Enteric Protozoan Research Articles

Frequency of enteric protozoan parasites among patients with gastrointestinal complaints in medical centers of Zahedan, Iran

We investigated the prevalence of intestinal protozoan parasites in patients with gastrointestinal complaints in medical centers in Zahedan, Iran. A total of 1562 stool samples was examined from July 2004 to January 2006 using microscopy (direct smear, formalin-ether concentration), xenic culture and PCR techniques. Four hundred and twenty-seven (27.3%) of the patients were infected with one or more intestinal parasites. Giardia lamblia (10.1%), Entamoeba coli (10%), E. hartmanni (1.7%), Blastocystis hominis (2.2%), Chilomastix mesnili (1.7%), Trichomonas hominis (0.7%), E. histolytica/E. dispar (0.51%) and Iodamoeba butschlii (0.45%) were the most prevalent protozoa detected with microscopy. Of the eight microscopy-positive E. histolytica/E. dispar samples, six were identified as E. dispar by PCR/gel electrophoresis, whereas E. histolytica was not detected at all. Although Zahedan is an area with poor hygiene located in a tropical area near the border of Pakistan and Afghanistan, the prevalence of E. histolytica and E. dispar here compared with other parasites and infectious diseases is unexpectedly low.

A survey of enteric bacteria and protozoans in fresh bovine faeces on New Zealand dairy farms

To determine the counts and/or prevalence in fresh bovine faeces of Escherichia coli, enterococci, Campylobacter, Salmonella, shiga toxin-producing E. coli (STEC), Giardia and Cryptosporidium, as inputs to numerical models designed to estimate microbial loadings on pasture grazed by cattle in New Zealand. In each season over one year, samples of freshly deposited bovine faeces were collected from four New Zealand dairy farms (n = 155), and enumerated for E. coli, enterococci, Campylobacter, Giardia and Cryptosporidium. They were also tested for the presence of Salmonella and STEC. The overall median bacterial counts (g(-1) wet weight) were E. coli- 5.9 x 10(6); enterococci - 1.3 x 10(4); Campylobacter- 3.9 x 10(5). All counts were highly variable within and between samplings, and few seasonal or regional patterns emerged. However, mean Campylobacter counts were consistently higher in spring. No Salmonella spp. was detected, and only two samples were positive for STEC. Cryptosporidium and Giardia were isolated from 5.2% and 4.5% of the samples, respectively, yielding low numbers of (oo)cysts (1-25 g(-1) and 1-17 g(-1), respectively). Fresh bovine faeces are a significant source of E. coli, enterococci and Campylobacter on New Zealand pastures, although numbers are likely to vary markedly between faecal samples. The study provides the first significant set of indicator and pathogen counts for one of the largest sources of faecal contamination of natural waters in New Zealand, and will be used to model these inputs.

New insights on classification, identification, and clinical relevance of Blastocystis spp.

Blastocystis is an unusual enteric protozoan parasite of humans and many animals. It has a worldwide distribution and is often the most commonly isolated organism in parasitological surveys. The parasite has been described since the early 1900s, but only in the last decade or so have there been significant advances in our understanding of Blastocystis biology. However, the pleomorphic nature of the parasite and the lack of standardization in techniques have led to confusion and, in some cases, misinterpretation of data. This has hindered laboratory diagnosis and efforts to understand its mode of reproduction, life cycle, prevalence, and pathogenesis. Accumulating epidemiological, in vivo, and in vitro data strongly suggest that Blastocystis is a pathogen. Many genotypes exist in nature, and recent observations indicate that humans are, in reality, hosts to numerous zoonotic genotypes. Such genetic diversity has led to a suggestion that previously conflicting observations on the pathogenesis of Blastocystis are due to pathogenic and nonpathogenic genotypes. Recent epidemiological, animal infection, and in vitro host-Blastocystis interaction studies suggest that this may indeed be the case. This review focuses on such recent advances and also provides updates on laboratory and clinical aspects of Blastocystis spp.

Correlation between CD4 counts of HIV patients and enteric protozoan in different seasons - an experience of a tertiary care hospital in Varanasi (India).

BackgroundProtozoan infections are the most serious among all the superimposed infections in HIV patients and claim a number of lives every year. The line of treatment being different for diverse parasites necessitates a definitive diagnosis of the etiological agents to avoid empirical treatment. Thus, the present study has been aimed to elucidate the associations between diarrhoea and CD4 counts and to study the effect of HAART along with management of diarrhoea in HIV positive patients. This study is the first of its kind in this area where an attempt was made to correlate seasonal variation and intestinal protozoan infestations.MethodsThe study period was from January 2006 to October 2007 wherein stool samples were collected from 366 HIV positive patients with diarrhea attending the ART centre, inpatient department and ICTC of S.S. hospital, I.M.S., B.H.U., Varanasi. Simultaneously, CD4 counts were recorded to assess the status of HIV infection vis-à-vis parasitic infection. The identification of pathogens was done on the basis of direct microscopy and different staining techniques.ResultsOf the 366 patients, 112 had acute and 254 had chronic diarrhea. The percentages of intestinal protozoa detected were 78.5% in acute and 50.7% in chronic cases respectively. Immune restoration was observed in 36.6% patients after treatment on the basis of clinical observation and CD4 counts. In 39.8% of HIV positive cases Cryptosporidium spp. was detected followed by Microsporidia spp. (26.7%). The highest incidence of intestinal infection was in the rainy season. However, infection with Cyclospora spp. was at its peak in the summer. Patients with chronic diarrhea had lower CD4 cell counts. The maximum parasitic isolation was in the patients whose CD4 cell counts were below 200 cells/μl.ConclusionThere was an inverse relation between the CD4 counts and duration of diarrhea. Cryptosporidium spp. was isolated maximum among all the parasites in the HIV patients. The highest incidence of infection was seen in the rainy season.

Giardia and Cryptosporidium in inflowing water and harvested shellfish in a Lagoon in Southern Italy

Giardia and Cryptosporidium spp. are important enteric protozoan pathogens for humans and animals, and have been found to contaminate water as well as edible shellfish all over the world. This is the first study to simultaneously investigate the presence of Giardia and Cryptosporidium in inflowing water and harvested shellfish in a geographically closed environment (Varano Lagoon, Southern Italy). Samples of treated wastewater were collected each month — at the outlet from the treatment plant, and downstream at the inlet into the lagoon — from the channels flowing into the Lagoon, together with specimens of Ruditapes decussatus and Mytilus galloprovincialis from shellfish-farms on the same lagoon. Giardia cysts were found by immunofluorescence (IF) microscopy in 16 out of 21 samples of treated wastewater and in 7 out of 21 samples from downstream water channels, and viable cysts were also detected by a β-giardin RT-PCR. G. duodenalis Assemblages A and B were identified by small ribosomal subunit (18S-rDNA) and triosephosphate isomerase (tpi)-PCR, followed by sequencing. Cryptosporidium oocysts were found by IF in 5 out of 21 wastewater samples, and in 8 out of 21 samples from water channels. Molecular analysis identified the zoonotic species Cryptosporidium parvum by oocyst wall protein (COWP)-PCR and sequencing. Higher concentrations of Giardia cysts than Cryptosporidium oocysts were registered in almost all wastewater and water samples. IF and molecular testing of shellfish gave negative results for both protozoa. Wastewaters carrying Giardia and Cryptosporidium (oo)cysts are discharged into the Lagoon; however, the shellfish harvested in the same environment were found to be unaffected, thus suggesting that physical, ecological and climatic conditions may prevent contamination of harvested shellfish.

Microscopy of 4 Pathogenic Enteric Protozoan Parasites: A Review

Nonimmunological microscopy procedures for Giardia lamblia, Cryptosporidium species, Cyclospora cayetanensis, and microsporidia are reviewed. In addition to the unstained wet preparation and the routine trichrome stain of the “routine” parasitology, the Gram stain, various modifications of the trichrome stain, the modified carbol fuchsin acid-fast stain, and several fluorescent variations of the acid-fast stain are also covered. The now-extensive literature on the newer techniques of the heated safranin stain, the use of autofluorescence, and the use of fluorescent brighteners, such as calcofluor white and related compounds, are reviewed. Emphasis is placed on the importance of the microscopist being aware of reported variations in both the staining protocols and their results. With increasing use being made of fluorescence microscopy in examination of specimens for the oocysts of cyclospora and the spores of microsporidium, an understanding of the effect of both excitation and barrier filters on the observed results of the procedures is emphasized.

Bacterial contamination of water used for holy water in Thailand

Many classes of pathogens excreted in feces are able to initiate waterborne infections (Leclerc et al. 2002). Fecal pathogens including enteric and aquatic bacteria, enteric viruses, and enteric protozoa are strongly resistant in the water environment and to most disinfectants (Leclerc et al. 2002). In developing tropical countries, gastrointestinal tract infections are common. Pathogenic organisms in water sources used for drinking are important causes of diarrhea.

Signaling role of NADPH oxidase in Entamoeba histolytica‐induced cell death of HT29 colonic epithelial cells

Entamoeba histolytica is an enteric tissue‐invasive protozoan parasite that causes amoebic colitis and occasionally liver abscess in humans. During tissue invasion, E. histolytica is exposed to elevated amounts of exogenous reactive oxygen species (ROS). ROS cause severe damage to biological macromolecules, leading to metabolic malfunctions. NADPH oxidase is one of the major sources of ROS in vasculature; it consists of a catalytic subunit, p22phox, p47phox, p67phox, and the small GTP binding protein Rac1. In this study, we investigated the roles of ROS and of NADPH oxidase in the E. histolytica‐induced cell death of HT29 cells. NADPH oxidase components were highly expressed in HT29 cells. When HT 29 cells were co‐incubated with E. histolytica, PI and tryphan blue stained cell portion markedly increased compared with results for cells incubated with medium alone. In addition, a large amount of intracellular ROS was detected after exposure to viable trophozoites. The treatment with diphenyleneiodonium chloride (DPI), an unspecific inhibitor of NADPH oxidase and rac1 siRNA strongly inhibited the E. histolytica‐induced ROS generation. Moreover, the E. histolytica‐induced cell death was effectively blocked by pretreatment of cells with DPI and rac1 siRNA. These results strongly suggest that NADPH oxidase‐generated ROS is essential for the E. histolytica‐induced HT29 cell death.

Prevalence of Cryptosporidium, Giardia and Isospora species infections in pet cats with clinical signs of gastrointestinal disease.

This study reports the prevalence of Cryptosporidium, Giardia and Isospora species in cats showing signs of gastrointestinal disease. Records from a United Kingdom commercial diagnostic laboratory between December 2003 and December 2005 were reviewed. Of 1355 cats, Cryptosporidium species oocysts were found in 13 cats (1%), Giardia species trophozoites in 74 (6%), and Isospora felis oocysts in 46 (3%). In a second group of 48 cats, prevalence of Giardia species was 15% using an immunoassay for detection of antigen compared to 4% detected with microscopy. Prevalence of Giardia (9%) and Isospora (9%) species was higher in cats less than 6 months old. Gender and breed did not affect prevalence. There was a trend for Cryptosporidium and Isospora species infections to be detected in late autumn and early winter. Regional differences in prevalence were not detected. None of these organisms show a characteristic pattern of clinical signs. This study demonstrates that enteric protozoal infection is common in domestic cats showing signs of alimentary disease.

Kinetic characterization of methionine γ‐lyases from the enteric protozoan parasite Entamoeba histolytica against physiological substrates and trifluoromethionine, a promising lead compound against amoebiasis

Methionine gamma-lyase (MGL) (EC 4.4.1.11), which is present in certain lineages of bacteria, plants, and protozoa but missing in mammals, catalyzes the single-step degradation of sulfur-containing amino acids (SAAs) to alpha-keto acids, ammonia, and thiol compounds. In contrast to other organisms possessing MGL, anaerobic parasitic protists, namely Entamoeba histolytica and Trichomonas vaginalis, harbor a pair of MGL isozymes. The enteric protozoon En. histolytica shows various unique aspects in its metabolism, particularly degradation of SAAs. Trifluoromethionine (TFM), a halogenated analog of Met, has been exploited as a therapeutic agent against cancer as well as against infections by protozoan organisms and periodontal bacteria. However, its mechanism of action remains poorly understood. In addition, the physiological significance of the presence of two MGL isozymes in these protists remains unclear. In this study, we compared kinetic parameters of the wild-type and mutants, engineered by site-directed mutagenesis, of the two MGL isotypes from En. histolytica (EhMGL1 and EhMGL2) for various potential substrates and TFM. Intracellular concentrations of l-Met and l-Cys suggested that these SAAs are predominantly metabolized by EhMGL1, not by EhMGL2. It is unlikely that O-acetyl-l-serine is decomposed by EhMGLs, given the kinetic parameters of cysteine synthase reported previously. Comparison of the wild-type and mutants revealed that the contributions of several amino acids implicated in catalysis differ between the two isozymes, and that the degradation of TFM is less sensitive to alterations of these residues than is the degradation of physiological substrates. These results support the use of TFM to target MGL.

Is there a role for routine duodenal biopsy in diagnosing giardiasis in a European population?

Objective. Giardia lamblia is the most common enteric protozoan world-wide. The prevalence varies internationally. Patients with giardiasis typically have diarrhoea but may have atypical symptoms when referred for endoscopy. The purpose of this study was to investigate whether a policy of routine duodenal biopsies increased the yield of diagnosing Giardia and whether treatment improved the patient's presenting symptoms. Material and methods. Our hospital endoscopy database was examined retrospectively, from January 2003 to January 2004, to identify patients who had undergone gastroscopy and targeted duodenal biopsies. Thereafter, routine duodenal biopsies were carried out prospectively on unselected adult patients referred for gastroscopy from January 2004 to April 2006. Patients diagnosed with Giardia were assessed for symptoms and response to treatment. Results. In the retrospective group, of the 5979 gastroscopies performed, 1464 duodenal biopsies were carried out; 5 patients had giardiasis, a prevalence of 0.08%, CI 0.01–0.16%. In the prospective group, 2000 gastroscopies with duodenal biopsies were performed; 9 patients had giardiasis, a prevalence of 0.45%, CI 0.16–0.74%. The difference in prevalence was 0.37%, CI 0.06–0.67%, which was statistically significant with a Yates’ χ2CC of 9.49, p<0.005. On comparing response to treatment, the Fisher exact test revealed a proportional difference that was not statistically significant. Conclusions. In a UK population, the prevalence of Giardia was low. Although a policy of routine duodenal biopsy resulted in more cases of Giardia being identified, this increase in detection did not correspond with an improvement in patients’ symptoms. This suggests that Giardia may be a coincidental finding. This study does not support the approach of routine duodenal biopsy in diagnosing giardiasis.

Oh my aching gut: irritable bowel syndrome, Blastocystis, and asymptomatic infection

Blastocystis is a prevalent enteric protozoan that infects a variety of vertebrates. Infection with Blastocystis in humans has been associated with abdominal pain, diarrhea, constipation, fatigue, skin rash, and other symptoms. Researchers using different methods and examining different patient groups have reported asymptomatic infection, acute symptomatic infection, and chronic symptomatic infection. The variation in accounts has lead to disagreements concerning the role of Blastocystis in human disease, and the importance of treating it. A better understanding of the number of species of Blastocystis that can infect humans, along with realization of the limitations of the existing clinical laboratory diagnostic techniques may account for much of the disagreement. The possibility that disagreement was caused by the emergence of particular pathogenic variants of Blastocystis is discussed, along with the potential role of Blastocystis infection in irritable bowel syndrome (IBS). Findings are discussed concerning the role of protease-activated receptor-2 in enteric disease which may account for the presence of abdominal pain and diffuse symptoms in Blastocystis infection, even in the absence of fever and endoscopic findings. The availability of better diagnostic techniques and treatments for Blastocystis infection may be of value in understanding chronic gastrointestinal illness of unknown etiology.

Blastocystis rattiContains Cysteine Proteases That Mediate Interleukin-8 Response from Human Intestinal Epithelial Cells in an NF-κB-Dependent Manner

Blastocystis is a ubiquitous enteric protozoan found in the intestinal tracts of humans and a wide range of animals. Evidence accumulated over the last decade suggests association of Blastocystis with gastrointestinal disorders involving diarrhea, abdominal pain, constipation, nausea, and fatigue. Clinical and experimental studies have associated Blastocystis with intestinal inflammation, and it has been shown that Blastocystis has potential to modulate the host immune response. Blastocystis is also reported to be an opportunistic pathogen in immunosuppressed patients, especially those suffering from AIDS. However, nothing is known about the parasitic virulence factors and early events following host-parasite interactions. In the present study, we investigated the molecular mechanism by which Blastocystis activates interleukin-8 (IL-8) gene expression in human colonic epithelial T84 cells. We demonstrate for the first time that cysteine proteases of Blastocystis ratti WR1, a zoonotic isolate, can activate IL-8 gene expression in human colonic epithelial cells. Furthermore, we show that NF-kappaB activation is involved in the production of IL-8. In addition, our findings show that treatment with the antiprotozoal drug metronidazole can avert IL-8 production induced by B. ratti WR1. We also show for the first time that the central vacuole of Blastocystis may function as a reservoir for cysteine proteases. Our findings will contribute to an understanding of the pathobiology of a poorly studied parasite whose public health importance is increasingly recognized.

Predominance of subtype 3 among Blastocystis isolates from a major hospital in Singapore

Blastocystis is an enteric protozoan parasite commonly found in humans and animals. Phylogenetic and genotypic analyses have shown that Blastocystis exhibits extreme genetic diversity, and humans are host to a number of zoonotic isolates. In the present study, the prevalence of Blastocystis in 276 stool samples from a hospital in Singapore was examined, and for the first time, riboprinting using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was used to determine the genetic diversity of the Blastocystis isolated from the Singapore population. The prevalence rate was determined to be 3.3% (9/276), and Blastocystis displaying two main ribotypes were isolated. As a comparison, we performed PCR-RFLP using two different published methodologies, and both methods allowed the isolates to be divided into two distinct groups based on their riboprint patterns. According to a recently proposed classification scheme, 78% (7/9) of the isolates were of subtype 3, while 22% (2/9) were subtype 1. The predominance of subtype 3 in an urbanized city state such as Singapore is in agreement with the idea that subtype 3 is a genotype of human origin.

Population Genetics Provides Evidence for Recombination in Giardia

Giardia lamblia (syn. Giardia intestinalis, Giardia duodenalis) is an enteric protozoan parasite with two nuclei, and it might be one of the earliest branching eukaryotes. However, the discovery of at least rudimentary forms of certain features, such as Golgi and mitochondria, has refuted the proposal that its emergence from the eukaryotic lineage predated the development of certain eukaryotic features. The recent recognition of many of the genes known to be required for meiosis in the genome has also cast doubt on the idea that Giardia is primitively asexual, but so far there has been no direct evidence of sexual reproduction in Giardia, and population data have suggested clonal reproduction. We did a multilocus sequence evaluation of the genotype A2 reference strain, JH, and five genotype A2 isolates from a highly endemic area in Peru. Loci from different chromosomes yielded significantly different phylogenetic trees, indicating that they do not share the same evolutionary history; within individual loci, tests for recombination yielded significant statistical support for meiotic recombination. These observations provide genetic data supportive of sexual reproduction in Giardia.

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In this issue

Cryptosporidium Oocysts in Drinking Water Supply of Chennai City, Southern India

AbstractCryptosporidium is an enteric parasitic protozoan capable of causing chronic diarrhea. One of the most common modes of transmission is through faeces‐contaminated water. This study determines the distribution of Cryptosporidium oocysts for the first time in Chennai City's drinking water supply. 199 drinking water samples were collected from ten zones of the city. In the water samples Cryptosporidium, a common pathogenic protozoan of the gastrointestinal tract, has been analyzed physico chemically as well as microbiologically for heterotrophic organisms and total coliforms (TC). The studies revealed that three zones of the city were highly contaminated with coliforms and parasitic protozoa. A statistical analysis was done to find any correlation between heterotrophic organisms, total coliforms, and oocysts. Even though a positive correlation exists between oocysts and bacteriological parameters, a regression equation shows that heterotrophic plate count (HPC) and total coliforms were only 20% responsible for the presence of oocysts. The level of Cryptosporidium oocysts isolated from the water samples may present a public health hazard although no major outbreaks have so far been reported in Chennai City. Routine surveillance of water quality throughout the city is needed to curb the pollutants.

Rab11B small GTPase regulates secretion of cysteine proteases in the enteric protozoan parasiteEntamoeba histolytica

Vesicular trafficking plays a pivotal role in the virulence of the enteric protozoan parasite Entamoeba histolytica. In the present study, we showed that one isotype of the small GTPase Rab11, EhRab11B, plays a central role in the secretion of a major virulence factor, cysteine proteases. EhRab11B did not colocalize with markers for the endoplasmic reticulum, early endosomes and lysosomes, but was partially associated with non-acidified vesicles in the endocytic pathway, likely recycling endosomes. Overexpression of EhRab11B resulted in a remarkable increase in both intracellular and secreted cysteine protease activity, concomitant with an augmentation of cytolytic activity as demonstrated by an increased ability to destroy mammalian cells. The oversecretion of cysteine proteases with EhRab11B overexpression was neither sensitive to brefeldin A nor specific to a certain cysteine protease species (e.g. CP1, 2 or 5), suggesting that these three major cysteine proteases are trafficked via an EhRab11B-associated secretory pathway, which is distinct from the classical brefeldin-sensitive pathway. Overexpression of EhRab11B also enhanced exocytosis of the incorporated fluid-phase marker, supporting the notion that it is involved in recycling. This is the first report demonstrating that Rab11 plays a central role in the transport and secretion of pathogenic factors.

PREVALENCE OF ENTERIC PROTOZOA IN HUMAN IMMUNODEFICIENCY VIRUS (HIV)–POSITIVE AND HIV-NEGATIVE MEN WHO HAVE SEX WITH MEN FROM SYDNEY, AUSTRALIA

A prospective, comparative study of the prevalence of enteric protozoa was determined among human immunodeficiency virus (HIV)- positive and HIV-negative men who have sex with men (MSM) in Sydney, Australia. A total of 1,868 patients submitted stool specimens; 1,246 were from MSM (628 HIV positive and 618 HIV positive) and 622 from non-MSM were examined over a 36-month period. A total of 651 (52.2%) stool specimens from MSM were positive for protozoa compared with 85 (13%) from non-MSM. There was a significant difference in the prevalence of Blastocystis hominis, Endolimax nana, Entamoeba histolytica/dispar complex, Entamoeba hartmanni, Iodamoeba butschlii, and Enteromonas hominis detected between MSM and non-MSM (P<0.001). The only notable difference between HIV-negative and HIV-positive MSM was that HIV-infected MSM were found to more likely have a Cryptosporidium parvum infection. Entamoeba histolytica was found in 3 patients, E. dispar in 25, and E. moshkovskii in 17, all of whom were MSM. When compared with a control group, MSM were significantly more likely to harbor intestinal protozoa and have multiple parasites present. The results of this study show high rates of enteric parasites persist in MSM and highlight the importance of testing for intestinal parasites in MSM. This is the first report of E. moshkovskii from MSM.

Two Rab7 isotypes, EhRab7A and EhRab7B, play distinct roles in biogenesis of lysosomes and phagosomes in the enteric protozoan parasite Entamoeba histolytica

Rab7 small GTPase plays a crucial role in the regulation of trafficking to late endosomes, lysosomes and phagosomes. While most eukaryotes encode a single Rab7, the parasitic protist Entamoeba histolytica possesses nine Rab7. In this study, to understand the significance of the presence of multiple Rab7 isotypes, a role of two representative Rab7 isotypes, EhRab7A and EhRab7B, was investigated. EhRab7B was exclusively localized to acidic vacuoles containing lysosomal proteins, e.g. amoebapore-A and cysteine protease. This lysosome localization of EhRab7B was in good contrast to EhRab7A, localized to a non-acidic compartment in steady state, and only partially colocalized with lysosomal proteins. Overexpression of EhRab7B resulted in augmentation of late endosome/lysosome acidification, similar to the EhRab7A overexpression. Expression of EhRab7B-GTP mutant caused dominant-negative phenotypes including decrease in late endosome/lysosome acidification and missecretion of lysosomal proteins, while EhRab7A-GTP enhanced acidification but did not affect either intracellular or secreted cysteine protease activity. Expression of either EhRab7B or EhRab7B-GTP mutant caused defect in phagocytosis, concomitant with the disturbed formation and disassembly of prephagosomal vacuoles, the compartment previously shown to be linked to efficient ingestion. Altogether, these data indicate that the two Rab7 isotypes play distinct but co-ordinated roles in lysosome and phagosome biogenesis.