=47) and amoebiasis by Entamoeba histolytica (n=3) or E. dispar (n=10) and apparently healthy subjects (n=24) were used for this purpose. Stool samples were aliquoted in five sub-samples and individually processed by each extraction method evaluated. Purified DNA samples were subsequently tested in PCR-based assays routinely used in our laboratory. The five compared methods yielded amplifiable amounts of DNA of the pathogens tested, although performance differences were observed among them depending on the parasite and the infection burden. Methods combining chemical, enzymatic and/or mechanical lysis procedures at temperatures of at least 56°C were proven more efficient for the release of DNA from Cryptosporidium oocysts.

Identification and genotyping of Giardia spp. and Cryptosporidium spp. isolates in aquatic birds in the Salburua wetlands, Álava, Northern Spain

Aquatic birds are known to be suitable hosts for a number of avian-specific species and genotypes of the enteric protozoan parasites Giardia and Cryptosporidium. Waterbirds have also been reported as sporadic carriers of species of both pathogens from human or domestic animal origin via environmental contamination. Because aquatic birds can shed substantial amounts of infective Giardia and Cryptosporidium (oo)cysts to the environment including surface waters intended for human consumption, this situation may pose a potential risk of waterbone zoonotic disease.A total of 265 waterbird faecal samples were collected from May 2014 to June 2015 at Salburua (Álava), one of the most valued continental wetlands in northern Spain. The detection of Giardia oocyst and Cryptosporidium oocysts was carried out by direct fluorescence microscopy and molecular (PCR and sequence analysis) methods targeting the small subunit ribosomal RNA gene of the parasites. Typing of Giardia duodenalis isolates at the sub-assemblage level was based on the specific amplification and sequencing of a partial fragment of the glutamate dehydrogenase gene.Overall, Giardia cysts and Cryptosporidium oocysts were detected in 22 (8.3%) and 6 (2.3%), respectively, of the 265 faecal samples analysed. The two only Giardia isolates characterized (one novel, one known) were assigned to the sub-assemblage BIV of G. duodenalis, none of them previously reported in Spanish human isolates. This finding raises doubts about the actual origin of the infection and whether waterbirds may serve as potential source of infective Giardia cysts to humans via waterborne transmission or through direct contact. The six Cryptosporidium isolates obtained were characterized as avian genotype III (n=4), duck genotype b (n=1), and goose genotype Id (n=1), all considered avian-specific and therefore of negligible risk of zoonotic infection.

Studies on Blood and Enteric Protozoans Infecting Camels At Behera Province, Egypt

This study was carried out to investigate the prevalence of blood and enteric protozoan parasites in freshly slaughtered camels at Behera Province, during the years of 2014 and 2015. 120 (66 male and 54 female) freshly slaughtered camels were examined, 94 (46 males and 48 females) (78.3%) were found to be infected with enteric protozoa, while 63 camels (24 males and 39 females) (52.5%) were found to be infected with blood protozoa. Concerning enteric protozoa, the highest rate was recorded for Diplodinium spp. (58%), followed by Cryptosporidium parvum. (24.16%), then Eimeria spp (13.33%). Eimaria spp. were E.camili (50%), E. dromedarii (31.25%) and, E. pellerdyi (18.75%). Giardia intestinalis, (5%) and Balantidium coli (4.16%). The prevalence of blood protozoa revealed that the highest rate was for Theileria spp., then Anaplasma marginale and Trypanosoma evansi 45.82, 15.8 and 1.66%, respectively. Regarding, seasonal dynamics of enteric protozoan parasites, Cryptosporidium parvum, Eimeria spp and Diplodinium spp showed their highest incidence during Winter, while Balantidium coli recorded the highest rate during Spring. Summer had the highest rate for Theileria spp. but there were abundance of Anaplasma marginale during spring and Autumn. Finally, Trypanosoma evansi were only found during winter and autumn.

Endoparasite survey of free-swimming baleen whales (Balaenoptera musculus, B. physalus, B. borealis) and sperm whales (Physeter macrocephalus) using non/minimally invasive methods.

A number of parasitic diseases have gained importance as neozoan opportunistic infections in the marine environment. Here, we report on the gastrointestinal endoparasite fauna of three baleen whale species and one toothed whale: blue (Balaenoptera musculus), fin (Balaenoptera physalus), and sei whales (Balaenoptera borealis) and sperm whales (Physeter macrocephalus) from the Azores Islands, Portugal. In total, 17 individual whale fecal samples [n = 10 (B. physalus); n = 4 (P. macrocephalus); n = 2 (B. musculus); n = 1 (B. borealis)] were collected from free-swimming animals as part of ongoing studies on behavioral ecology. Furthermore, skin biopsies were collected from sperm whales (n = 5) using minimally invasive biopsy darting and tested for the presence of Toxoplasma gondii, Neospora caninum, and Besnoitia besnoiti DNA via PCR. Overall, more than ten taxa were detected in whale fecal samples. Within protozoan parasites, Entamoeba spp. occurred most frequently (64.7%), followed by Giardia spp. (17.6%) and Balantidium spp. (5.9%). The most prevalent metazoan parasites were Ascaridida indet. spp. (41.2%), followed by trematodes (17.7%), acanthocephalan spp., strongyles (11.8%), Diphyllobotrium spp. (5.9%), and spirurids (5.9%). Helminths were mainly found in sperm whales, while enteric protozoan parasites were exclusively detected in baleen whales, which might be related to dietary differences. No T. gondii, N. caninum, or B. besnoiti DNA was detected in any skin sample. This is the first record on Giardia and Balantidium infections in large baleen whales.

Crystal Structure Analysis of Wild Type and Fast Hydrolyzing Mutant of EhRabX3, a Tandem Ras Superfamily GTPase from Entamoeba histolytica

The enteric protozoan parasite, Entamoeba histolytica, is the causative agent of amoebic dysentery, liver abscess and colitis in human. Vesicular trafficking plays a key role in the survival and virulence of the protozoan and is regulated by various Rab GTPases. EhRabX3 is a catalytically inefficient amoebic Rab protein, which is unique among the eukaryotic Ras superfamily by virtue of its tandem domain organization. Here, we report the crystal structures of GDP-bound fast hydrolyzing mutant (V71A/K73Q) and GTP-bound wild type EhRabX3 at 3.1 and 2.8Å resolutions, respectively. Though both G-domains possess “phosphate binding loop containing nucleoside triphosphate hydrolases fold”, only the N-terminal domain binds to guanine nucleotide. The relative orientation of the N-terminal domain and C-terminal domain is stabilized by numerous inter-domain interactions. Compared to other Ras superfamily members, both the GTPase domains displayed large deviation in switch II perhaps due to non-conservative substitutions in this region. As a result, entire switch II is restructured and moved away from the nucleotide binding pocket, providing a rationale for the diminished GTPase activity of EhRabX3. The N-terminal GTPase domain possesses unusually large number of cysteine residues. X-ray crystal structure of the fast hydrolyzing mutant of EhRabX3 revealed that C39 and C163 formed an intra-molecular disulfide bond. Subsequent mutational and biochemical studies suggest that C39 and C163 are critical for maintaining the structural integrity and function of EhRabX3. Structure-guided functional investigation of cysteine mutants could provide the physiological implications of the disulfide bond and could allow us to design potential inhibitors for the better treatment of intestinal amebiasis.

Development of a nucleic Acid extraction procedure for simultaneous recovery of DNA and RNA from diverse microbes in water.

Drinking and environmental water samples contain a diverse array of constituents that can interfere with molecular testing techniques, especially when large volumes of water are concentrated to the small volumes needed for effective molecular analysis. In this study, a suite of enteric viruses, bacteria, and protozoan parasites were seeded into concentrated source water and finished drinking water samples, in order to investigate the relative performance of nucleic acid extraction techniques for molecular testing. Real-time PCR and reverse transcription-PCR crossing threshold (CT) values were used as the metrics for evaluating relative performance. Experimental results were used to develop a guanidinium isothiocyanate-based lysis buffer (UNEX buffer) that enabled effective simultaneous extraction and recovery of DNA and RNA from the suite of study microbes. Procedures for bead beating, nucleic acid purification, and PCR facilitation were also developed and integrated in the protocol. The final lysis buffer and sample preparation procedure was found to be effective for a panel of drinking water and source water concentrates when compared to commercial nucleic acid extraction kits. The UNEX buffer-based extraction protocol enabled PCR detection of six study microbes, in 100 L finished water samples from four drinking water treatment facilities, within three CT values (i.e., within 90% difference) of the reagent-grade water control. The results from this study indicate that this newly formulated lysis buffer and sample preparation procedure can be useful for standardized molecular testing of drinking and environmental waters.

Unexpected finding of feline-specific Giardia duodenalis assemblage F and Cryptosporidium felis in asymptomatic adult cattle in Northern Spain

Giardia duodenalis and Cryptosporidium spp. are common enteric protozoan parasites in production animals, including cattle. Typically, both the clinical outcome of these infections and the distribution of G. duodenalis assemblages and Cryptosporidium species are age-dependent, with the occurrence of diarrhoeal disease being mainly associated with young animals and sub-clinical, low intensity infections being predominantly found in adult animals.To investigate the prevalence and genetic diversity of G. duodenalis and Cryptosporidium spp. in asymptomatic adult cattle, a total of 362 faecal samples were collected in four farms in the province of Álava, Northern Spain, between November 2011 and December 2012. The presence of G. duodenalis was estimated by real-time PCR, and the assemblages were determined by multilocus sequence-based genotyping of the glutamate dehydrogenase and β-giardin genes of the parasite. Detection and identification of Cryptosporidium species was carried out by sequencing of a partial fragment of the small-subunit ribosomal RNA gene.Overall, G. duodenalis and Cryptosporidium spp. were detected in 68 (18.8%) and 45 (12.4%), respectively, of the 362 animals tested. Strikingly, four isolates representing two novel sub-types of G. duodenalis assemblage F were identified at the gdh, but not the bg, locus. This is the first report describing the presence of the feline-specific G. duodenalis assemblage F in bovine isolates. Additionally, five (three novel and two known) sub-types of G. duodenalis assemblage E were also identified at the gdh locus and a single one (assigned to sub-assemblage EII) at the bg locus. Of nine Cryptosporidium isolates, four (including a novel sub-type) were assigned to the cat-specific C. felis, two were typed as C. bovis, and the remaining three were only characterized at the genus level. Data presented here provide epidemiological and molecular evidence demonstrating that the host range specificity of G. duodenalis assemblages and Cryptosporidium species may be wider than previously anticipated.

Modulation of endogenous Cysteine Protease Inhibitor (ICP) 1 expression in Entamoeba histolytica affects amoebic adhesion to Extracellular Matrix proteins

Entamoeba histolytica is an enteric tissue-invading protozoan parasite that causes amoebic colitis and occasionally liver abscess in humans. During tissue invasion, amoebic adhesion to host components is an important event for host cell death leading to successful invasion and infection. Among amoebic virulence factors, Gal/GalNAc lectin is known to be major adhesion factor to host cells. In this study, we investigated the role of amoebic secreted CP (Cysteine Proteases) in amoebic adhesion to extracellular matrix (ECM) protein using CP inhibitor and E. histolytica strains in which the endogenous inhibitor of cysteine protease (ICP) 1 gene was overexpressed (ICP1+) or repressed by antisense small RNA-mediated gene silencing (ICP1−). We found that pretreatment of wild-type amoebae with CP inhibitor E64, or thiol-group modifiers such as diamide and N-Ethylmaleimide resulted in a significant decrease in adhesion to laminin and collagen ECM proteins. Furthermore, ICP1+ strain, with a reduction of secreted CP activity, exhibited reduced ability by 40% to adhere to laminin. In contrast, ICP1− strain, with a 1.9-fold increase of secreted CP activity, showed a two-fold increase in amoebic adherence to laminin compared to the control strain. In addition, total amount of secreted CP5 was decreased in ICP1+ amoeba. Conversely, total amount of secreted CP1 and mature-form CP5 were increased in ICP1− amoeba. We also found that ICP1 was secreted into extracellular milieu. These results suggest that secreted CP activity by E. histolytica may be an important factor affecting adhesion to host proteins, and regulation of CP secretion by ICP plays a major role in pathogenesis. This study provides insight into the CP-mediated tissue pathogenesis in amoeba-invaded lesions during human amoebiasis.

Amoebic PI3K and PKC is required for Jurkat T cell death induced by Entamoeba histolytica.

The enteric protozoan parasite Entamoeba histolytica is the causative agent of human amebiasis. During infection, adherence of E. histolytica through Gal/GalNAc lectin on the surface of the amoeba can induce caspase-3-dependent or -independent host cell death. Phosphorylinositol 3-kinase (PI3K) and protein kinase C (PKC) in E. histolytica play an important function in the adhesion, killing, or phagocytosis of target cells. In this study, we examined the role of amoebic PI3K and PKC in amoeba-induced apoptotic cell death in Jurkat T cells. When Jurkat T cells were incubated with E. histolytica trophozoites, phosphatidylserine (PS) externalization and DNA fragmentation in Jurkat cells were markedly increased compared to those of cells incubated with medium alone. However, when amoebae were pretreated with a PI3K inhibitor, wortmannin before being incubated with E. histolytica, E. histolytica-induced PS externalization and DNA fragmentation in Jurkat cells were significantly reduced compared to results for amoebae pretreated with DMSO. In addition, pretreatment of amoebae with a PKC inhibitor, staurosporine strongly inhibited Jurkat T cell death. However, E. histolytica-induced cleavage of caspase-3, -6, and -7 were not inhibited by pretreatment of amoebae with wortmannin or staurosporin. In addition, we found that amoebic PI3K and PKC have an important role on amoeba adhesion to host compartment. These results suggest that amebic PI3K and PKC activation may play an important role in caspase-independent cell death in Entamoeba-induced apoptosis.

Ligand heterogeneity of the cysteine protease binding protein family in the parasitic protist Entamoeba histolytica

Lysosomal soluble proteins are targeted to endosomes and lysosomes by specific receptors resident in the endoplasmic reticulum and/or the Golgi apparatus. The enteric protozoan parasite Entamoeba histolytica has a novel class of lysosomal targeting receptors, named the cysteine protease binding protein family (CPBF). Among 11 CPBFs (CPBF1–11), ligands for three members, CPBF1, CPBF6 and CPBF8, were previously shown to be cysteine proteases, α- and γ- amylases, and β-hexosaminidase and lysozymes, respectively. To further understand the heterogeneity of the ligands of CPBFs, we attempted to isolate and identify the ligands for other members of CPBFs, namely CPBF2, 3, 4, 5, 7, 9, 10 and 11, by immunoprecipitation and mass spectrometric analysis. We found that CPBF2 and CPBF10 bound to α-amylases while CPBF7 bound to β-hexosaminidases. It is intriguing that cysteine protease are exclusively recognised by CPBF1, whereas three α-amylases and β-hexosaminidases are redundantly recognised by three and two CPBFs, respectively. It was shown by bioinformatics analysis and phylogenetic reconstruction that each CPBF contains six prepeptidase carboxyl-terminal domains, and the domain configuration is evolutionarily conserved among CPBFs. Taken together, CPBFs with unique and conserved domain organisation have a remarkable ligand heterogeneity toward cysteine protease and carbohydrate degradation enzymes. Further structural studies are needed to elucidate the structural basis of the ligand specificity.

Solution structure of a putative FKBP-type peptidyl-propyl cis–trans isomerase from Giardia lamblia

The FK506 Binding Protein (FKBP) super-family was named for the ability of these proteins to bind to the immunosuppressive drug FK506, a 23-member macrolide lactone. Immunosuppression is achieved when the proteinFK506 complex binds to calcineurin to form a larger complex that blocks signal transduction in the T-lymphocyte transduction pathway (Gothel and Marahiel 1999). FKBP proteins also have peptidyl-proline cis–trans isomerases (PPIase) activity that catalyzes the interconversion of the peptidyl-prolyl imide bond between the cis and trans configuration in peptide and protein substrates (Gothel and Marahiel 1999). The isomerase activity is unrelated to the immunosuppressive effects when bound to FK506, but, is essential for the proper folding of many proteins and a number of PPIases act as chaperones. Because proper protein folding is often necessary for proper biochemical function, FKBP proteins are indirectly associated with many diverse biologically processes including trafficking, cell-cycle regulation, development, and signal transduction (Gothel and Marahiel 1999). It has been observed that the parasite Trypanosoma cruzi secretes a FKBP-type protein upon infecting its host, leading to the suggestion that some parasites may use these proteins as virulence factors (Moro et al. 1995). Cultures of the parasite responsible for malaria, Plasmodium falciparum, are sensitive to FK506 (Bell et al. 1994). Regardless of the potential role of FKBP-type proteins as parasite virulence factors, the many critical biologically functions dependent on FKBP-type proteins makes this super-family of proteins good drug targets (Moro et al. 1995; Alag et al. 2010). The most common parasitic protist in the United States is Giardia lamblia (Krappus et al. 1994): the etiological agent responsible for giardiasis, an infection of the small intestines that often causes diarrhea. While giardiasis is generally not life-threatening, in developing countries this enteric protozoan parasite contributes to malnutrition and underperformance in school, and consequently, has been added to the ‘‘Neglected Disease Initiative’’ list by the World Health Organization (Savioli et al. 2006). Recently, the G. lamblia genome was sequenced (Morrison et al. 2007) and among the 6,470 genes encoded in the five chromosomes is a putative, 109-residue FKBP-type PPIase, Gl-FKBP. To assist structure-based design of a more palatable, single-dose treatment for giardiasis targeting GlFKBP, we used NMR-based methods to determine its structure in solution. The structure of Gl-FKBP and the G. W. Buchko S. N. Hewitt W. C. Van Voorhis P. J. Myler Seattle Structural Genomics Center for Infectious Disease, Seattle, WA, USA

DETECTION OF HUMAN INTESTINAL PROTOZOA BY USING MULTIPLEX ALLELE SPECIFIC POLYMERASE CHAIN REACTION (MAS-PCR) In New Damietta City

Enteric protozoa continue to be the most commonly encountered parasitic diseases causing significant morbidity and mortality in developing regions of the world affecting millions of people. This study assessed the use of Multiplex Allele Specific Polymerase Chain Reaction (MAS-PCR) assay and microscopy for detection and identification of common pathogenic protozoan parasites in New Damietta city of North Delta region, Egypt. During Jun 2013 until Sept 2013, fresh stool samples were obtained from 249 patients up to 65 years of age attending the internal clinic of the Damietta University Hospital and those visiting their general practitioner (GP) of outpatient clinics because of gastrointestinal symptoms. Stool samples collected was preserved at -200C for DNA extraction whilst the remaining was preserved in sodium acetate-acetic acid formalin and concentrated using the formol-ether technique for microscopic examination. DNA extracts were analyzed with the multiplex allele specific Polymerase Chain Reaction (MAS-PCR) for pathogenic protozoan parasites. The diagnostic results obtained using a multiplex allele specific PCR for the detection of E. histolytica/dispar, G. lamblia and C. parvum/C. hominis were compared with these obtained by routine microscopy of faecal samples from patients. 69 samples were positive by MAS-PCR assays, 9 cases of G. intestinalis infection, 34 cases of D. fragilis infection, 3 cases of E. histolytica infection, 17 cases E.dispar and 6 cases of Cryptosporidium infection in the clinical samples. By microscopy, only 32 samples were positive for one or more of the enteric protozoa, 5cases of G. intestinalis infection, 9cases of D. fragilis infection, 13 cases of E. histolytica infection, and 1 cases of Cryptosporidium infection in the clinical samples. However, there are no cases of E.dispar observed. Mixed infections were detected in 4 samples. The sensitivities varied from 58% for D. fragilis to 47% for E. histolytica, 35% for Giardia, and 30% for Cryptosporidium, while the specificities also varied from 97% for E. histolytica to 99% for D. fragilis and 100% for E.dispar . No cross-reactivity was detected in stools samples containing various bacterial, viral, and other protozoan species. This present study showed relatively high rates of protozoa infections in the study patients. The study has also demonstrated that the multiplex real time PCR assay was more sensitive compared to microscopy in the diagnosis of the intestinal protozoa parasites and thus, molecular methods must be considered the diagnostic methods of choice for enteric protozoan parasites. Keywords: Human intestinal protozoa, Stools specimens, Microscopy, Multiplex Allele Specific Polymerase Chain Reaction (MAS-PCR), Diagnosis.

DETECTION AND DIFFERENTIATION OF HUMAN PROTOZOAN PARASITES IN STOOL SPECIMENS BY USING MULTIPLEX ALLELE SPECIFIC POLYMERASE CHAIN REACTION (MAS-PCR) In New Damietta City, Egypt

Enteric protozoa continue to be the most commonly encountered parasitic diseases causing significant morbidity and mortality in developing regions of the world affecting millions of people. This study assessed the use of Multiplex Allele Specific Polymerase Chain Reaction (MAS-PCR) assay and microscopy for detection and identification of common pathogenic protozoan parasites in New Damietta city of North Delta region, Egypt. Between Jun 2013 until Sept 2013, fresh stool samples were obtained from 249 patients up to 65 years of age attending the internal clinic of the Damietta University Hospital and those visiting their general practitioner (GP) of outpatient clinics because of gastrointestinal symptoms. Stool samples collected was preserved at -200C for DNA extraction whilst the remaining was preserved in sodium acetate-acetic acid formalin and concentrated using the formol-ether technique for microscopic examination. DNA extracts were analyzed with the multiplex allele specific Polymerase Chain Reaction (MAS-PCR) for pathogenic protozoan parasites. The diagnostic results obtained using a multiplex allele specific PCR for the detection of E. histolytica/dispar, G. lamblia and C. parvum/C. hominis were compared with these obtained by routine microscopy of faecal samples from patients. 69 samples were positive by MAS-PCR assays , 9 cases of G. intestinalis infection, 34 cases of D. fragilis infection, 3 cases of E. histolytica infection, 17 cases E.dispar and 6 cases of Cryptosporidium infection in the clinical samples. By microscopy, only 32 samples were positive for one or more of the enteric protozoa, 5cases of G. intestinalis infection, 9cases of D. fragilis infection, 13 cases of E. histolytica infection, and 1 cases of Cryptosporidium infection in the clinical samples. However, there are no cases of E.dispar observed. Mixed infections were detected in 4 samples. The sensitivities varied from 58% for D. fragilis to 47% for E. histolytica, 35% for Giardia, and 30% for Cryptosporidium, while the specificities also varied from 97% for E. histolytica to 99% for D. fragilis and 100% for E.dispar . No cross-reactivity was detected in stool samples containing various other bacterial, viral, and protozoan species. This present study showed relatively high rates of protozoa infections in the study patients. The study has also demonstrated that the multiplex real time PCR assay was more sensitive compared to microscopy in the diagnosis of the intestinal protozoa parasites and thus, molecular methods must be considered the diagnostic methods of choice for enteric protozoan parasites. Keywords: Human intestinal protozoa, Stool specimens, Microscopy, Multiplex Allele Specific Polymerase Chain Reaction (MAS-PCR), Diagnosis.

Host Immune Response to Intestinal Amebiasis

Entamoeba histolytica is an invasive enteric protozoan parasite that causes amebiasis. Globally, diarrheal disease is second only to pneumonia as a leading cause of death in children under five, and intestinal amebiasis is one of the top ten causes of severe diarrhea in the developing world. Amebiasis is more common in malnourished children, a state that afflicts approximately one-third of children in the developing world. In the critical first year of life, 11% of Bangladeshi infants living in poverty suffer from E. histolytica diarrhea [1], [2]. There is currently no vaccine for this devastating disease, thus an understanding of the human immune response toward the parasite would greatly enhance the ability to develop effective immunotherapies. The host deploys a series of immune defenses against the parasite as it invades the colon. The ameba, however, has developed complex strategies to evade host defenses and promote its own survival. Here, we summarize the dynamics of the interaction of parasite with host and its importance in the pathogenesis of amebiasis (Figure 1). Figure 1 A. Host Immune Response to Intestinal Amebiasis. Innate Immunity Stomach acid serves as an important first line of defense against enteropathogens through its ability to kill acid-sensitive microorganisms. However, infectious amebic cysts are highly resistant and survive passage through the acidic environment of the stomach. In the intestine, the next layer of innate defense may be the mucus layer, which is thought to act as a protective barrier, preventing E. histolytica from invading intestinal epithelial cells (IECs). Mucin, a major constituent of the intestinal mucus layer, is a glycoprotein secreted by goblet cells and submucosal glands. Mucin glycoproteins bind to and inhibit the Gal/GalNAc adherence lectin of the parasite, preventing in vitro adherence and killing of CHO cells [3]. Trophozoites, however, can disrupt the mucus layer and intestinal barrier by secreting cysteine proteases (CPs) and glycosidases to allow for penetration of the colonic mucosa. Specifically, E. histolytica cysteine protease-A5 (EhCP-A5) degrades mucin-2 (MUC2) and extracellular matrix (ECM) proteins [4]. The importance of cysteine proteases was demonstrated by an ex vivo human intestinal model, where EhCP-A5–silenced parasites failed to penetrate into the colonic lamina propria [5]. IECs exposed to E. histolytica trophozoites secrete potent chemokines, such as IL-8, resulting in immune cell recruitment and infiltration of the lamina propria and intestinal epithelium [6]. Neutrophils are one of the first immune cells to respond to amebic invasion. Neutrophils activated by interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), or lipopolysaccharides (LPS) carry out amebicidal activity in vitro by releasing reactive oxygen species (ROS) [7], [8]. Depletion of neutrophils with anti-Gr-1 antibodies resulted in exacerbated intestinal disease in murine models, supporting the protective role of neutrophils in amebiasis [9]. It should be noted, however, that anti-Gr-1 antibodies can deplete other granulocytes such as eosinophils. Macrophages also play a crucial role in the host response against intestinal amebiasis. Macrophages are amebicidal after stimulation with IFN-γ or TNF-α [10], [11]. Several amebic antigens are known to activate these cells via pattern recognition receptors. Toll-like receptor (TLR)-2 expression in macrophages is upregulated when exposed to the Gal/GalNAc lectin of E. histolytica, triggering pro-inflammatory cytokine production via NF-κB activation [12]. Macrophages that lack TLR-2 and TLR-4 displayed impaired response to E. histolytica lipopeptidophosphoglycan (LPPG), suggesting that pattern recognition is essential to the immune response [13]. Additionally, E. histolytica DNA can activate macrophages through interacting with TLR-9 [14]. Amebicidal activity of macrophages is contributed to by the production of nitric oxide (NO) from L-arginine, which is mediated by macrophage nitric oxide synthase. Inducible nitric oxide synthase (iNOS)–deficient mice were more susceptible to amebic liver abscess and to E. histolytica–induced hepatocytic apoptosis, implicating a critical role for NO in the host defense against amebiasis [15].

Enteric Protozoan Parasites in Stray Cats in Kuwait with Special References to Toxoplasmosis and Risk Factors Affecting Its Occurrence

In Kuwait, stray cats were surveyed for enteric protozoan infection using fecal examination and their sera were tested for Toxoplasma gondii IgG using indirect hemagglutination test (IHAT) as well as for feline immunodeficiency virus (FIV) antibodies and feline leukaemia virus (FeLV) antibodies using ELISA. Out of 240 fecal samples examined 22 (9.2%) were found to be infected with oocysts of four species of coccidian protozoa. Isopspora felis was the most predominant enteric protozoan parasite (7.1%), followed by T. gondii (2.1%), I. rivolta (1.6), Sarcocystis was only found in one case (0.4%). Juvenile cats ( 6 months old) had higher infection rate with oocyst of enteric protozoa than older cats (p-value 0.001). Sero-survey of 240 stray cats revealed that 19.6% were positive to T. gondii IgG. Toxoplasma sero-positivity was observed in higher number of adults compared to young cats suggests that with age the risk of exposure to T. gondii increases. While concurrent retroviral infections were not found to be associated with increased risk for developing T. gondii antibodies.

Specific and quantitative detection and identification of Cryptosporidium hominis andC. parvum in clinical and environmental samples

Cryptosporidium is an enteric protozoan parasite that is resistant to inactivation by commonly used drinking water disinfectants. Between 2004 and 2010, it was responsible for 60% of all waterborne protozoan parasitic outbreaks reported worldwide. Most sporadic infections in humans and almost all outbreaks are caused by Cryptosporidium parvum and Cryptosporidium hominis. We report the development and validation of a quantitative qPCR assay using minor groove binder (MGB)-probes targeting a unique Cryptosporidium specific protein-coding gene, that directly detects, quantitates and identifies C. hominis and C. parvum in environmental and faecal samples. An internal amplification control (IAC) was also developed and included in this assay. The qPCR assay was compared with an 18S nested PCR assay for sensitivity and specificity. The analytical sensitivity for the qPCR assay was 1 oocyst and 1–10 oocysts for the 18S assay. Evaluation of analytical specificity of the qPCR assay revealed no cross-reactions with other genera and detected all C. parvum and C. hominis isolates correctly. The diagnostic sensitivity and specificity of the qPCR was 100% compared to 96.9% and 98.4%, respectively for the 18S assay. The qPCR assay was also highly reproducible with RSD (relative standard deviation) values of 1.4–9.4%, when the assay was performed by four different technicians. When tested on water samples, the qPCR assay was more sensitive than the 18S assay, detecting positives in 37 of 138 water samples compared to 35 for the 18S locus. This qPCR assay should be a valuable tool for the detection and differentiation of C. hominis and C. parvum in both clinical and environmental samples.

Genotype Analysis of Giardia lamblia Isolated From Children in Ahvaz, Southwest of Iran

Background: Giardia lamblia is an enteric protozoan parasite, which infects human and a wide range of vertebrate hosts. Objectives: The aim of this study was to investigate genotypes of G. lamblia from children fecal samples in Ahvaz, South West of Iran by PCR-RFLP method. Materials and Methods: Fecal samples were collected from 58 children who were positive for G. lamblia. DNA extractions were performed by QIAamp Stool Mini Kit. DNA were evaluated by semi nested PCR-RFLP assay, targeting the glutamate dehydrogenase (gdh) gene, which was used to distinguish within and between genotypes A and B. Results: Fifty samples (86%) were confirmed by semi-nested PCR. Genotype analysis among 50 isolates indicated 5 (10%) and 8 (16%) assemblages AII and B, respectively. Mixed Infections with both assemblages AII and B were also detected in 37 (74%) cases. Conclusions: Current study indicated the molecular characterization of G. lamblia in southwest of Iran. Postulated sources of contamination by accidental discharge of sewage effluent and faecal contamination from animals may contribute to this high rate of mixed infection in the current study. Further studies are needed to underestand the source and route of infection better.

DETECTION OF GIARDIA LAMBLIA, CRYPTOSPORIDIUM SPP. AND ENTAMOEBA HISTOLYTICA IN CLINICAL STOOL SAMPLES BY USING MULTIPLEX REAL-TIME PCR AFTER AUTOMATED DNA ISOLATION

Diagnosis of intestinal parasites in stool samples is generally still carried out by microscopy; however, this technique is known to suffer from a low sensitivity and is unable to discriminate between certain protozoa. In order to overcome these limitations, a real-time multiplex PCR was evaluated as an alternative approach for diagnosing Giardia lamblia, Cryptosporidium spp. and Entamoeba histolytica in stool samples. Therefore, a total of 631 faecal samples were analysed both by microscopy as well as by real-time PCR following automated DNA extraction. Results showed that real-time PCR exhibited sensitivity and specificity of both 100%, whereas traditional microscopy exhibited sensitivity and specificity of 37.5% and 99.8% respectively.As real-time PCR provides simple, sensitive and specific detection of these three important pathogenic protozoan parasites, this technique, rather than microscopy, has become our diagnostic method of choice for the detection of enteric protozoan parasites for the majority of patients.