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.     

Freeze-fracture and cytochemical studies on the in vitro cyst form of reptilian Blastocystis pythoni

After cultured cysts are osmotically shocked by treating with distilled water, there is an exponential increase in the cyst form of Blastocystis pythoni; this was demonstrated by an immunofluorescence antibody assay against the culture organisms. In 11-day-old cultures of B. pythoni, 68.8% of the organisms (= 2.2 x 10(8) cysts/ml) were in the cyst form. Examination of thin sections of cysts revealed many similarities to the cyst forms of Blastocystis obtained from fecal samples in previous investigations. Freeze-fracture images of the plasma membrane of non-cyst cells also revealed a similar distribution of the intramembrane particles (IMP) when compared to non-cysts of B. hominis, while the plasma membrane of the cyst form showed practically no IMP. The size and morphology of particle-rich small depressions and smooth small protrusions observed on the P face and E face of non-cyst cells, respectively, were similar to endocytic sites reported for B. hominis. In the present study glycogen was cytochemically demonstrated at the ultrastructural level by an alkaline bismuth staining method in both cyst and non-cyst cells.     

Blastocystis hominis: frequency of infection in ambulatory patients from the northern section of Santiago, Chile]

In July 1989-July 1990 period, a coproparasitological study of 6,162 ambulatory patients from the northern section of Santiago, was undertaken. Out of the total number of the studied individual, 88.6% were children, (51.4% females and 48.6% males). The global frequency of infection by B. hominis was 30.4%. In relation to age. Blastocystis hominis was found in: 13.1% of the group of children (1 month-2 years); 34.1% of the pre-school children, 45.4% in the school-children and 43.0% in the adults. B. hominis was frequently detected in association with other parasites and/or commensals, and observed alone in the 6.0% of the studied patients.     

Phylogenetic position of Blastocystis hominis and of stramenopiles inferred from multiple molecular sequence data

Blastocystis hominis, a parasite of the human intestine, has recently been positioned within stramenopiles by the small subunit rRNA phylogeny. To further confirm its phylogenetic position using multiple molecular sequence data, we determined the nucleotide sequences putatively encoding small subunit ribosomal RNA, cytosolic-type 70-kDa heat shock protein, translation elongation factor 2, and the non-catalytic 'B' subunit of vacuolar ATPase of B. hominis (HE87-1 strain). Moreover, we determined the translation elongation factor 2 sequence of an apicomplexan parasite, Plasmodium falciparum, that belongs to alveolates. The maximum likelihood analyses of small subunit rRNA and cytosolic-type 70-kDa heat shock protein clearly demonstrated that B. hominis (HE87-1 strain) is positioned within stramenopiles, being congruent with the previous small subunit rRNA analysis, including the sequences of B. hominis (Nand strain) and a Blastocystis isolate from guinea pig. Although no clear resolution among major eukaryotic groups was obtained by the individual phylogenies based on the four molecules analyzed here, a combined analysis of various molecules, including these, clearly indicated that Blastocystis/stramenopiles are the closest relatives of alveolates.     

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.     

Cytochrome-free mitochondria of an anaerobic protozoan--Blastocystis hominis

Isolated mitochondria of the anaerobic protozoan Blastocystis hominis were subjected to spectral analysis, color, catalase, and peroxidase tests and found to be completely negative for cytochrome enzymes, catalase, and peroxide. Based on the absence of cytochrome enzymes, the possible evolution of B. hominis mitochondria from anaerobic bacteria is postulated.     

Cytochrome-Free Mitochondria of an Anaerobic Protozoan—Blastocystis hominis

ABSTRACT. Isolated mitochondria of the anaerobic protozoan Blastocystis hominis were subjected to spectral analysis, color, catalase, and peroxidase tests and found to be completely negative for cytochrome enzymes, catalase, and peroxide. Based on the absence of cytochrome enzymes, the possible evolution of B. hominis mitochondria from anaerobic bacteria is postulated.     

Generation Time and Growth Rate of the Human Intestinal Parasite Blastocystis hominis

ABSTRACT. Blastocystis hominis , an anaerobic intestinal protozoan parasite of man, has a generation time (GT) in axenic culture of 8.5–19.4 h, depending on the strain tested. Average GT of the eight strains was 11.7 h. Zero growth time cell counts of 5.0 × 10⁵/ml to 2.0 × 10⁶/ml rose in 3–5 days to 1 × 10⁷ or 1 × 10⁸ cells/ml. The GT was determined for the 24-h period during which the most rapid growth occurred; about 2% of the B. hominis cells were in division during this time. Division under the culture conditions provided was by binary fission, the usual mode for B. hominis in vitro as well as in vivo. Division times were determined also by direct observation of individual dividing cells in slide cultures. These were usually ca. 40–60 min but sometimes as low as 20 min.     

Infectivity of different genotypes of human Blastocystis hominis isolates in chickens and rats

Most Blastocystis hominis isolates from humans are believed to be potentially zoonotic. This is because B. hominis isolates found in a variety of other host species have been found to have identical or relatively similar genotypes to those found in human isolates. However, the transmission of human B. hominis isolates to other animals has not been confirmed experimentally. In this study, the infectivity associated with several unique human Blastocystis genotypes (subtypes 2, 3, 4 and 7) was therefore investigated by infecting chickens and rats with two isolates of each subtype experimentally. The results showed that one isolate of subtype 4 and one isolate of subtype 7 was capable of infecting both chickens and rats, while two isolates of subtype 2, another isolate of subtype 4, and another isolate of subtype 7 could only infect chickens. Conversely, two isolates of subtype 3 failed to infect either of the animals. These results confirmed that several genotypes from human isolates could infect chickens and/or rats, indicating that chickens and rats are suitable experimental animal models for studying the zoonotic potential of human Blastocystis isolates.     

Ultrastructure of Blastocystis hominis in human stool samples.

A study of the ultrastructure of Blastocystis hominis in human stools found morphological differences between the organisms seen and those present in laboratory cultures. B. hominis found in stool samples showed little morphological variation with storage time before fixation, but were consistently smaller (approximately 5 microns in diameter), with a thicker surface coat than the cultured organisms. The large central vacuole, characteristic of the cultured organisms, and accepted as standard morphology of B. hominis, was rarely observed in organisms present in stool samples. Instead, a number of small vacuoles, or possibly a network of interconnected vacuoles, were noted. After short-term culture, organisms from these samples appeared with the typical vacuolated morphology. No large vacuoles were present in organisms obtained at colonoscopy. These results suggest that the vacuolated form as previously described may be an artefact of culture conditions, and that the form of B. hominis present in the gastrointestinal tract is avacuolar.     

Blastocystis hominis--a potential intestinal pathogen.

The parasite Blastocystis hominis has been found in 10% to 18% of stool specimens submitted to microbiology laboratories. Controversy exists as to whether this organism can cause illness in humans. We have reviewed the records of 65 symptomatic patients with B hominis in their stool. We conclude that B hominis is a potential pathogen that may or may not require drug therapy depending on the overall clinical circumstances, the severity of symptoms, and the presence of other pathogenic organisms.     

Detection of Blastocystis hominis in unpreserved stool specimens by using polymerase chain reaction

Blastocystis hominis is a common enteric parasite of worldwide distribution. Its pathogenetic potential has not yet been established, although numerous case reports suggest that B. hominis may cause the development of various gastrointestinal symptoms and disorders. The detection of the parasite in stool specimens is conventionally done by microscopy of direct smears, fecal concentrates, or permanently stained smears; however, morphology-based diagnosis is problematic. The aim of this study was to develop and evaluate a polymerase chain reaction (PCR) technique for the direct detection of B. hominis in human stool samples. Primers were based on small subunit ribosomal DNA and able to detect > or =32 parasites/200 mg stool artificially spiked with cultured B. hominis. In the evaluation of 43 clinical specimens, the PCR was tested against the formol ethyl acetate concentration technique (FECT) and a culture technique, proving 100% test specificity and a significantly higher sensitivity than the FECT. The PCR method is recommended for screening clinical specimens for B. hominis infection and for use in prevalence studies.     

Ultrastructural variation of Blastocystis hominis stocks in culture

An ultrastructural study of 10 different Blastocystis hominis stocks was undertaken. Three distinct morphological forms, vacuolar, granular and amoeboid, were distinguished. Numerous variations in the organelles and general cell structure were observed between stocks. B. hominis displayed considerable size variation in the vacuolar forms, ranging from 4 to 63 micron. Thickness and density of the surface coat varied between different stocks. Beneath the surface coat the bilaminar cell membrane displayed electron-dense pits. The nature and quantity of the vacuolar contents varied, and in the granular form four morphologically different inclusions were seen. The organelles which showed the greatest variation between stocks were the mitochondria, varying in shape, electron-density, type of cristae and presence of inclusions. There was minimal variation between stocks with regard to endoplasmic reticulum, Golgi complex and nuclei. Budding of material between the cytoplasm and central vacuole was observed in some stocks. Indications of phagocytic behaviour of B. hominis were seen in the amoeboid form and in the vacuolar form of one stock.     

Molecular comparative studies among Blastocystis isolates obtained from humans and animals

This study compared specific polymerase chain reaction (PCR) primers and phylogenetic tree analysis of restriction fragment length polymorphism using the small subunit ribosomal RNA gene in various Blastocystis populations. A phylogenetic tree was constructed using 12 restriction enzymes and a sample pool of 22 isolates, including 2 reference strains and Proteromonas lacertae as an outgroup. The analysis showed that the 22 isolates could be separated into 7 clusters. Four of the 7 clusters were mixed groups that comprised isolates from both humans and nonhuman hosts. The other 3 clusters contained isolates from humans or nonhuman hosts only. The phylogenetic analysis also showed that B. hominis isolates from geographical separated areas did not necessarily cluster in the genetically different groups. The results of genetic homology and phylogenetic tree analysis among Blastocystis isolates from humans and animals indicated that all isolates from animals appear to be B. hominis. Polymerase chain reaction amplifications using previously described and newly defined specific primers mirrored the clusters obtained by the phylogenetic tree analysis. Our results show that primer PCR can be used as a powerful tool for the typing of Blastocystis populations.     

Incidence of infection by intestinal parasites among school children in Santiago, Chile, 1988-1989

In December 1988-February 1989 period, a survey on enteroparasitic infections in children (mean age 8.7 years; males 54.4% and females 45.6%) from five schools located in Santiago, Chile, was undertaken. To each of these individuals, three samples were obtained in order to perform in them the following examinations: co-proparasitological study, modified Ziehl-Neelsen staining for detecting Cryptosporidium sp. and cellulose adhesive test for Enterobius vermicularis. Parasitic elements were found in the following percentages: Blastocystis hominis 51.8, Enterobius vermicularis 39.9, Giardia lamblia 32.1, Entamoeba histolytica 7.8, Hymenolepis nana 2.1 and Ascaris lumbricoides 0.4, Cryptosporidium sp. were not observed. The present study would be demonstrating that enteroparasites maintain their endemic condition among school children from Santiago, including B. hominis which had not been searched previously.     

Blastocystis hominis in patients at the Ruiz y Paez University Hospital from Bolivar City, Venezuela]

Blastocystis hominis is a polymorphic protozoan of discussed taxonomic position, which is currently associated with human intestinal disease. In order to determine the prevalence of the microorganism in a sample of hospitalized patients, a study was carried out from november 1996 to april 1997 on 100 adult patients of both sexes aged 20 to 79 years at the "Ruíz y Páez" University Hospital of Bolivar city, Venezuela. A coproparasitological study was carried out using direct examination and Faust method. Infection by parasites and/or commensals was demonstrated in 48 patients. The most frequent agent was B. hominis with a prevalence of 42.0%. We did not find a statistically association between sex (P > 0.05) or age (X2 = 3.52; d.f; = 3) and B. hominis infection. B. hominis was most frequently identified as the single parasite (88.1%), and with a number of less than 5 cells per 400X microscopic field (73.8%). The infection was more common in patients with base chronic-immunosuppressive diseases, the major one being cancer. Diarrhea was observed in 27.0% of cases. Due to its high prevalence, especially as a single agent, together with the particular immunological characteristics of the patients studied, a potential pathogenic role of the opportunistic type is suggested for B. hominis.     

Identification of Blastocystis hominis by colonic brush cytology. A case report.

Blastocystis hominis is a unicellular organism the pathogenic potential of which in humans remains unclear. It may be identified during a workup for gastrointestinal symptoms, usually in stool examined for ova and parasites. We describe a case in which B hominis was identified by cytologic examination in a patient with Crohn's disease who underwent colonoscopy and brushing of a transverse colon stricture. The morphologic features of this organism are described and contrasted with those of the uninucleate cyst form of Entamoeba histolytica.     

Seasonal variation of intestinal protozoa infections in outpatients of the north section of Santiago, Chile. 1995-1996]

Formalin preserved fecal samples from 6,058 and 5,863 outpatients were examined for intestinal parasites during 1995 and 1996 respectively. Prevalence rates of infections by intestinal protozoa in both years were similar. By age group (0-9, 10-19 and > 20 years old) Blastocystis hominis was observed in 18.6-19.3, 37.0-31.1 and 25.3-25.4% in 1995-1996 respectively. Prevalence of Giardia intestinalis infections decreased from 16.6-17.4% in the 0-9 year-old children group to 4.1-4.5% in patients over 20 years. Overall percentages of infection by Entamoeba histolytica varied between 4.2 and 10.9. Rates of infections by G. intestinalis, E. histolytica, and Entamoeba coli observed during rainy-cold months (april-september) of the year versus drywarmy period (october-march) were the same. On the contrary, more cases of B. hominis infection 25.8% versus 18.2% (this difference being statistically significant, p > 0.001) were observed during rainy-cold months of the year.     

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.     

Epidemiological survey of Giardia spp. and Blastocystis hominis in an Argentinian rural community

The aim of this study was to relate personal data, socio-cultural and environmental characteristics, and the presence of symptoms/signs with the frequencies of Giardia spp. and Blastocystis hominis among a rural population in Buenos Aires Province, Argentina. Of the surveyed population (350), 3.7% were infected with only Giardia spp. or 22.9% with B. hominis, and 2.3% were infected with both protozoa. The frequency of infection according to sex; 6.1% of males were infected and 1.6% of females by Giardia spp., 26.7% and 19.5% by B. hominis, and 2.4% and 2.2% by both parasites, respectively. Giardia spp. was detected in only three adults (over 14 years), but B. hominis was more frequent in adults than in children. The prevalences of these protozoa in this community are lower than those reported by other Argentinean studies, which is probably associated with the low density of the studied population (5.95 inhab/km2). Statistical analysis revealed that a male sex, flooding of the home, the use of a latrine, and an abdominal pain were correlated with the presence of these parasites, which indicate the importance of these factors in rural communities.