Coproscopy and molecular screening for detection of intestinal protozoa.

BACKGROUND: Intestinal parasitosis is one of several health concerns about immigrants who travel from endemic to non-endemic regions. Reliable rapid sensitive diagnostic tools, for use in non-endemic regions, are urgently required to enable frequent assessment of immigrant workers in jobs where risk of local transmission is a particular concern (e.g. food-handlers). We assessed the burden of intestinal protozoa in newly arrived immigrants and those applying for renewal of work permits in Qatar (n = 735), by both microscopic examination of stool samples and by Real Time PCR methodology. RESULTS: Prevalence was considerably higher using RT-PCR compared with coproscopy (Blastocystis hominis: 65.2 vs 7.6%; Giardia duodenalis: 14.3 vs 2.9%; Entamoeba histolytica: 1.6 vs 1.2%). Dientamoeba fragilis was sought only by RT-PCR (prevalence of 25.4%). Prevalence of G. duodenalis was significantly higher in male subjects, associated with blue collar workers and declined over time. Prevalence of B. hominis varied significantly with region of origin of subjects with highest values recorded among African immigrants. Prevalence of D. fragilis also varied with region of origin of subjects, and was lower in young female subjects and in renewal applicants compared with first-time applicants for work permits. CONCLUSIONS: We strongly recommend that, henceforth, intestinal protozoa should be screened by RT-PCR, with a particular focus on frequent assessment of immigrant food-handlers.

Ultrastructural insights into morphology and reproductive mode of Blastocystis hominis.

To understand well the morphology and reproductive mode of Blastocystis hominis, with the help of transmission electron microscopy and scanning electron microscopy the ultrastructural details of B. hominis from fresh diarrheal specimens and cultured strains were observed. In both fecal samples and culture conditions, there were vacuolar and granular forms. In diarrhea, it exists in multivacuolar, avacuolar, and amoeboid forms. In the in vitro culture, vacuolar form could transform to granular form. The most commonly noticed structure on the cell surface was surface coat with diversity in appearance (the funiform, lamellar, filiform, and floccose in different thickness) and distributions. Three modes of reproduction were confirmed, they were binary fission, plasmotomy, and budding. Under the impact of host's response, the ultrastructures of surface coat, nucleus, and mitochondrion-like organelle sometimes changed.

In vivo encystation of Blastocystis hominis.

Blastocystis from infected stools of a person who showed chronic symptoms of abdominal discomfort and diarrhea were examined over a 6-month period, using transmission electron microscopy, for the ultrastructural changes from vacuolar to cystic stage. The study confirms the irregular shedding phenomenon of the organism previously reported, and for the first time, records sequential changes in encystation in stools collected over a time period. The study also confirms the existence of a precystic stage which has an immature cell wall consisting of a layer of a homogenous electron-dense mass surrounding the cell which acts as a intermediatory stage between the vacuolar and cystic stage.

Evaluation of the nitric oxide activity against Blastocystis hominis in vitro and in vivo.

The effect of exogenous nitric oxide (NO) on growth, viability and ultra-structural of B. hominis was assessed in vitro by sodium nitrite (NaNO2) in 0.6 mM, 0.8 mM & 1 mM concentrations. The viability of B. hominis was identified using neutral red stain. The role of NO as an endogenous oxidant was assessed by identifying its level in cecum tissue, ileum tissue, blood and stool elutes of mice infected with B. hominis symptomatic human isolates using reactive nitrogen assay compared to control. In vitro study revealed that NaNO2 inhibited the growth and decreased viability of B. hominis with minimal lethal concentra-tion dose 1 mM on the 4th day while, minimal effects were detected with 0.6 and 0.8 mM. Transmission electron microscopy study proved that apoptotic-like features were observed in growing axenic culture of B. hominis upon exposure to NaNO2. These changes were not only found on the vacuolar (central body) form but also they were detected on granular, multi-vacuolar and cyst forms. In vivo study proved that high levels of NO were found in infected mice compared to low changes in control group. The high levels were in cecum tissue particularly. The mean levels of NO among infected mice were 211.8 +/- 20.7 microM in cecum, 90.4 +/- 11.6 microM in ileum, 60.1 +/- 4.7 microM in blood and 63.6 +/- 7.3 microM in stool elutes while, the mean levels of NO in control mice were 70.2 +/- 3.1 in cecum, 67.8 +/- 4.7 microM in ileum, 30.9 +/- 4.2 microM in blood and 28.1 +/- 2.9 microM in stool elutes. The differences were statistically highly significant. NO-donor drugs proved useful in treatment and increase the host resistance to B. hominis.

Evidence of plasmotomy in Blastocystis hominis.

Blastocystis hominis has been regarded as an enigmatic parasite as many aspects of its basic biology remain uncertain. Many reproductive processes have been suggested for the organism; however, to date, only the binary fission has been proven. Plasmotomy is one of the modes of reproduction previously suggested to be seen in in vitro cultures. The present study provides trichrome and acridine orange staining evidence for the existence of nucleic acid suggestive of division of nucleus into multinucleate forms with the respective cytoplasm dividing giving rise to two or three progeny B. hominis. Transmission electron micrographs further confirmed that these daughter cells had respective surrounding surface coat, mitochondria, and vacuoles.

[Is Blastocystis hominis an opportunist agent?]. / Blastocystis hominis firsatçi bir patojen mi?

Despite its high prevalence throughout the world, major issues about Blastocystis hominis remain unresolved, including fundamental areas such as taxonomy and pathogenicity. Sequences of the SSUrRNA gene place Blastocystis in the stramenophiles. Analysis of the elongation factor 1-alpha gene, however, indicates similarity to Entamoeba histolytica. There is considerable morphological variability and karyotype diversity, and it appears that more than one species is present in humans and animals. In culture, three major forms predominate: vacuolar, granular, and ameboid. The vacuolated form (usually 10 to 30 mum) was most frequently detected in fecal specimens. The prevalence of Blastocystosis in humans appears to be higher in developing countries (30% to 50%) than in developed countries (1.5% to 10%), and has been associated with travel. B. hominis is the most common parasite isolated from stool specimens in symptomatic and asymptomatic persons in a variety of settings. Isolates resembling B. hominis have been described in a variety of mammals, birds, reptiles, and even insects. The significance of this human infection is uncertain.

Amoeboid form of Blastocystis hominis - a detailed ultrastructural insight.

The amoeboid form of Blastocystis hominis has been reported infrequently, and its morphological descriptions have yielded conflicting and confusing reports. In the present study, we used the amoeboid forms seen predominantly in symptomatic patients infected with Blastocystis to provide detailed descriptions on the fine surface structure and intracellular morphology. Scanning electron microscopy revealed the irregular shape of the amoeboid form, with an intercalated fibrillar structure and a highly convoluted surface with deep indentations and projected pseudopodia. Transmission electron microscopy showed the existence of two types of amoeboid forms of B. hominis in in vitro culture, one with a large central vacuole containing tiny electron-dense particles while the other contains multiple small vacuoles in the cytoplasm. A surface coat with varying thickness surrounded the amoeboid form, which also showed prominent, extended pseudopodia of varying shape. Irregularly shaped mitochondrion-like organelles with prominent cristae, lipid inclusions, and multiple vacuoles were frequently seen in close proximity with the pseudopodia. The characteristic nucleus with a crescentic band of electron-dense chromatin material was also seen.

[Study on the biological characteristic of Blastocystis hominis: morphology, mode of reproduction and the relation to bacteria].

OBJECTIVE: To observe the reproductive modes of Blastocystis hominis and study the relation between this protozoa and bacteria. METHODS: Using the Iodine and Haematoxylin staining, the morphology of B. h from patients and RPMI 1640 medium were observed. The B. h positive mucous diarrheal specimens were cultured and identified any possible known pathogenic intestinal bacteria. B. h and colibacillus were co-cultured to observe the interaction between them. RESULTS: Four modes of reproduction for B. h were confirmed: binary fission, endodyogeny, multiple fission and budding. The fact that there was no other intestinal pathogens in half of the B. h positive specimens suggested B. h may cause disease independently. B. h and colibacillus were restrained each other. CONCLUSION: B. h reproduces in at least four modes. B. h could be a pathogen and its pathogenesis may be related to micro-ecological changes.

Protozoan programmed cell death--insights from Blastocystis deathstyles.

Programmed cell death (PCD) is an essential process in the growth and development of multicellular organisms. However, accumulating evidence indicates that unicellular eukaryotes can also undergo PCD with apoptosis-like features. The protozoan parasite Blastocystis hominis has been reported to exhibit both apoptotic and non-apoptotic features of PCD when exposed to a variety of stimuli. Recent observations of PCD pathways in Blastocystis suggest that this protozoan, as is the case with its multicellular counterparts, possesses complex cell-death mechanisms.

Isolation and characterization of the mitochondrion-like organelle from Blastocystis hominis.

Blastocystis hominis in an unusual protozoan parasite of the human intestinal tract. Previous studies have described the presence of mitochondrial-like structures despite the anaerobic nature of the organism. In this study, we describe a simple and rapid technique to isolate and characterize mitochondrion-like organelles (MLO) from B. hominis. The parasite was disrupted using glass beads and the MLO were collected and purified using a sucrose gradient. Negative staining and transmission electron microscopy of the isolated organelles showed mitochondrial-like structures. B. hominis cells were stained with rhodamine 123 and MitoLight to show the presence of transmembrane potential of the MLO. DAPI staining of the cells suggested the presence of DNA in the MLO. Though brief reports have been made in literature, this study is the first to describe a technique for the isolation of the MLO from this organism. Using this technique of isolation, major metabolic functions of the organelle, their associated macromolecules and intra-mitochondrial location can be extensively studied. The role of MLO in this anaerobic protozoan can be widely investigated using this protocol.

[Study on morphology of Blastocystis hominis in culture and from diarrhea patients].

OBJECTIVE: To study morphology of different stage Blastocystis hominis (B. h) for establishing a base in the research of life cycle and pathogenicity of B. h and providing information for clinical laboratory. METHODS: B. h from diarrheal patients was continuously cultured in LES medium, and morphology of B. h was studied with iodine and iron hematoxylin staining under light microscope. RESULTS: The vacuolar, granular, amoeboid and cyst forms of B. h and transformation among the forms were observed microscopically. CONCLUSION: Among different forms, the vacuolar and granular forms were often seen clinically and the vacuolar form can transform to cysts.

Recent advances in Blastocystis hominis research: hot spots in terra incognita.

Despite being discovered more than 80 years ago, progress in Blastocystis research has been gradual and challenging, due to the small number of laboratories currently working on this protozoan parasite. To date, the morphology of Blastocystis hominis has been extensively studied by light and electron microscopy but all other aspects of its biology remain little explored areas. However, the availability of numerous and varied molecular tools and their application to the study of Blastocystis has brought us closer to understanding its biology. The purpose of this review is to describe and discuss recent advances in B. hominis research, with particular focus on new, and sometimes controversial, information that has shed light on its genetic heterogeneity, taxonomic links, mode of transmission, in vitro culture and pathogenesis. We also discuss recent observations that B. hominis has the capacity to undergo programmed cell death; a phenomenon similarly reported for many other unicellular organisms. There are still many gaps in our knowledge of this parasite. Although there is a growing body of evidence suggesting that B. hominis can be pathogenic under specific conditions, there are also other studies that indicated otherwise. Indeed, more studies are warranted before this controversial issue can be resolved. There is an urgent need for the identification and/or development of an animal model so that questions on its pathogenesis can be better answered. Another area that requires attention is the development of methods for the transfection of foreign/altered genes into B. hominis in order to facilitate genetic experiments.

Programmed cell death in a human intestinal parasite, Blastocystis hominis.

Although programmed cell death (PCD) has been associated with multicellular organisms, there have been more reports of its presence in some protozoans. Our study shows the existence of PCD in an intestinal protozoan, Blastocystis hominis. Light and electron microscopy, biochemical and flow cytometry studies showed apoptosis-like death in B. hominis cells exposed to a cytotoxic monoclonal antibody (MAb 1D5). B. hominis cells displayed key morphological and biochemical features of apoptosis, namely, nuclear condensation and in situ fragmentation, reduced cytoplasmic volume, some externalization of phosphatidylserine and maintenance of plasma membrane integrity. No oligonucleosomal DNA laddering was observed in gel electrophoresis. This study supports earlier observations that the cellular machinery that is required to carry out PCD may have existed before the advent of multicellularity. Our study also ascribes a novel function for the B. hominis central vacuole in apoptosis; it acts as a repository where apoptotic bodies are stored before being released into the extracellular space.

Do Blastocystis hominis colony forms undergo programmed cell death?

Ultrastructural observations were made on colony forms of the protozoan parasite, Blastocystis hominis. Cross-sections of entire colonies were observed by TEM. Cells within a colony were heterogeneous in morphology, consisting of vacuolar, amoeboid, multivacuolar and unusual forms. Dying cells appeared to be in the process of fragmenting into numerous membranebound vesicles, giving rise to empty spaces within the colony. Interestingly, older colonies appeared to show cell fragmentation which resulted in larger, membranebound structures. Numerous cytoplasmic inclusions were present in the central vacuole of some cells, with many containing mitochondria. Amoeboid forms were observed to harbour small membrane-bound vesicles in endosome-like compartments. Other unusual features included margination of chromatin material and distinct blebbing of nuclei. These ultrastructural features suggest that B. hominis colony forms perhaps undergo a form of programmed cell death.

[Morphological and ultrastructural observation of Blastocystis hominis].

OBJECTIVE: To observe the morphology and ultrastructure of Blastocystis hominis. METHODS: Morphological observation was made with 4-5 days cultured B. hominis by light microscopy, and similar material fixed with 4% glutaraldehyde was used for transmission electron microscopy. RESULTS: Several forms of B. hominis were observed including vacuolar, granular, amebic, multifission and cystic forms. The multiplication patterns of B. hominis included both binary fission and sporogony. Under transmission electron microscope, the nuclei, mitochondria, rough endoplasmic reticula and lysomes were observed in addition to lipid droplets in its cytoplasm, and glycogen in the central vacuole. CONCLUSION: The central vacuole of vacuolar form may be related to the storage of the excreta. The amebic form of B. hominis might be pathogenic.

Blastocystis hominis: A simplified, high-efficiency method for clonal growth on solid agar.

Colony growth of protozoan parasites in agar can be useful for axenization, cloning, and viability studies. This is usually achieved with the pour plate method, for which the parasite colonies are situated within the agar. This technique has been described for Giardia intestinalis, Trichomonas vaginalis, and Entamoeba and Blastocystis species. Extracting such colonies can be laborious. It would be especially useful if parasites could be grown on agar as colonies. These colonies, being exposed on the agar surface, could be conveniently isolated for further investigation. In this study, we report the successful culture of B. hominis cells as colonies on solid agar. Colonies were enumerated and the efficiency of plating was determined. It was observed that B. hominis could be easily cultured on agar as clones. The colonies were dome-shaped and mucoid and could grow to 3 mm in diameter. Flow cytometric analyses revealed that parasite colonies remained viable for up to 2 weeks. Viable colonies were conveniently expanded in liquid or solid media. Scanning electron microscopy revealed that each colony consists of two regions; a dome-shaped, central core region and a flattened, peripheral region. Older colonies possessed numerous strand-like surface coat projections. This study provides the first report of clonal growth of B. hominis on agar and a simple, effective method for cloning and expansion of B. hominis cells.

Blastocystis hominis: origin and significance of vacuolar and granular forms.

Using supravital neutral red staining and light microscopy, individual Blastocystis organisms, subcultured from clinical isolates in modified monophasic Robinson's medium, were followed over various periods on glass slides. A rapid transition from uniformly stained to granular and vacuolated forms preceded the organism's death as evidenced by pale staining and Brownian motion in the cell's interior. Granular and vacuolar forms of Blastocystis may indicate degenerative changes in individual cells, fixation artifact, or both.

Scanning electron microscopy of the surface coat of Blastocystis hominis.

Scanning electron microscopy of Blastocystis hominis showed that its outer coat has a fibrillar structure and individual fibrils may extend up to 5 microm from the periphery of the parasite. The surface coat remains intact during cell division. Bacteria are often seen adhering to it, but for the first time a trophozoite of Chilomastix mesnili was also seen in this position. It is postulated that breakdown of attached organisms may provide nutrients for Blastocystis.

Postcystic development of Blastocystis hominis.

A concentrated suspension of Blastocystis hominis cysts was inoculated into Jones' medium and removed after 24 h for study of their development at the ultrastructural level. The parasite divides in the cyst, and up to three daughter cells can be seen. During this process the cyst wall dissolves, leaving behind thin membranous remnants. Excystation occurs mostly by the emergence of the daughter cells through an aperture in the outer fibrillar coat. Before excystation the vacuolar and the granular stages form and the daughter cells develop their own fibrillar layer.

Development of Blastocystis hominis cysts into vacuolar forms in vitro.

The development of cysts of Blastocystis hominis isolated from human feces by the Ficoll-Paque concentration method and cultured in Jones' medium containing 10% horse serum is described. The morphological changes were studied by light and transmission electron microscopy at different intervals for up to 48 h. The cysts developed into a large number of vacuolar forms within 24 h, and binary fission was the only mode of reproduction observed.