[Blastocystis hominis- parasites or commensals? ]. / Blastocystis hominis - komensal czy patogen?

Blastocystis hominis (B. hominis) is a cosmopolitan pro- tozoa which parasitizes the human large intestine. This parasite had been considered to be commensal of the large intestine for a long time, because even an intense invasion may be asymptomatic. However, this species is now being regarded as a parasitic organism. In this paper the latest data concerning the epidemiology, diagnostics and treatment of B. hominis invasion have been cited and discussed.

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.

[A preliminary study on preservation methods of Blastocystis hominis].

OBJECTIVE: To observe the effect of different cryoprotective agents and temperature factors on the viability of Blastocystis hominis so as to explore the ideal method for preservation of B. hominis. METHODS: B. hominis agents were obtained from a patient's fecal specimen. Having washed by normal saline and divided into tubes, the samples were cryopreserved in -20 degrees C refrigerator or in -196 degrees C liquid nitrogen with 10% DMSO, 40% glycerol and 15% ethylene glycol respectively. The thawed B. hominis agents were then used for culture. By trypan blue staining and microscopy, the viability and proliferation of those resuscitative cells were investigated. RESULTS: B. hominis survived for 3 weeks at 18 degrees C-20 degrees C while less than 1 week at 4 degrees C-6 degrees C. When stored in -20 degrees C refrigerator or liquid nitrogen with cryoprotective agents, they survived for more than 3 months. The cryopreservation with 40% glycerol at -196 degrees C for 6 months resulted in 41.7% viability of the revivified cells. Cleavage cells were easily observed after culturing for 72 hours. CONCLUSION: Preserving B. hominis in liquid nitrogen with 40% glycerol is an optimal cryopreservation protocol.

Morphology and reproductive mode of Blastocystis hominis in diarrhea and in vitro.

The in vitro culture of Blastocystis hominis in Roswell Park Memorial Institute (RPMI) 1,640 medium containing 20% calf serum is described. The morphological and reproductive mode studies of B. hominis in symptomatic patients' faeces and in further RPMI 1,640 medium-cultured samples were undertaken by light microscopy using iodine staining and hematoxylin staining. Three distinct morphological forms, vacuolar, granular and amoeboid, were distinguished in faeces and in vitro cultures. The cystic form was detected in long-term cultures. Five modes of reproduction, namely, binary fission, endodyogeny, plasmotomy, budding and schizogeny were observed.

Predominance of amoeboid forms of Blastocystis hominis in isolates from symptomatic patients.

Blastocystis hominis is one of the most common human parasites that inhabit the intestinal tract. Conflicting reports continue to exist regarding the existence and the functional role of the amoeboid forms in the life cycle of the parasite. The present study investigates the presence of these forms in 20 isolates obtained from ten symptomatic and asymptomatic patients respectively. A total of 10,000 parasite cells per ml from each isolate were inoculated into three culture tubes each containing 3 ml of Jones' medium supplemented with 10% horse serum, incubated at 37 degrees C. The contents were examined daily for 10 days. Irregular and polymorphic amoeboid forms with multiple extended pseudopodia were observed in all isolates from symptomatic patients, while none of the isolates from asymptomatic patients showed the presence of the amoeboid forms. The amoeboid forms were initially noted on day 2 and the percentages increased from 2% to 28%, with peak percentages from day 3 to day 6. Transmission electron microscopy revealed two types of amoeboid forms; one containing a large central vacuole completely filled with tiny electron-dense granules, and the other which revealed multiple small vacuoles within the central body. The cytoplasm contained strands of electron-dense granules resembling rough endoplasmatic reticulum, which is suggestive of active protein synthesis. The surface coat of the amoeboid form surrounding the parasite showed uneven thickness. Acridine orange stained the central body yellow and the periphery orange, indicating activity at the level of nucleic acids. The amoeboid form could either be an indicator of pathogenicity of B. hominis, or the form likely to contribute to pathogenicity and be responsible for the symptoms seen in patients.

Programmed cell death in Blastocystis hominis occurs independently of caspase and mitochondrial pathways.

We demonstrated previously that a cytotoxic monoclonal antibody (MAb) 1D5 elicits a programmed cell death (PCD) response in Blastocystis hominis and showed that caspase-3-like protease influences but is not essential for PCD in MAb 1D5-treated B. hominis. We also showed that mitochondrial dysregulation played a role in cell death. In the current study, we further analyzed the signaling pathways involved in PCD mediated by MAb 1D5. B. hominis cells were treated with MAb 1D5 or control MAb 5, either with or without pretreatment with a pan-caspase inhibitor, zVAD.fmk, and/or a mitochondrial transition pore blocker, cyclosporine A (CA). Flow cytometric examination of cell size, mitochondrial membrane potential (delta psi(m)), caspase activation and in situ DNA fragmentation showed that zVAD.fmk and CA, used independently or in combination, failed to inhibit MAb 1D5-mediated PCD. Interestingly, cell exposure to either inhibitor resulted in partial inhibition of DNA fragmentation while combined exposure of cells to inhibitors abolished DNA fragmentation completely. This study sheds new light on the conserved nature of PCD pathways in parasitic protozoa and is also the first report describing caspase- and mitochondria-independent cell death pathways in a protozoan parasite.

Caspase-3-like protease influences but is not essential for DNA fragmentation in Blastocystis undergoing apoptosis.

Blastocystis hominis undergo apoptosis after treatment with a cytotoxic monoclonal antibody (MAb), 1D5, by mechanisms that are not fully understood, although our previous study demonstrated that caspase-3-like protease activity is involved. To elucidate the mechanism of MAb 1D5-induced apoptosis, we inhibited Blastocystis caspase-3-like protease to investigate if there would be a concomitant decrease in in situ DNA fragmentation. However, MAb 1D5-induced apoptosis, evidenced by DNA fragmentation, was not completely blocked by pretreating with specific caspase-3 inhibitor, Ac-DEVD-CHO, indicating that caspase-independent apoptotic pathways might also be involved. Our results also revealed that the treatment with MAb 1D5 resulted in the loss of mitochondrial membrane potential (deltapsim), independent of Ac-DEVD-CHO pretreatment. In conclusion, this study demonstrates that MAb 1D5-induced apoptosis in B. hominis is not wholly dependent on caspase-3-like protease activity and is associated with mitochondrial dysregulation. This is the first report showing evidence for complex apoptotic pathways in a unicellular parasite.

Metronidazole induces programmed cell death in the protozoan parasite Blastocystis hominis.

Previous studies by the authors have shown that the protozoan parasite Blastocystis hominis succumbed to a cytotoxic monoclonal antibody with a number of cellular and biochemical features characteristic of apoptosis in higher eukaryotes. The present study reports that apoptosis-like features are also observed in growing cultures of axenic B. hominis upon exposure to metronidazole, a drug commonly used for the treatment of blastocystosis. Upon treatment with the drug, B. hominis cells displayed key morphological and biochemical features of programmed cell death (PCD), viz. nuclear condensation and nicked DNA in nucleus, reduced cytoplasmic volume, externalization of phosphatidylserine and maintenance of plasma membrane integrity with increasing permeability. This present study also supports the authors' previously postulated novel function for the B. hominis central vacuole in PCD; it acts as a repository where apoptotic bodies are stored before being released into the extracellular space. The implications and possible roles of PCD in B. hominis are discussed.

Blastocystis hominis: evidence for caspase-3-like activity in cells undergoing programmed cell death.

We have shown previously that the human intestinal protozoan, Blastocystis hominis, undergoes apoptosis-like programmed cell death (PCD) when exposed to a cytotoxic monoclonal antibody (mAb), 1D5. In the present study, ELISA and immunoblot assays employing chicken anti-CPP32 antibody suggest that caspase-3-like antigens exist in B. hominis. Using colorimetric and flow cytometric assays for caspase-3 activity, we also observed an increase in activity between 1 h and 6 h after exposure to mAb 1D5, with greatest activity at 6 h. These findings suggest that caspase-3-like proteases play an important role in B. hominis undergoing PCD, similar to the phenomenon in higher eukaryotic organisms.

Blastocystis hominis: un parásito de patogenicidad dudosa / Blastocystis hominis: a parasite of uncertain pathogenicity

Se comentan las opiniones de varios autores sobre la naturaleza del Blastocystis hominis y que abarcan desde su primer hallazgo microscópico hasta su actual ubicación taxonómica. Se señalan los criterios que fueron utilizados para su clasificación en el reino protista. Además, se analizan las complejas formas de división que hasta ahora se conocen y se describen las tres imágenes microscópicas más aceptadas entre los expertos, y que pueden ser halladas en los exámenes enteroparasitológicos. Se presentan dos láminas: una con esquemas de la morfología, y la otra, con fotomicrografías del Blastocystis hominis. Se discute la incertidumbre acerca de las hipótesis que vinculan a este protozoario con la enfermedad y la necesidad de consensuar los criterios en los informes de los resultados del laboratorio parasitológico. Finalmente, se analiza la efectividad de la metodología utilizada en el diagnóstico de este parásito

Blastocystis hominis: un parásito de patogenicidad dudosa / Blastocystis hominis: a parasite of uncertain pathogenicity

Se comentan las opiniones de varios autores sobre la naturaleza del Blastocystis hominis y que abarcan desde su primer hallazgo microscópico hasta su actual ubicación taxonómica. Se señalan los criterios que fueron utilizados para su clasificación en el reino protista. Además, se analizan las complejas formas de división que hasta ahora se conocen y se describen las tres imágenes microscópicas más aceptadas entre los expertos, y que pueden ser halladas en los exámenes enteroparasitológicos. Se presentan dos láminas: una con esquemas de la morfología, y la otra, con fotomicrografías del Blastocystis hominis. Se discute la incertidumbre acerca de las hipótesis que vinculan a este protozoario con la enfermedad y la necesidad de consensuar los criterios en los informes de los resultados del laboratorio parasitológico. Finalmente, se analiza la efectividad de la metodología utilizada en el diagnóstico de este parásito (AU)

Survival of Blastocystis hominis clones after exposure to a cytotoxic monoclonal antibody.

Our previous studies have shown that monoclonal antibodies (MAbs) to Blastocystis hominis react mainly with carbohydrate epitopes, while 1 MAb (1D5) reacts specifically with a protein of 30.5 kDa. In the present study, 3 monoclonal antibodies (1D5, 1E7 and 4F7) were used in immunogold localization. 1E7 and 4F7 were found to react primarily with the surface coat, while 1D5 was plasma membrane-specific. In the presence of complement, only 1D5 exhibited a cytotoxic effect on B. hominis whereas 1E7 and 4F7 did not, suggesting that the surface coat of B. hominis could serve as an immunological barrier against host antibodies. Using a recently described agar plating method, only 1D5 exhibited significant (P < 0.01) complement-independent cytotoxicity to B. hominis, inhibiting colony growth at low concentrations. Parasites that had been exposed to 1D5 were morphologically smaller than those that were not exposed to this MAb. Colonies that grew in the presence of 1D5 were isolated and grown in liquid medium containing increasing amounts of the cytotoxic MAb. Two clones that grew well in liquid medium containing 1D5 were also able to develop into colonies in soft agar. This study has shown that the 30.5 kDa protein found on the plasma membrane of B. hominis is a functionally important protein and that not all cells within a certain population would be susceptible to the cytotoxic effects of 1D5. These findings suggest that a heterogenous population exists in continuously maintained cultures of B. hominis.

Description of an improved method for Blastocystis hominis culture and axenization.

An improved method for Blastocystis hominis culture and axenization was developed in the present study. Stool samples were cultured in prereduced Boeck-Drbohlav NHI modified medium (with several modifications) supplemented with antibiotics (0.4% ampicillin, 0.1% streptomycin, 0.0006% amphotericin B). Axenization was performed by the combination of partial purification of B. hominis by Ficoll-metrizoic acid gradient and inoculation in fresh medium containing active antibiotics against remaining bacteria. A total of 25 strains were obtained by this procedure. The time required for axenization ranged between 3 and 5 weeks. The generation time of axenic strains ranged from 6.6 to 12.1 h (mean +/- SD 110.0 +/- 1.8 h) and the mean number of generations was 2.5 +/- 0.6 h per 24 h. The size of vacuolar and ameboid forms found in stools and in culture was similar. The special formulation of the medium used reduced the generation time and did not modify the cellular size as compared with fecal forms.

Blastocystis hominis revisited.

Blastocystis hominis is a unicellular organism found commonly in the intestinal tract of humans and many other animals. Very little is known of the basic biology of the organism, and controversy surrounds its taxonomy and pathogenicity. There morphological forms (vacuolar, granular, and ameboid) have been recognized, but recent studies have revealed several additional forms (cyst, avacuolar, and multivacuolar). The biochemistry of the organism has not been studied to any extent, and organelles and structures of unknown function and composition are present in the cells. Several life cycles have been proposed but not experimentally validated. The form used for transmission has not been defined. Infections with the organism are worldwide and appear in both immunocompetent and immunodeficient individuals. Symptoms generally attributed to B. hominis infection are nonspecific, and the need for treatment is debated. If treatment appears warranted, metronidazole is suggested as the drug of choice, although failures of this drug in eradicating the organism have been reported. Infection is diagnosed by light microscopic examination of stained smears or wet mounts of fecal material. Most laboratories identify B. hominis by observing the vacuolar form, although morphological studies indicate that other forms, such as the cyst form and multivacuolar form, also should be sought for diagnosis.

The diagnosis of Blastocystis hominis cysts in human faeces.

Blastocystis hominis cysts in faeces can be concentrated on Ficoll column. They are pleomorphic in shape having a mean diameter of 6.65 microns (S.D. = 2.32). They may or may not have a membranous layer surrounding them. The membranous layer tends to come off with pressure or dying of the specimen and corresponds to the fibrillar layer observed under the electron microscope. The Ficoll concentration technique is also useful for concentrating other protozoan cysts.

Colony formation of Blastocystis hominis in soft agar.

This is the first description of a method for growing axenized Blastocystis hominis as colonies in petri dishes containing soft agar. Blastocystis cells cultured in two types of agar appeared to show different colonial morphologies as well as differing colony yields. Microscopic examination of the colonies revealed many amoeboid and giant cells. Many cells were also shown to possess thin filament-like structures that appeared to stretch across the central vacuole.

Morphological diversity of Blastocystis hominis in sodium acetate-acetic acid-formalin-preserved stool samples stained with iron hematoxylin.

The objective of this investigation was to study the morphological characteristics of Blastocystis hominis in sodium acetate-acetic acid-Formalin-preserved stool samples. Routinely processed samples were examined for morphological detail, including size, shape, nuclear detail, and central body characteristics. Morphological findings revealing the importance of recognizing B. hominis in the diagnostic laboratory are described.