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

International Journal for Parasitology 32 (2002) 789–804 www.parasitology-online.com

Invited review

Recent advances in Blastocystis hominis research: hot spots in terra incognita Kevin S.W. Tan*, Mulkit Singh, Eu Hian Yap Department of Microbiology, Faculty of Medicine, National University of Singapore, 5 Science Drive 2, Singapore, Singapore 117597 Received 12 September 2001; received in revised form 14 December 2001; accepted 18 December 2001

Abstract 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. q 2002 Australian Society for Parasitology Inc. Published by Elsevier Science Ltd. All rights reserved. Keywords: Blastocystis; Taxonomy; Genetic diversity; Morphology; Culture; Immunobiology; Pathogenesis; Programmed cell death; Apoptosis

1. Introduction Blastocystis hominis is a fascinating intestinal protozoan parasite first described in the early 1900s (Alexeieff, 1911; Brumpt, 1912). However, it was Charles Zierdt’s numerous studies of this organism (spanning predominantly the 1970s and 1980s) that first attracted the attention of biologists and clinicians. Early workers were unable to classify B. hominis and erroneously described it as the cyst of a flagellate, vegetable material, yeast and fungus (Zierdt, 1991). Its protistan features were subsequently described by Zierdt et al. (1967), based on morphological and physiological criteria. Blastocystis hominis is probably the most common human gut protozoan in the world, with .50% prevalence in developing countries (Stenzel and Boreham, 1996). Its clinical relevance is currently uncertain and shrouded in controversy with numerous conflicting reports on its ability to cause disease. This is one of the major impetuses for the recent surge of interest in this parasite. * Corresponding author. Tel.: 165-874-6780; fax: 165-776-6872. E-mail address: [email protected] (K.S.W. Tan).

Despite the efforts of a handful of laboratories to study B. hominis, until recently, only its morphology had been extensively described (Stenzel and Boreham, 1996). We now have more detailed information on the various morphological forms from numerous light and electron microscopic (EM) studies. Many other aspects of Blastocystis biology remain virtually unexplored territories. Very little is known about its mode of transmission, pathogenicity, culture characteristics, taxonomy, life-cycle, biochemistry and molecular biology. Substantial progress in B. hominis research in recent years has provided us with, or brought us closer to, answers to some of the fundamental questions regarding Blastocystis biology. Blastocystis research has been challenging for a variety of reasons. (1) There is a lack of critical research partly because of the relatively small number of laboratories currently studying the parasite. (2) The existence of genetic and antigenic diversity among morphologically identical parasites complicates the comparison and verification of data. (3) The lack of a suitable animal model hinders investigations into its immuno- and patho-biology. (4) The scarcity of well-characterised genes and proteins in this parasite

0020-7519/02/$20.00 q 2002 Australian Society for Parasitology Inc. Published by Elsevier Science Ltd. All rights reserved. PII: S 0020-751 9(02)00005-X

790

K.S.W. Tan et al. / International Journal for Parasitology 32 (2002) 789–804

discourages study of its cell and molecular biology. This review focuses on recent work that has contributed to a better understanding of B. hominis biology, describes current research findings and discusses future research prospects. For an extensive account of the parasite’s history, the reader is referred to Zierdt’s review (1991) while prior information on the morphology and biology of B. hominis may be found in Stenzel and Boreham’s (1996) review.

2. Taxonomy 2.1. Taxonomy and phylogenetic affinities Even though discovered more than 80 years ago, Blastocystis still remains a mysterious parasite with little-understood taxonomic links. The protistan features of this organism were revealed by the observation that it possessed one or more nuclei, had smooth and rough endoplasmic reticulum (ER), Golgi complex and mitochondrion-like organelles; it failed to grow on fungal media and was not killed by antifungal drugs; and some antiprotozoan drugs showed activity against this parasite (Zierdt, 1988, 1991). The latter author classified the organism initially as a sporozoan based on morphology, cultural characteristics and modes of division and later reclassified it as a sarcodine, albeit with insufficient evidence. Stenzel and Boreham (1996), in the most up-to-date review of Blastocystis, lamented, that “recent debates on the systematics of the protists have not included the genus Blastocystis in any suggested classifications”. As if in response to this, there has been a surge of interest in this area in the last few years. The taxonomy and phylogenetic affinities of Blastocystis have been analysed by comparison of the parasite’s ssrRNA gene sequences with those from different eukaryotes. By analysis of partial ssrRNA sequences, Johnson et al. (1989) showed that B. hominis is not monophylectic with the yeasts (Saccharomyces), fungi (Neurospora), sarcodines (Naegleria, Acanthamoeba and Dictyostelium) or sporozoans (Sarcocystis and Toxoplasma). Based on this data, Johnson et al. (1989) concluded that B. hominis is unrelated to the yeasts and could be placed in an outer group of the clade that links ciliates and Apicomplexa. In a separate study (Silberman et al., 1996) the complete Blastocystis ssrRNA gene was sequenced and it showed that Blastocystis could be placed within the Stramenopiles. The Stramenopiles, synonymous with Cavalier-Smith’s Heterokonta (Cavalier-Smith, 1997), are defined as ‘a complex and heterogenous evolutionary assemblage that includes unicellular and multicellular protists, with both heterotrophic and photosynthetic representatives’. This diverse group includes slime nets, water moulds and brown algae. The study indicated that Blastocystis was, surprisingly, phylogenetically related to the flagellate Proteromonas. These two organisms share certain life-cycle traits. They are gut endosymbionts of vertebrates and encyst to an environmentally resistant form that

appears to allow transmission between hosts. However, unlike Proteromonas, Blastocystis does not possess flagella and tubular hairs. According to a revised classification of the six-kingdom system of life (Cavalier-Smith, 1998), Blastocystis is not a fungus or protozoan as previously assumed and because it does not possess cilia, it is placed in a newly created Class Blastocystea in the Subphylum Opalinata, Infrakingdom Heterokonta, Subkingdom Chromobiota, Kingdom Chromista. This classification makes Blastocystis the first chromist known to parasitise humans. The elongation factor-1a (EF-1a) gene, a highly conserved gene suitable for use as a phylogenetic marker, has recently been employed for molecular studies in Blastocystis. Nakamura et al. (1996) analysed the amino acid sequence of Blastocystis hominis EF-1a, deduced from a cDNA clone, and from a phylogenetic reconstruction analysis using a maximum likelihood method of protein phylogeny, confirmed that B. hominis should not be included within the fungal lineages and suggested that it diverged before Trypanosoma, Euglena, Dictyostelium and other eukaryotes. However, due to low bootstrap probabilities, it was not possible to determine the relationships between Blastocystis, Entamoeba, Plasmodium and Tetrahymena with statistical significance. Ho et al. (2000) examined the nucleotide sequences of the EF-1a gene of Blastocystis and studied the genetic-relatedness of this parasite from humans and animals. A phylogenetic analysis of this gene from 13 isolates of Blastocystis with other eukaryotic EF-1a sequences revealed that the parasite diverged within the same group and the isolates all belonged to the same genus. In contrast to the phylogenetic data based on ssrRNA gene sequences, this study showed that Blastocystis branched off together with Entamoeba histolytica. This apparent discrepancy may be explained statistically by the low bootstrap value (58.1) used to group Blastocystis with E. histolytica. Other possibilities exist for the variation in affinities. They may be due to the choice of genes for analysis. The ssrRNA genes have been known to possess drastic G 1 C content variation among species (Hasegawa and Hashimoto, 1993), which may give rise to misleading trees and hence other genes (e.g. EF-1a, EF-2 and RNA polymerase III large subunit) with less extreme biases have been suggested to be better candidates for phylogenetic studies. Other possible factors contributing to the discrepancy include inadequate species sampling, relatively few informative positions, mutational saturation and long branch attraction phenomenon (Philippe and Laurent, 1998; Roger et al., 1999). Indeed, it is evident that more studies need to be carried out to clarify the issue of Blastocystis phylogenetic affinities. The analysis of multiple candidate genes and larger numbers of isolates should help place Blastocystis in a more precise taxonomic framework. 2.2. Cryptic genetic diversity Numerous morphologically similar Blastocystis isolates have been described from humans and various animals.

K.S.W. Tan et al. / International Journal for Parasitology 32 (2002) 789–804

Various studies utilising sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting (Kukoschke and Mu¨ ller, 1991; Boreham et al., 1992; Mu¨ ller, 1994; Tan et al., 2001b), isoenzyme analysis (Mansour et al., 1995; Gericke et al., 1997), electrophoretic karyotyping (Upcroft et al., 1989; Ho et al., 1994; Carbajal et al., 1997), random amplified polymorphic DNA (RAPD) (Yoshikawa et al., 1998; Init et al., 1999) and restriction fragment length polymorphism (RFLP) analysis (BohmGloning et al., 1997; Clark, 1997; Hoevers et al., 2000; Yoshikawa et al., 2000; Ho et al., 2001) have shown that there is considerable antigenic and genetic heterogeneity among B. hominis isolates within and among geographical regions, suggesting that several strains or species of this parasite exist. Pulsed field gel electrophoresis (PFGE) is a commonly employed technique for chromosome separation and the study of karyotypic diversity. Using this method, significant diversity was revealed among 15 B. hominis isolates (Carbajal et al., 1997). From the study, 11 karyotypic profiles could be clustered into three karyotypes. In contrast, PFGE of five B. hominis isolates from Singapore revealed broadly similar karyotypic patterns (Ho et al., 1994). This discrepancy may be a statistical problem due to the smaller sample size of the Singapore study. RAPD has been used to distinguish isolates of Blastocystis. Yoshikawa et al. (1996) analysed polymerase chain reaction (PCR) banding patterns of a strain (HE87-1) from a patient in Japan, an isolate (CK86-1) from a chicken and the Nand strain (ATCC strain) of B. hominis. The Japanese and the chicken strain shared similar bands, raising the possibility that the chicken isolate may be a zoonotic strain of B. hominis or that certain B. hominis strains are crossinfective. Both strains had common bands with the reference Nand strain. Another human isolate (B) from Singapore did not share any banding patterns with the Nand strain, suggesting that it is an intraspecific variant of B. hominis. The genetic diversity in B. hominis has also been shown by use of RFLP analysis of the ssrRNA gene and of the elongation factor-1a. Clark (1997) examined the sequence variation in the ssrRNA gene in 30 randomly selected isolates of B. hominis using RFLP analysis of PCR-amplified ssrRNA. This method allowed for a rapid comparison of the different isolates. The RFLP patterns obtained with 11 different restriction enzymes were termed riboprints and the different isolates obtained by this method were grouped into ribodemes (genotypes). Seven distinct ribodemes were obtained with varying frequencies. A dendrogram constructed by phylogenetic analysis showed that the seven ribodemes fell into two distinct lineages. Two of the isolates had riboprints identical to that of a guinea pig isolate, indicating that some human Blastocystis infections may be of zoonotic origin, as had been alluded to by other authors (Yoshikawa et al., 1996). Clark (1997) suggested that this genetic diversity in B. hominis might be responsible

791

for the controversial role of Blastocystis in producing disease in humans; isolates vary in their pathogenic role in humans. However, a study on a large number (113) of isolates suggested otherwise (Bohm-Gloning et al., 1997). Their analysis on the RFLP patterns, of PCR-amplified ssrRNA gene, using three restriction enzymes showed the existence of five subgroups none of which were significantly associated with intestinal disease. It is difficult to draw conclusions about the association between genotypes and disease based on only three restriction enzymes. Perhaps the use of more discriminatory enzymes in subsequent studies is required before this important issue can be resolved. More recently, Hoevers et al. (2000) examined the genetic diversity in 14 isolates obtained from four different geographical locations by similar RFLP analysis of ssrRNA. The RFLP patterns allowed them to identify 12 genotypes but they did not find any correlation between geographic origins of B. hominis and RFLP banding patterns. In a comparative study with riboprinting (Ho et al., 2001) it was discovered that RFLP analysis of elongation factor-1a (EF-1a) gene could also be useful for determining genetic diversity among Blastocystis isolates. In concurrence with the terminology in RFLP studies with ssrRNA, the term elfaprints was designated for the RFLP patterns generated from EF-1a and elfatypes (genotypes) for the isolates with similar elfaprints. Elfaprinting was shown to be useful in distinguishing five B. hominis Singapore isolates from an isolate obtained in Pakistan. From the above studies, it is apparent that extensive genetic diversity exists among B. hominis isolates. The genotypes generated from each study can be useful references for studying taxonomic relationships among morphologically identical parasites. Hoevers et al. (2000) had suggested that in order to optimise the usefulness of these observations, such as for effective comparison of data, some form of standardisation is required. Reference strains and standardised panel of restriction enzymes should be used in future studies. 2.3. Speciation Many Blastocystis isolates resembling B. hominis have been described from primates, rodents, birds, reptiles, amphibians and even insects like the cockroach (Boreham and Stenzel, 1993; Chen et al., 1997a; Teow et al., 1992; Yoshikawa et al., 1998). Species from animals have been characterised by morphological criteria and karyotypic differences. Different species in chickens, duck and geese have been described on the basis of morphological differences (studies of Belova and coworkers, cited by Stenzel and Boreham, 1996). Teow et al. (1991) described a species, Blastocystis lapemi, from a sea snake as a result of growth characteristics and a karyotypic profile that differed from B. hominis. Chen et al. (1997b) and Singh et al. (1996) also used karyotypic analysis to describe Blastocystis ratti from rats and new species from reptiles, respectively. The designation of reptilian Blastocystis species was based on broadly

792

K.S.W. Tan et al. / International Journal for Parasitology 32 (2002) 789–804

dissimilar karyotypic profiles and growth conditions when compared with human isolates. The rat isolates, when compared with human ones, also revealed distinctly dissimilar profiles and had the unique property of containing numerous cystic forms in axenised cultures (Chen et al., 1999). Thus, karyotypic differences, when used in conjunction with other discriminatory methods (growth conditions, cell types, gene sequences), can be useful for species designation. Opinions vary amongst researchers as to the validity of these techniques in describing new species. There is a great deal of morphological variation in organisms grown in culture from those seen in faeces. Furthermore, ultrastructural studies on the morphological stages have not been helpful in designating species. Yoshikawa et al. (1998) questioned the use of karyotypic patterns to describe new species from reptilian hosts (Teow et al., 1991; Singh et al., 1996). They cited the extensive karyotypic heterogeneity amongst clinical isolates of Giardia lamblia and Trypanosoma cruzi indicating that karyotypic differences may not be enough for speciation in Blastocystis. They advocated the use of PCR-based RAPD to distinguish species and strains of Blastocystis. The use of karyotypic patterns for speciation of Blastocystis was somewhat supported by studies (Ho et al., 2000, 2001) on the EF-1a gene of Blastocystis. A comparative study on the deduced amino acid sequences of a PCR product, amplified from the coding region of the EF-1a gene, of seven isolates of Blastocystis from rats and reptiles showed them to be different from one another and from B. hominis. Furthermore, when the ssrRNA and EF-1a genes were used in a PCR-based RFLP analysis of genetic heterogeneity in Blastocystis, it was shown that the banding patterns (ribodemes and elfatypes) of the animal isolates were distinctly different from those of B. hominis (Ho et al., 2001). The banding patterns of the rat Blastocystis, B. ratti, were similar among the rat isolates and different from that of B. hominis. All four reptilian isolates studied had different banding patterns from one another, indicating that they belonged to different species. Although these observations appear to validate the earlier use of PFGE as a tool in speciation of Blastocystis, there are a number of interesting observations that need to be accounted for. The B. ratti ribodeme (three isolates) was identical to B. hominis ribodeme 3 in Clark’s (1997) study, which also included a guinea pig isolate. Also, one of the B. hominis isolates (S) in Ho et al.’s (2001) study had a similar riboprint with Clark’s ribodeme 1 and a Japanese B. hominis isolate (HE87-1). HE87-1 was observed to share very similar RAPD patterns with a chicken isolate (Yoshikawa et al., 1996). These observations suggest that some genotypes of Blastocystis are not host-specific and therefore some caution is warranted when deciding if a particular Blastocystis isolate is truly of human or animal origin. Also, the abovementioned ribodemes (1 and 3) are distant from the bulk of the other human Blastocystis isolates and may therefore

suggest that other mammals represent a huge reservoir for infection of humans (Clark, 1997). In a separate study, Snowden et al. (2000) analysed the RFLP of ssrRNA genes belonging to eight Blastocystis isolates that were isolated from various animals (cow, goat, sheep, guinea pig and rhea). Five genotypes were identified in the isolates; multiple genotypes were found in isolates from a single animal host and multiple host species shared a single genotype. Thus, their data further indicate that Blastocystis may not be host specific or may be cross-infective among certain host types. Studies involving more isolates and other conserved genes should help clarify taxonomic relationships among isolates. 2.4. Conclusions In summary, research over the last few years has shown that Blastocystis is a ubiquitous parasite of the animal kingdom. Its taxonomy is still not fully understood although recent studies have linked it closely with either the Stramenopiles or the amoebae. In Cavalier-Smith’s treatise it currently stands as a member of the Heterokont group of organisms under the Kingdom Chromista. The species in humans, B. hominis, exhibits a great deal of genetic heterogeneity. Based on limited current data, there does not seem to be any association between distinct genotypes and geographical origin or pathogenicity. In light of the extensive genetic variation observed in B. hominis (Silberman et al., 1996; Clark, 2000), the establishment of distinct specific names for the various genotypes should be considered. Several studies have indicated human-to-human, animalto-human and animal-to-animal transmissions. Molecular techniques such as karyotypic typing, RAPD and RFLP analyses have helped to characterise the different genotypes of Blastocystis. The analyses of ssrRNA and elongation factor-1a genes have been found useful in such studies. 3. Morphology 3.1. Vacuolar and granular forms Blastocystis hominis is a polymorphic organism and the four forms commonly described in literature are the vacuolar, granular, amoeboid and cyst forms. The vacuolar form, also referred to as the central vacuole form, varies greatly in size, from 2 to 200 mm in diameter, with average diameters of 4–15 mm (Stenzel and Boreham, 1996). A surface coat of varying thickness is found surrounding most cells. The plasma membrane of the parasite contains coated pits, which appear to have a function in endocytosis (Stenzel et al., 1989). Vacuolar forms are spherical and characterised by a large central vacuole, which may occupy 90% of the cell’s volume. The central vacuole usually contains fine granular or flocculent material of varying electron density and appears to have a storage function (Boreham and Stenzel, 1993). The presence of this large structure results in a

K.S.W. Tan et al. / International Journal for Parasitology 32 (2002) 789–804

thin band of peripheral cytoplasm. It is within this region that the nucleus and organelles such as mitochondria, Golgi apparatus and ER reside. The larger organelles are usually clustered in thickened portions of the cytoplasm, usually located at opposite ends of the cell. In some instances, portions of the cytoplasm have been observed to invaginate into the central vacuole, resulting in the deposition of cytoplasmic material (Suresh et al., 1995) and organelles such as mitochondria (Stenzel et al., 1991; Pakandl, 1999; Tan et al., 2001a; Nasirudeen et al., 2001a) within membranebound structures. Interestingly, we have recently observed that B. hominis cells undergoing programmed cell death appear to accumulate such membrane-bound structures, reminiscent of apoptotic bodies in higher organisms, in the central vacuole (Tan et al., 2001a; Nasirudeen et al., 2001a) (see Section 7.1). The vacuolar form is predominant in axenised liquid cultures and is also commonly observed, albeit as a smaller form (,5 mm), in fresh faecal samples. The granular form has been suggested to arise from the vacuolar form and the transition induced by a variety of factors (Stenzel and Boreham, 1996). These include increased serum concentrations in the culture medium, transfer of cells to a different culture medium, axenisation and addition of certain antibiotics (Stenzel and Boreham, 1996). The granular form shares many similarities with the vacuolar form except that numerous granules are found within the thin band of peripheral cytoplasm or, more commonly, within the central vacuole. There is considerable morphological variation in the types of granules within the central vacuole. These may be myelin-like inclusions, small vesicles, crystalline granules and lipid droplets (Dunn et al., 1989). There have also been suggestions that the central vacuole functions in schizogony and endodyogeny with certain reproductive granules representing progeny of Blastocystis (Zierdt et al., 1967; Zierdt, 1991; Suresh et al., 1994). However, there has been some debate as to whether these are bona fide progeny of B. hominis as, ultrastructurally, they appear similar to granule types reported previously. One possibility for the confusion is that many of these observations were also made by conventional light microscopy, which can lead to inaccurate or biased interpretation. For example, it had been suggested (Boreham and Stenzel, 1993) that the less commonly seen multivacuolar form may be misconstrued as a cell undergoing schizogony because the multiple vacuoles can resemble progeny. With the advent of improved live imaging systems, such as deconvolution and/or restoration microscopy, subcellular structures can be observed at very high resolving powers (,0.2 mm). Using fluorescent markers and time-course assays, such systems should be helpful in confirming if certain granular structures reported earlier are indeed progeny of B. hominis. 3.2. Amoeboid form The amoeboid form of B. hominis has been reported infre-

793

quently and its morphological descriptions have yielded conflicting and confusing reports (McClure et al., 1980; Dunn et al., 1989; Zierdt, 1991; Tan et al., 1996b). It has been observed in older cultures, cultures treated with antibiotics and occasionally in faecal samples (Zierdt, 1973). An early TEM study (Zierdt and Tan, 1976) described the amoeboid form as oval, with one or two large pseudopods but without a cell membrane. It is rather peculiar that a cell can survive without a plasma membrane and it is more likely that this was an artefact of sample preparation, as suggested by Boreham and Stenzel (1993). Dunn et al. (1989) provided detailed ultrastructural descriptions of the amoeboid form by TEM, although it would have been helpful if there were light micrographs to corroborate their observations. The amoeboid forms were rather small (2.6– 7.8 mm) and possessed extended pseudopodia. Engulfed bacteria were observed in lysosome-like structures within the cell. Curiously, a central vacuole, Golgi complex, surface coat and mitochondria were not seen. We have observed that colony growth of B. hominis cells induced the formation of numerous amoeba-like forms (Tan et al., 1996b). These cells were, by both light microscopy and TEM, shown to be irregular in outline and possessed distinct pseudopod-like extensions (Tan et al., 1996c, 2001a). In contrast to Dunn et al.’s study, our TEM study revealed a central vacuole, numerous Golgi bodies, ER and mitochondria within the pseudopod-like cytoplasmic extensions, suggesting that these cells are involved in highly active, energy-requiring processes. The cells also appeared to contain, within lysosome-like organelles, cellular fragments of degenerating neighbouring cells, suggesting a role of this form in endocytosis. It is currently unclear if the amoeboid forms isolated from colonies are artefacts of culture or represent forms that would also be found in vivo. It is interesting to note that the colony-induced amoeboid forms of Trichomonas vaginalis had been postulated to represent the same forms that adhere to vaginal epithelial cells (Scott et al., 1995). With our new knowledge of extensive genetic diversity amongst B. hominis isolates, it is conceivable that the conflicting descriptions from the few studies may have arisen from isolate differences. There is scant information on the differentiation of the amoeboid form or its role in the life-cycle of the parasite. It has been suggested that the amoeboid form is an intermediate between the vacuolar and cyst forms and that it ingests bacteria to provide nutrition for the encystment process (Singh et al., 1995). However, Stenzel and Boreham (1996) had postulated that it arose from the morphologically similar avacuolar form (see Section 3.4). None of these have been proven although the presence of amoeboid forms in colonies does suggest that these had arisen from the vacuolar forms used for inoculating the agar plates. The role of the amoeboid form is clearer. The presence of ingested particulate matter, be they bacteria (Boreham and Stenzel, 1993; Stenzel and Boreham, 1996) or cellular debris (of dead neighbouring cells) (Tan, 1999, PhD thesis, National

794

K.S.W. Tan et al. / International Journal for Parasitology 32 (2002) 789–804

University of Singapore; Tan et al., 2001a) in this form does point towards a nutritive or regulatory role (see Section 7.1). 3.3. Cyst form Despite numerous descriptions of the vacuolar and granular forms since the early 1900s, the existence of a morphologically distinct cystic form was confirmed and described relatively recently (Mehlhorn, 1988; Stenzel and Boreham, 1991; Moe et al., 1997, 1999; Zaman et al., 1997a, 1998, 1999b). The delay in these findings was probably attributed to the smaller size (3–5 mm) and distinct appearance of the cyst, which could also be easily confused with faecal debris. Furthermore, these faecal cysts are seldom seen in axenised cultures. Faecal cysts are spherical to ovoid and are protected by a multilayered cyst wall. Internal cellular contents include one to four nuclei, multiple vacuoles and glycogen and lipid deposits (Stenzel and Boreham, 1996; Zaman et al., 1997a; Moe et al., 1999). Such cysts are commonly surrounded by a loose fibrillar layer that appears to shed as they mature (Zaman et al., 1997a). Blastocystis cysts isolated from animal faecal material revealed distinct morphological differences when compared with faecal cysts of B. hominis. Relatively larger cysts, measuring up to 15 mm in diameter, were isolated from Macaca monkeys whilst multiple individual cysts enclosed by a single fibrillar layer were observed from chicken faecal material (Stenzel et al., 1997). The authors suggested that the disparate morphologies of the cyst forms could indicate the presence of different Blastocystis species. Viability analysis of B. hominis cysts revealed that these do not lyse in water (Zaman et al., 1995), are able to survive at room temperature for up to 19 days, but are fragile at extremes of heat and cold, and in common disinfectants (Moe et al., 1996). In contrast to the cystic forms, the vacuolar and granular forms are sensitive to temperature changes, hypertonic and hypotonic environments and exposure to air (Matsumoto et al., 1987; Zierdt, 1991). Thus, the cystic stage is the transmissible form of the parasite. We have observed that alteration of serum concentration resulted in an increase in cystic forms of B. ratti (Chen et al., 1999) when cultured axenically in Iscove’s modified Dulbecco’s medium (IMDM). However, mass encystation of vacuolar forms with 30% horse serum was achieved for some, but not all, B. ratti isolates suggesting that other factors specific to different isolates were contributing to the encystation process. To date, in vitro encystation of B. hominis, to produce the faecal cyst morphological type, has not been described and we were unable to induce cyst formation with the method employed for B. ratti (unpublished observations). However, taking into account that the genotype of B. ratti was identical to Clark’s B. hominis ribodeme 3 (see Section 2.3), it is also possible that different genotypes of B. hominis possess varying susceptibilities to in vitro encystation. We have, however, reported previously a method to induce, in B. hominis, the formation of ‘in vitro

cysts’ that are morphologically distinct from the faecal cyst and resemble granular forms except for the presence of a thick osmiophilic cyst wall-like structure. These were able to withstand hypotonic shock (Villar et al., 1998) and also infect laboratory rats via oral inoculation (Suresh et al., 1994). When vacuolar forms were cultured in encystation medium, the proportion of ‘in vitro cysts’ to vacuolar forms increased with time (Villar et al., 1998). Whether such cysts exist in vivo has been a matter of debate (Stenzel and Boreham, 1996) and more work needs to be done to resolve this issue. To avoid confusion, subsequent studies involving B. hominis cysts should indicate if the cysts were faecal- or in vitro-derived morphological types. The stepwise differentiation of cysts (faecal-derived morphological type) to vacuolar forms (excystation) has been elegantly described by time-course TEM (Moe et al., 1999; Chen et al., 1999). It showed that the faecal cyst of B. hominis and B. ratti develop, rather similarly and dramatically, into vacuolar forms within 24 h of inoculation into the growth media. At the 1 h time point, the cysts appeared typical (see this section, first paragraph). However, at 3 h, multiple small vacuoles, with many harbouring inclusions, accumulated throughout the cytoplasm. The cysts gradually lose their cyst wall around 6 h, eventually replacing it with a thick surface coat. This disintegration of the cyst wall appeared to allow for the enlargement of the parasite from the initial 5 mm to about 7–9 mm at 9 h. This cell enlargement occurred concurrently with the apparent aggregation of the multiple small vacuoles of the cyst at the central region of the cell, with these vacuoles eventually coalescing to form the central vacuole. This coalescence resulted in the deposition of the abovementioned vacuolar inclusions into the central vacuole, resulting in a granular form-like appearance. By 12 h, typical vacuolar and granular forms were apparent and binary fission was observed. The parasite may also undergo division whilst still surrounded by cyst wall material and up to four daughter cells may be observed within a cyst (Chen et al., 1999; Zaman et al., 1999b). 3.4. Other forms Besides the four abovementioned forms, there have also been descriptions of some other forms isolated from intestines and fresh faecal material. The description of cells isolated from a colonoscopy sample was distinctly different from the vacuolar and granular forms (Stenzel et al., 1991) but was similar to an earlier study of B. hominis obtained from a patient producing large volumes of diarrhoeal fluid (Zierdt and Tan, 1976). These were termed avacuolar forms for their lack of a central vacuole. In contrast to culture forms, these cells were smaller (approximately 5 mm in diameter) and lacked a surface coat. Differences in the morphology of the mitochondria were also noted. Interestingly, these avacuolar cells (Stenzel et al., 1991) could not be established in culture and so it remains to be seen if these were indeed Blastocystis cells to begin with. Multivacuolar

K.S.W. Tan et al. / International Journal for Parasitology 32 (2002) 789–804

forms have been reported in fresh human faecal material (Stenzel et al., 1991; Stenzel and Boreham, 1996). These relatively small cells (5–8 mm in diameter) contain multiple small vacuoles varying in size and content and possessed thick surface coats. However, small vacuolar forms were also clearly discernable from the micrographs indicating that the multivacuolar form is not the sole form in faecal material. In a separate study (Lanuza et al., 1997) involving 81 isolates, vacuolar and, in some instances, amoeboid forms were the only Blastocystis cell types seen in faecal samples. The avacuolar and multivacuolar forms have been suggested to represent in vivo stages of the parasite while the larger vacuolar and granular forms predominate in in vitro culture (Boreham and Stenzel, 1993). This needs further confirmation due to the small number of studies that have reported these forms. The multivacuolar forms may well represent intermediate developmental stages of faecal cysts as they undergo differentiation into vacuolar forms, with the coalescence of the described multiple vacuoles forming the central vacuole. The disappearance of the multivacuolar forms in short-term in vitro culture and the subsequent appearance of the vacuolar forms (Stenzel et al., 1991) suggest that these represent transient stages in the B. hominis life-cycle. 3.5. Unusual structures of uncertain function 3.5.1. Surface coat A fine fibrillar layer, commonly referred to as a surface coat, usually surrounds the vacuolar and granular forms. It is probably the same structure that surrounds the cyst form (Zaman et al., 1997a) but there have been conflicting reports as to whether the amoeboid form possesses one (Tan and Zierdt, 1973; Dunn et al., 1989; Tan, 1999, PhD thesis). It varies in thickness and morphology and is usually thicker in cells examined from fresh faecal matter as compared with that of cells from in vitro culture (Boreham and Stenzel, 1993). A number of studies have shown that the surface coat of B. hominis does contain carbohydrates (Yoshikawa et al., 1995a; Lanuza et al., 1996, Tan et al., 1996a) although no quantitative analyses were carried out. Lanuza et al. (1996) used a panel of FITC-labelled lectins in their study and observed that the surface coat had components containing alpha-d-mannose, alpha-d-glucose, N-acetyl-d-glucosamine, alpha-l-fucose, chitin and sialic acid. The surface coat appears to be continuously synthesised and shed in the environment (Zaman et al., 1997b). Scanning EM has revealed that the surface coat of B. hominis had a spongy, lace-like appearance and, in some cases, could reach lengths of about 10 mm (Zaman et al., 1999a; Tan et al., 2000). Bacteria may be found in close association with the surface coat (Silard and Burghelea, 1985; Dunn et al., 1989; Zaman et al., 1997b, 1999a). One of these studies (Zaman et al., 1997b) revealed that bacteria attached to the surface coat show loss of electron density, suggesting that it may serve as an entrapment mechanism for nutritive purposes. The

795

surface coat has also been postulated to function as a mechanical and chemical barrier against host innate and acquired immune responses (Boreham and Stenzel, 1993). This hypothesis was supported by our observation that monoclonal antibodies (mAb) that bind extensively to B. hominis surface coat did not inhibit growth whereas a particular mAb specific for a plasma-membrane protein was cytotoxic to the parasite (Tan et al., 1997). 3.5.2. Mitochondria or hydrogenosomes? Blastocystis hominis is an anaerobe (Stenzel and Boreham, 1996) and the presence of mitochondria-like organelles in this parasite is rather unusual. It has been suggested that the mitochondria of B. hominis are, in fact, hydrogenosomes (Boreham and Stenzel, 1993; Stenzel and Boreham, 1996) because biochemical analyses have shown that a number of typical mitochondrial enzymes were absent from B. hominis (Zierdt, 1986, 1988). Hydrogenosomes were first described in trichomonads (Lindmark and Mu¨ ller, 1973; Lindmark et al., 1975) and have been identified in a broad phylogenetic range of organisms. These organelles have been observed in rumen dwelling ciliates (Yarlett et al., 1981; Lloyd et al., 1989; Paul et al., 1990), free-living ciliates (Fenchel and Finlay, 1991) and rumen fungi (Yarlett et al., 1986; O’Fallon et al., 1991). All organisms known to contain hydrogenosomes are either anaerobic or aerotolerant anaerobes, and all appear to lack mitochondria (Johnson et al., 1995). Hydrogenosomes differ from mitochondria in that they lack cytochromes, the tricarboxylic acid cycle and oxidative phosphorylation (Palmer, 1997). TEM of B. hominis cells cultured in the presence of the reducing agent, sodium thioglycollate, revealed that the mitochondria had transformed into numerous structures that, surprisingly, resembled the hydrogenosomes of Trichomonas vaginalis (Tan, 1999, PhD thesis). These hydrogenosome-like structures were bound by a double membrane, appeared as vesicles 0.5–1.0 mm in diameter and contained a fine granular matrix. A possible test to confirm if the hydrogenosome-like structures seem in B. hominis are indeed hydrogenosomes is to detect the presence of its marker enzyme, hydrogenase. This can be achieved by cytochemical tests which detect hydrogenase activity, as has been shown for other protozoa (Zwart et al., 1988; Paul et al., 1990). Considering the growing evidence for the presence of hydrogenosomes in a wide variety of anaerobic organisms, it is not unlikely that the mitochondria of B. hominis are, in reality, hydrogenosomes. 4. In vitro culture Polyxenic or monoxenic B. hominis cultures generally grow well in Jones’s (1946) or Boeck and Drbohlav’s (1925) inspissated egg medium. Once axenised, B. hominis shows luxuriant growth in enriched monophasic media such as minimum essential medium (MEM) or IMDM that have been supplemented with 10% horse serum and pre-reduced

796

K.S.W. Tan et al. / International Journal for Parasitology 32 (2002) 789–804

for 48 h prior to culture (Boreham and Stenzel, 1993; Ho et al., 1993). There have been some new methods for the culture of B. hominis, particularly in the area of colony growth and axenisation. The ability to grow microorganisms as colonies on solid or semisolid medium has been enormously useful in viability, genetic and biochemical studies because it provides a convenient means of generating and analysing clonal populations. Culture of protozoa as discrete colonies in or on agar/agarose has been established for T. vaginalis (Hollander, 1976), entamoebae (Gillin and Diamond, 1978), Giardia intestinalis (Gillin and Diamond, 1980) and trypanosomatids (Tanuri et al., 1981; Carruthers and Cross, 1992). Colony formation of B. hominis in soft agar was reported relatively recently (Tan et al., 1996b) and was achieved by plating parasites in a mixture of Bacto agar, IMDM and 10% horse serum. Addition of the reducing agent, sodium thioglycollate, improved yields significantly (efficiency of plating ranging from 71 to 98%) and clonal growth was achievable (Tan et al., 1996c). Individual colonies were easily expanded in liquid cultures, providing a method for isolation and expansion of discrete parasite clones. The colony growth method was successfully employed for screening surface-reactive mAb for cytotoxic effects (Tan et al., 1997) and for the axenisation of Blastocystis species isolated from a human, a reptile and laboratory rats (Chen et al., 1997b; Ng and Tan, 1999). More recently, we have described an improved, high-efficiency method for clonal growth of B. hominis on solid agar (Tan et al., 2000). This method was better than the previously described ones as the colonies were now exposed on the agar surface, which made it easier for enumeration and manipulation. The buff-coloured colonies grew to 2 mm in diameter by 7 days post-inoculation and clones were easily expanded in liquid cultures. With the aid of fluorescent viability dyes and by flow cytometry, these colony forms were shown to be viable for 14 days. This is in contrast to liquid cultures, which enter death phase around day 5 (Ho et al., 1993; Lanuza et al., 1997). Such colony growth had allowed for subculturing to be carried out once in 2 weeks instead of weekly. Axenisation of Blastocystis spp. is often achieved by antibiotic treatment (Zierdt and Williams, 1974; Kukoschke and Mu¨ ller, 1991; Ho et al., 1993; Teow et al., 1992; Lanuza et al., 1997; Ng and Tan, 1999) and not by harsher physical methods because of the inherent fragility of the culture forms. There have been reports of unsuccessful axenisation (Boreham and Stenzel, 1993; Lanuza et al., 1997) indicating that in some instances, B. hominis may require bacteria for survival. This is somewhat supported by the observation that once axenised, certain Blastocystis isolates grew slower than when cultured in the presence of accompanying bacteria (Chen et al., 1997b). Another problem encountered by earlier workers was the lengthy periods required to axenise cultures (usually over a month). This can usually be shortened by methods, such as differential centrifugation,

that aid in the physical separation of the parasites from the bacterial bulk, when used in concert with conventional antibiotic treatment. The combination of Ficoll-metrizoic acid gradient and addition of antibiotics was successfully employed to axenise 25 out of 81 B. hominis isolates in an average time of 3 weeks (Lanuza et al., 1997). We have also observed that axenisation was made possible by initial reduction of bacterial load using antibiotics followed by colony growth to isolate pure parasite colonies (Chen et al., 1997b; Ng and Tan, 1999). The cryopreservation protocol described previously (Zierdt, 1991) involved the addition of dimethyl sulphoxide and demanded extreme care. In contrast, glycerol supplemented with foetal calf serum (FCS) was shown to improve yields to about 90% (Suresh et al., 1998). However, this study involved only a single isolate of B. hominis and more work therefore needs to be done before concluding that this formulation would be suitable for other Blastocystis spp. or B. hominis isolates.

5. Immunobiology 5.1. Host immune response Very little is known about the host immune response to B. hominis. A lack of humoral immune response was reported by Chen et al. (1987) who had used Western blot to detect only IgG in sera from only four patients. IgG against B. hominis antigens was subsequently shown, by enzyme linked immunosorbent assay (ELISA), to be elevated in 25 of 28 infected humans in a later study (Zierdt et al., 1995), although the threshold dilution was reported as 1/ 50. Hussain et al. (1997) reported significantly increased IgG2 antibody levels to B. hominis in patients with irritable bowel syndrome, suggesting a possible link between B. hominis and irritable bowel syndrome. The authors suggested that the IgG2 subclass antibody was induced in response to surface coat fragments of the parasite that had been transported, via Peyer’s patch M cells, from the gut lumen to lymphocytes and macrophages. From these few studies, it is evident that B. hominis can elicit a humoral immune response in humans. However, little else is known about innate and acquired immunity to blastocystosis. To better understand the host immune response to B. hominis infection, a suitable animal model must be found. Mice are generally ideal for such studies as a considerable variety of murine-related reagents and technologies exist. The Giardia muris-mouse model of giardiasis (Roberts-Thomson et al., 1976) has provided a useful tool for the study of the immune response to Giardia infection (Faubert, 2000). Unfortunately, mice, hamsters and rabbits are not naturally infected with Blastocystis (Chen et al., 1997a). Rats, however, have been observed to be especially susceptible to Blastocystis infections (Chen et al., 1997a) and would perhaps be useful animal models.

K.S.W. Tan et al. / International Journal for Parasitology 32 (2002) 789–804

5.2. Antigenic diversity Antigenic heterogeneity has been shown to exist among B. hominis isolates (Kukoschke and Mu¨ ller, 1991; Boreham et al., 1992; Mu¨ ller, 1994; Lanuza et al., 1999; Tan et al., 2001b) and was facilitated by techniques such as SDSPAGE, immunoblotting, immunodiffusion and immunofluorescence. Depending on the study, the differences may be discrete (i.e. no cross-reactivity) (Kukoschke and Mu¨ ller, 1991; Boreham et al., 1992; Mu¨ ller, 1994; Tan et al., 2001b) or partial (Lanuza et al., 1999). Such diversity, as has also been shown genetically, reinforces the emerging concept that B. hominis is biologically heterogeneous and strongly suggests that there are more than one species of Blastocystis in humans. It has been postulated that serotypes differ in their pathogenic potential, but the data have been contradictory. Support for the hypothesis came from a single study (Lanuza et al., 1999) that showed that 18 isolates of B. hominis could be classified into two related antigenic groups. Patients suffering from chronic diarrhoea clustered within group 1 while those suffering from acute diarrhoea clustered within group 2. Unfortunately, this study lacked details on the statistical significance of the associations. In a larger study involving 61 B. hominis isolates (Mu¨ ller, 1994), four distinct serogroups were identified using the immunodiffusion assay but none of them were found to be significantly correlated with intestinal disease. This issue would be more readily resolved if and when a suitable animal model is found. Whatever the case, such antigenic diversity should be taken into consideration when developing serological diagnostic tests for B. hominis infections. 5.3. Monoclonal antibodies There have been two reports on the production and characterisation of mAb against B. hominis (Yoshikawa et al., 1995b; Tan et al., 1996a). Yoshikawa et al. (1995b) analysed a panel of eight mAbs. The majority of the mAbs was IgM and, by immunoEM, was observed to bind to surface coat epitopes. Only two of the mAbs showed cross-reactivity with a Blastocystis spp. isolated from a chicken. The other six B. hominis-reactive mAbs were surface coat-specific while the two cross-reactive mAbs bound to material within the central vacuole. The authors suggested that antigenic components on the surface coat are strain- or species-specific while those of the central vacuole were not. mAbs against B. hominis were also raised and characterised in our laboratory (Tan et al., 1996a). Most of these IgM mAb reacted with multiple carbohydrate epitopes, as evidenced by immunoblot, concanavalin A binding and periodate sensitivity assays. These antigens were later shown by immunogold EM to be located on the surface coat (Tan et al., 1997). However, one particular periodate-resistant surface-reactive mAb was cytotoxic to the parasite, as shown by a significant decrease in colony yields when compared with cells exposed to the other mAbs

797

(Tan et al., 1997, 2001b). This mAb (1D5) appeared to bind to a 30 kDa plasma membrane-associated protein. Interestingly, the death process that ensued appeared distinctly apoptotic (see Section 7.1). Despite the cytotoxicity of 1D5, surviving clones within a particular isolate could be generated that, after several culture passages in the presence of the mAb, showed almost complete resistance to its killing effect. Our recent data suggest that the 1D5 epitope is species- and isolate-specific, as there was cross-reactivity only with local B. hominis isolates but none against a B. hominis isolate from Pakistan and a panel of rat and reptilian Blastocystis spp. (Tan et al., 2001b). Collectively, the data suggest that B. hominis is predisposed to exist in a heterogeneous state (i.e. 1D5-susceptible or -resistant cells) in long-term axenic cultures and the 30 kDa surface protein appears to be functionally important to, but is not present on all, Blastocystis isolates. Unlike 1D5, surface coat carbohydrate-reactive mAbs were not cytotoxic to the parasite despite extensive binding (Tan et al., 1997). In other studies, (Hussain et al., 1997; Kaneda et al., 2000) it has been observed that the major immune response in infected patients is probably against surface coat carbohydrates. Taken together, the data suggest that the surface coat of B. hominis may serve as an immunological barrier for the parasite. Another interesting observation is that the surface coat of B. hominis appears to be continuously synthesised and shed into the extracellular environment (Zaman et al., 1997b). These phenomena may have a functional role in vivo. The shed surface coat glycoproteins may act as an immunological ‘smokescreen’ by promoting the generation of ineffective B cells that produce non-cytotoxic or non-neutralising antibodies against surface coat carbohydrate epitopes. This model for the host immune response to B. hominis needs to be proven experimentally.

6. Clinical aspects Blastocystis hominis is commonly found in healthy individuals as well in patients with gastrointestinal symptoms. In general, prevalence of infection is higher in developing than in developed countries (Stenzel and Boreham, 1996). Within communities, groups from lower socio-economic levels who suffer from poor environmental hygiene, due largely to lack of water supply, sewerage and waste removal services are at greater risk of infection (Borda et al., 1996; Wilairatana et al., 1996; Cirioni et al., 1999; Guignard et al., 2000; Taamasri et al., 2000). Increased risk of infection may also be associated with the workplace. In a recent survey conducted in Malaysia, Rajah Salim et al. (1999) showed that people working closely with animals were at higher risk of acquiring Blastocystis infection. About 41% of local animal handlers from two research institutions, a zoo and an abattoir were infected compared with a rate of 17% in a control group of high-rise city dwellers. The authors

798

K.S.W. Tan et al. / International Journal for Parasitology 32 (2002) 789–804

suggested that the animal handlers probably acquired the infection via the faecal-oral route. Foreign workers, such as the Peace Corp volunteers are also at risk of acquiring the infection when working in areas where this parasite is endemic (Herwaldt et al., 2001). The ability of B. hominis to cause disease is currently a matter of intense debate. Most of the confusion has come from numerous clinical and epidemiological studies that either implicate or exonerate the parasite as a cause of intestinal disease. Even case-control studies involving subjects with or without symptoms gave equivocal results. In a study involving expatriates and tourists in Nepal, there was no obvious difference in the prevalence of B. hominis in those with (30%) and without (36%) diarrhoea, suggesting that it was not an important agent causing diarrhoea in this population (Shlim et al., 1995). In contrast, Jelinek et al. (1997) found that B. hominis could be a possible intestinal pathogen in German travellers to the tropics. There is, however, growing evidence to suggest that immunocompromised individuals, particularly patients with AIDS, are more likely to suffer Blastocystis-related diarrhoeal illness (Brites et al., 1997; Ok et al., 1997; Devera et al., 1998; Germani et al., 1998; Ghosh et al., 1998; Cirioni et al., 1999; Prasad et al., 2000; Tasova et al., 2000), although other reports suggest otherwise (Brandonisio et al., 1999; Cimerman et al., 1999). No significant difference, 19.6% compared with 15–16% respectively, was observed in Blastocystis infection between HIV-positive immunocompetent patients and in AIDS patients (Junod, 1995). Similar observations were made in Germany where 38% of 262 HIV patients had Blastocystis in their stools (Albrecht et al., 1995). Patients with AIDS (46%) were more likely to carry B. hominis than those in the earlier stages of HIV infection (32%) but an association with clinical symptoms was not evident. Infection generally resolved spontaneously. Isolation of B. hominis does not justify treatment even in symptomatic, severely immunocompromised patients. Therapy should be limited to patients with persistent unexplained symptoms after a thorough evaluation and a complete screening for alternative aetiologies including the use of endoscopic procedures and careful examination of multiple specimens. In immunocompetent individuals, B. hominis infections have been implicated in a variety of specific and non-specific intestinal disorders such as diarrhoea, abdominal discomfort, anorexia and flatulence (Boreham and Stenzel, 1993). There were two reports on the possible association of B. hominis infection with irritable bowel syndrome, a noninflammatory bowel disease also characterised by similar signs and symptoms (Hussain et al., 1997; Giacometti et al., 1999). The first study showed that irritable bowel syndrome patients had significantly higher IgG2 antibody titres against B. hominis antigens, suggesting that the host immune response was directed primarily against carbohydrate antigens. The second study reported that a significant proportion of patients with irritable bowel syndrome

harboured B. hominis in their stools when compared with those suffering from non-irritable bowel syndrome associated gastrointestinal disease. However, it is unclear from these studies if B. hominis is the primary aetiological agent in irritable bowel syndrome as it is just as possible that a disruption of the microbial flora in irritable bowel syndrome patients had provided conditions for B. hominis to thrive in. Some key findings were revealed in recent experimental infections of BALB/c mice with B. hominis (Moe et al., 1996, 1997). It was shown conclusively that faecal cysts were responsible for the external transmission of B. hominis. Among the different forms of the parasite, the faecal cyst is undoubtedly the most resistant to environmental changes (Moe et al., 1996; Zaman, 1996). Young immunocompetent mice were susceptible to oral inoculation of faecal cysts and the parasites could be detected in the faeces by day 2 postinoculation, with infections clearing after approximately 2 weeks (Moe et al., 1997). There appeared to be an agerelated susceptibility to infection, with juvenile mice being more susceptible than the adult mice, and 8-week old adult mice being completely resistant to infection even when given relatively higher doses of parasites. The infected mice displayed weight loss and lethargy. At necropsy, these mice revealed distention of the caecum and colon. Interestingly, histological examination of the cecum and colon revealed intense inflammatory-cell infiltration, oedematous lamina propria and mucosal sloughing, suggesting that the parasite did cause pathogenesis in the infected mice. This is in contrast to a recent Japanese study, which showed that colonoscopy of seven positive human cases revealed no pathogenic intestinal lesions (Horiki et al., 1997). In a related study, a self-limiting myonecrosis was produced when B. hominis was injected i.m. in mice (Moe et al., 1998). The self-limiting and mildly virulent nature of these infections in healthy mice may reflect the course of similar infections in humans. However, such observations in experimentally infected mice may also be explained by the observation that mice do not naturally harbour Blastocystis (Chen et al., 1997a) and may therefore be merely displaying innate resistance to infection. Despite growing evidence that B. hominis has pathogenic potential, the true nature of its virulence is currently unconfirmed due to the limited number of studies. The possibility that virulent and avirulent strains of Blastocystis exist has been frequently suggested because B. hominis is antigenically (Kukoschke and Mu¨ ller, 1991; Boreham et al., 1992; Tan et al., 2001b) and genetically (Boreham et al., 1992; Yoshikawa et al., 1996; Carbajal et al., 1997; Clark, 1997; Yoshikawa et al., 1998; Init et al., 1999) heterogeneous. This hypothesis has yet to be tested and until the pathogenic status of B. hominis has been clarified we are in agreement with Stenzel and Boreham (1996) that it would be prudent to consider this protozoan a potential pathogen. Many laboratories currently diagnose B. hominis infections by looking for the presence of vacuolar forms in faeces and the amoeboid form in diarrhoeal stools (Carbajal et al.,

K.S.W. Tan et al. / International Journal for Parasitology 32 (2002) 789–804

1997). However, there are cases where the faecal cyst would predominate in faecal samples (Boreham et al., 1996), and laboratory personnel should also be trained to recognise these smaller forms. Culture and concentration methods have been shown to increase the sensitivity of detection (Zaman and Khan, 1994; Suresh et al., 1997). Though currently unavailable, diagnostic antibodies to cystic and vacuolar form antigens would also be useful for identification.

7. Current observations, implications and prospects 7.1. Programmed cell death Perhaps one of the most puzzling findings in recent years is the observation that certain protozoan parasites displayed morphological and biochemical features of programmed cell death or apoptosis. Until recently, programmed cell death was thought to exist only in multicellular organisms, whereby this death process serve a variety of important functions viz. in regulating cell numbers and in growth and development. Necrotic cell death usually results in cell lysis and release of cytotoxic intracellular contents, injuring surrounding cells and tissues. Programmed cell death, however, restricts this damage by dismantling cells into smaller, intact, membrane-bound structures. Since death is the final outcome of programmed cell death, it would seem a priori that this would serve no adaptive function for unicellular organisms. However, reports of programmed cell death in bacteria (Lewis, 2000), yeast (Frohlich and Madeo, 2000) and both free-living (Maercker et al., 1999; Vardi et al., 1999) and parasitic protozoa (Ameisen et al., 1995; Welburn et al., 1997; DosReis and Barcinski, 2001) strongly suggest that the cellular programmed cell death machinery is highly conserved and probably existed before the advent of multicellularity. What therefore is the purpose of programmed cell death in the unicellular organism? In Trypanosoma brucei, programmed cell death was shown to regulate growth of the procyclic form in the insect midgut (Welburn et al., 1997). Under in vitro starving conditions, it was observed that a distinct proportion of T. brucei cells would undergo programmed cell death; the authors suggest that the cells’ demise would provide more resources (e.g. nutrients) for the remaining cells. Exposure of these protozoa to concanavalin A induced apoptosis, resulting in surface membrane vesiculation, DNA fragmentation and migration of chromatin to the nuclear periphery (Welburn et al., 1996), all hallmark phenotypes of apoptosis in the metazoa. Taken together, these observations suggest that unicellular organisms have evolved altruistic mechanisms for survival; sacrificing dying or damaged cells for the benefit of the population as a whole (Heussler et al., 2001). The concept that many unicellular microbes live and die in complex communities that in many ways resemble a multicellular organism led us to investigate if colony

799

forms of B. hominis exhibit programmed cell death-like features (Tan et al., 2001a). Entire colonies, that had been fixed in situ and processed for TEM, revealed that cells, especially those in the central region of the colonies, had undergone bizarre fragmentation into numerous membranebound structures. These small structures, usually containing organelles, were frequently seen, extracellularly, in regions where intact cells had presumably resided. We believe that these membrane-bound structures are analogous to apoptotic bodies described in mammalian cells and serve to reduce dying or unwanted cells into smaller harmless packages. Other characteristic apoptotic features, such as nuclear and chromatin condensation were also observed. In a separate study, we observed that exposure of a particular surface-reactive mAb (1D5) cytotoxic to B. hominis (Tan et al., 1997) resulted in numerous morphological and biochemical features of apoptosis (Nasirudeen et al., 2001a). We employed several methods commonly used to assay for apoptosis in mammalian cells. Blastocystis hominis cells exposed to 1D5 had reduced cell size and increased cell density, evidenced by flow cytometry and light microscopy. TEM observations of such cells revealed condensation and clumping of chromatin material along the nuclear membrane, reduction in cell size and increase in electron density. Interestingly, in some cells, we observed the apparent deposition of apoptotic bodies into, and subsequent release from, the central vacuole. We propose that among other postulated functions, the central vacuole serves as a repository for apoptotic bodies. Perhaps the most conclusive apoptotic feature in the 1D5-exposed cells was the significant increase in DNA fragmentation while maintaining plasma membrane integrity, as shown by TUNEL and FDA/PI permeability assays, respectively (Nasirudeen et al., 2001a). To date, all the evidence for programmed cell death in protozoan parasites comes from morphological and cytochemical approaches with a scarcity of information regarding the genes and proteins involved. As such, our laboratory is currently focussing on the caspase family of genes and proteins, which are evolutionarily conserved from worm to human and play a crucial role in the apoptotic activation pathway (Budihardjo et al., 1999). Our recent experiments have shown that caspase 3-like proteins do exist in B. hominis and activity is significantly increased during apoptotic death (Nasirudeen et al., 2001b) and we are presently isolating the caspase-3 gene homolog by peptide sequence homology alignments and PCR. These findings add B. hominis to the growing list of unicellular organisms that exhibit programmed cell death features and, also provide us with the opportunity to exploit the parasite for the identification and characterisation of novel molecules involved in the programmed cell death pathway. If certain regulators of programmed cell death were conserved among the parasitic protozoa but are sufficiently divergent from humans, then these may be potential drug targets useful for activating the pathogen’s apoptosis pathway/s.

800

K.S.W. Tan et al. / International Journal for Parasitology 32 (2002) 789–804

7.2. New tools for research Technological advances have led to significant progress in many aspects of biological research. The use of flow cytometry, once restricted to the study of cells of the immune system, has also been shown to be useful for the analysis of protozoa (Vesey et al., 1994). The flow cytometer records deflection patterns of laser beams after they hit individual cells flowing in a steady stream of carrier fluid. Various properties such as DNA content, cell size, cell granularity and fluorescence intensity (if fluorescent probes are used) can be obtained rapidly and analysed objectively for large numbers of cells. This technique has been enormously useful in encystation, apoptosis and viability studies of Blastocystis and there is much potential for applications in other aspects of Blastocystis research. The recent completion of the human genome project and accessibility to numerous completed and partially finished genomes of multicellular and unicellular organisms provides an amazing resource for gene and protein analyses. Newly sequenced Blastocystis genes and proteins can be compared against extensive genome databases in the search for functional homologs. Conversely, information from genes or proteins from other organisms can be exploited when screening B. hominis genomic or cDNA libraries for particular genes of functional significance. Unfortunately, the absence of established systems for transient and/or stable transfection of foreign DNA into Blastocystis will hinder functional studies of such genes. Transfection systems have been described for a number of other protozoa (Benzel et al., 2000; Brooks et al., 2000; Dos Santos and Buck, 2000; Yee et al., 2000) and so it is likely that development in Blastocystis would be feasible. Multigene families such as the tubulin and rDNA genes have been successfully used as targets for stable DNA integration in other protozoa (Wirtz et al., 1999) and these may also be similarly useful for Blastocystis transfections.

contradictory. There are also still a number of unanswered questions. What is the life-cycle of the parasite? Although there have been a number of proposals (Zierdt, 1991; Singh et al., 1995; Stenzel and Boreham, 1996), not one has been experimentally demonstrated. Does the extensive genetic diversity observed among various B. hominis isolates (Clark, 1997) constitute different species of varying pathogenic potential? Until a suitable animal model is found, this question will be difficult to answer. Blastocystis appears to grow best in strictly anaerobic conditions, yet why does the parasite contain numerous mitochondria? These questions reveal that much more work needs to be done before we can understand even the basic biology of the parasite. The conflicting data in many aspects (taxonomy, morphology, pathogenesis, life-cycle) of Blastocystis biology is rather unsettling. To limit the problem, there should be more collaboration between the small number of laboratories currently involved in Blastocystis research. Information from the cell and molecular biology of numerous wellstudied protozoa would be useful for the Blastocystis researcher. Research into B. hominis should continue to progress unabated if we can harness the numerous technological and database resources available to the modern day biologist and apply it to the study of this interesting protozoan parasite. Acknowledgements The authors wish to thank Professor George A.M. Cross, Rockefeller University, for critical reading of the manuscript and helpful discussions. Special thanks are also due to Dr Tamara Horton for her help in improving the form and content of this manuscript. Research carried out in our laboratories was funded by generous research grants, RP 890354 and RP 3992340, from the National University of Singapore. References

8. Concluding remarks In the half a decade or so after Stenzel and Boreham’s last review (1996), there has been substantial headway made in understanding the biology of B. hominis. We have now a clearer picture of its morphology and mode of transmission. The new and improved methods for culture and axenisation will be useful for current and future Blastocystis researchers. Blastocystis hominis has been shown to be amenable to flow cytometric analysis and this has been a powerful tool for analysing various properties of large numbers of the parasite. The presence of programmed cell death in B. hominis will provide an interesting opportunity to study and characterise genes and proteins involved in the apoptotic pathway. Despite a surge of information on its taxonomic links, epidemiology and pathogenesis in recent years, the data are still surprisingly inconclusive and in certain cases,

Albrecht, H., Stellbrink, H.J., Koperski, K., Greten, H., 1995. Blastocystis hominis in human immunodeficiency virus-related diarrhea. Scand. J. Gastroenterol. 30, 909–14. Alexeieff, A., 1911. Sur la nature des formations dites ‘kystes de Trichomonas intestinalis’. C.R. Soc. Biol. 71, 296–8. Ameisen, J.C., Idziorek, T., Billaut-Mulot, O., Loyens, M., Tissier, J.-P., Potentier, A., Ouaissi, A., 1995. Apoptosis in a unicellular eukaryote (Trypanosoma cruzi): implication for evolutionary origin and role of programmed cell death in the control of cell proliferation, differentiation and survival. Cell. Death. Differ. 2, 285–300. Benzel, I., Weise, F., Wiese, M., 2000. Deletion of the gene for the membrane-bound acid phosphatase of Leishmania mexicana. Mol. Biochem. Parasitol. 111, 77–86. Boeck, W.C., Drbohlav, J., 1925. The cultivation of Entamoeba histolytica. Am. J. Hyg. 5, 371–407. Bohm-Gloning, B., Knobloch, J., Walderich, B., 1997. Five subgroups of Blastocystis hominis isolates from symptomatic and asymptomatic patients revealed by restriction site analysis of PCR-amplified 16 Slike rDNA. Trop. Med. Int. Health 2, 771–8.

K.S.W. Tan et al. / International Journal for Parasitology 32 (2002) 789–804 Borda, C.E., Rea, M.J., Rosa, J.R., Maidana, C., 1996. Intestinal parasitism in San Cayetano, Corrientes, Argentina. Bull. Pan. Am. Health Organ. 30, 227–33. Boreham, P.F.L., Stenzel, D.J., 1993. Blastocystis in humans and animals: morphology, biology, epizootiology. Adv. Parasitol. 32, 1–70. Boreham, P.F.L., Upcroft, J.A., Dunn, L.A., 1992. Protein and DNA evidence for two demes of Blastocystis hominis from humans. Int. J. Parasitol. 22, 49–53. Boreham, R.E., Benson, S., Stenzel, D.J., Boreham, P.F.L., 1996. Blastocystis hominis infection. Lancet 348, 272–3. Brandonisio, O., Maggi, P., Panaro, M.A., Lisi, S., Andriola, A., Acquafredda, A., Angarano, G., 1999. Intestinal protozoa in HIV-infected patients in Apulia, South Italy. Epidemiol. Infect. 123, 457–62. Brites, C., Barberino, M.G., Bastos, M.A., Sampaio Sa, M., Silva, N., 1997. Blastocystis hominis as a potential cause of diarrhea in AIDS patients: a report of six cases in Bahia, Brazil. Braz. J. Infect. Dis. 1, 91–94. Brooks, D.R., McCulloch, R., Coombs, G.H., Mottram, J.C., 2000. Stable transformation of trypanosomatids through targeted chromosomal integration of the selectable marker gene encoding blasticidin S deaminase. FEMS Microbiol. Lett. 186, 287–91. Brumpt, E., 1912. Blastocystis hominis n sp. et formes voisines. Bull. Soc. Pathol. Exot. 5, 725–30. Budihardjo, I., Oliver, H., Lutter, M., Luo, X., Wang, X., 1999. Biochemical pathways of caspase activation during apoptosis. Annu. Rev. Cell Dev. Biol. 15, 269–90. Carbajal, J.A., Del Castillo, L., Lanuza, M.D., Villar, J., Borra´ s, R., 1997. Karyotypic diversity among Blastocystis hominis isolates. Int. J. Parasitol. 27, 941–5. Carruthers, V.B., Cross, G.A.M., 1992. High-efficiency clonal growth of bloodstream- and procyclic-form Trypanosoma brucei on agarose plates. Proc. Natl Acad. Sci. USA 89, 8818–21. Cavalier-Smith, T., 1997. Sagenista and Bigyra, two phyla of heterotrophic heterokont Chromists. Archiv fu¨ r Protisten 148, 253–67. Cavalier-Smith, T., 1998. A revised six-kingdom system of life. Biol. Rev. Camb. Philos. Soc. 73, 203–66. Chen, J., Vaudry, W.L., Kowalewska, K., Wenman, W., 1987. Lack of serum immune response to Blastocystis hominis. Lancet 2, 1021 (Letter). Chen, X.Q., Singh, M., Ho, L.C., Moe, K.T., Tan, S.W., Yap, E.H., 1997a. A survey of Blastocystis species in rodents. Lab. Anim. Sci. 47, 91–94. Chen, X.Q., Singh, M., Ho, L.C., Tan, S.W., Ng, G.C., Moe, K.T., Yap, E.H., 1997b. Description of Blastocystis species from Rattus norvegicus. Parasitol. Res. 83, 313–8. Chen, X.Q., Singh, M., Howe, J., Ho, L.C., Tan, S.W., Yap, E.H., 1999. In vitro encystation and excystation of B. ratti. Parasitology 118, 151–60. Cimerman, S., Cimerman, B., Lewi, D.S., 1999. Prevalence of intestinal parasitic infections in patients with acquired immunodeficiency syndrome in Brazil. Int. J. Infect. Dis. 3, 203–6. Cirioni, O., Giacometti, A., Drenaggi, D., Ancarani, F., Scalise, G., 1999. Prevalence and clinical relevance of Blastocystis hominis in diverse patient cohorts. Eur. J. Epidemiol. 15, 389–93. Clark, G.C., 1997. Extensive genetic diversity in Blastocystis hominis. Mol. Biochem. Parasitol. 87, 79–83. Clark, G.C., 2000. Cryptic genetic variation in parasitic protozoa. J. Med. Microbiol. 49, 489–91. Devera, R., Azacon, B., Jimenez, M., 1998. Blastocystis hominis in patients at the Ruiz y Paez University Hospital from Bolivar City. Venezuela Bol. Chil. Parasitol. 53, 65–70 (in Spanish). Dos Santos, W.G., Buck, G.A., 2000. Simultaneous stable expression of neomycin phosphotransferase and green fluorescence protein genes in Trypanosoma cruzi. J. Parasitol. 86, 1281–8. DosReis, G.A., Barcinski, M.A., 2001. Apoptosis and parasitism: from the parasite to the host immune response. Adv. Parasitol. 49, 133–61. Dunn, L.A., Boreham, P.F.L., Stenzel, D.J., 1989. Ultrastructural variation of Blastocystis hominis stocks in culture. Int. J. Parasitol. 19, 43–56. Faubert, G., 2000. Immune response to Giardia duodenalis. Clin. Microbiol. Rev. 13, 35–54.

801

Fenchel, T., Finlay, B.J., 1991. The biology of free-living anaerobic ciliates. Eur. J. Protistol. 26, 201–15. Frohlich, K.U., Madeo, F., 2000. Apoptosis in yeast – a monocellular organism exhibits altruistic behaviour. FEBS Lett. 473, 6–9. Gericke, A., Burchard, G., Knobloch, J., Walderich, B., 1997. Isoenzyme patterns of Blastocystis hominis patient isolates derived from symptomatic and healthy carriers. Trop. Med. Int. Health 2, 245–53. Germani, Y., Minssart, P., Vohito, M., Yassibanda, S., Glaziou, P., Hocquet, D., Berthelemy, P., Morvan, J., 1998. Etiologies of acute, persistent, and dysenteric diarrheas in adults in Bangui, Central African Republic, in relation to human immunodeficiency virus serostatus. Am. J. Trop. Med. Hyg. 59, 1008–14. Ghosh, K., Ayyaril, M., Nirmala, V., 1998. Acute GVHD involving the gastrointestinal tract and infestation with Blastocystis hominis in a patient with chronic myeloid leukaemia following allogeneic bone marrow transplantation. Bone Marrow Transplant. 22, 1115–7. Giacometti, A., Cirioni, O., Fiorentini, A., Fortuna, M., Scalise, G., 1999. Irritable bowel syndrome in patients with Blastocystis hominis infection. Eur. J. Clin. Microbiol. Infect. Dis. 18, 436–9. Gillin, F.D., Diamond, L.S., 1978. Clonal growth of Entamoeba histolytica and other species of Entamoeba in agar. J. Protozool. 25, 539–43. Gillin, F.D., Diamond, L.S., 1980. Clonal growth of Giardia lamblia trophozoites in a semisolid agarose medium. J. Parasitol. 66, 350–2. Guignard, S., Arienti, H., Freyre, L., Lujan, H., Rubinstein, H., 2000. Prevalence of enteroparasites in a residence for children in the Cordoba Province. Argentina Eur. J. Epidemiol. 16 (3), 287–93. Hasegawa, M., Hashimoto, T., 1993. Ribosomal RNA trees misleading? Nature 361, 23. Herwaldt, B.L., de Arroyave, K.R., Wahlquist, S.P., de Merida, A.M., Lopez, A.S., Juranek, D.D., 2001. Multiyear prospective study of intestinal parasitism in a cohort of Peace Corps volunteers in Guatemala. J. Clin. Microbiol. 39, 34–42. Heussler, V.T., Kuenzi, P., Rottenberg, S., 2001. Inhibition of apoptosis by intracellular parasites. Int. J. Parasitol. 31, 1166–76. Ho, L.C., Singh, M., Suresh, G., Ng, G.C., Yap, E.H., 1993. Axenic culture of Blastocystis hominis in Iscove’s modified Dulbecco’s medium. Parasitol. Res. 79, 614–6. Ho, L.C., Singh, M., Suresh, K., Ng, G.C., Yap, E.H., 1994. A study of the karyotypic patterns of Blastocystis hominis by pulsed-field gradient electrophoresis. Parasitol. Res. 80, 620–2. Ho, L.C., Armugam, A., Jeyaseelan, K., Yap, E.H., Singh, M., 2000. Blastocystis elongation factor-1 alpha: genomic organization, taxonomy and phylogenetic relationships. Parasitology 121, 135–44. Ho, L.C., Jeyaseelan, K., Singh, M., 2001. Use of elongation-1 alpha gene in a polymerase chain reaction-based restriction-fragment-length polymorphism analysis of genetic heterogeneity among Blastocystis species. Mol. Biochem. Parasitol. 112, 287–91. Hoevers, J., Holman, P., Logan, K., Hommel, M., Ashford, R., Snowden, K., 2000. Restriction-fragment-length polymorphism analysis of smallsubunit rRNA genes of Blastocystis hominis isolates from geographically diverse human hosts. Parasitol. Res. 86, 57–61. Hollander, D.H., 1976. Colonial morphology of Trichomonas vaginalis in agar. J. Parasitol. 62, 826–8. Horiki, N., Maruyama, M., Fujita, Y., Yonekura, T., Minato, S., Kaneda, Y., 1997. Epidemiologic survey of Blastocystis hominis infection in Japan. Am. J. Trop. Med. Hyg. 56, 370–4. Hussain, R., Jaferi, W., Zuberi, S., Baqai, R., Abrar, W., Ahmed, A., Zaman, V., 1997. Significantly increased IgG2 subclass antibody levels to Blastocystis hominis in patients with irritable bowel syndrome. Am. J. Trop. Med. Hyg. 56, 301–5. Init, I., Mak, J.W., Lokman Hakim, S., Yong, H.S., 1999. Strain differences in Blastocystis isolates as detected by a single set of polymerase chain reaction primers. Parasitol. Res. 85, 131–4. Jelinek, T., Peyerl, G., Loscher, T., von Sonnenburg, F., Nothdurft, H.D., 1997. The role of Blastocystis hominis as a possible intestinal pathogen in travellers. J. Infect. 35, 63–66. Johnson, A.M., Thanou, A., Boreham, P.F.L., Baverstock, P.R., 1989. Blas-

802

K.S.W. Tan et al. / International Journal for Parasitology 32 (2002) 789–804

tocystis hominis: phylogenetic affinities determined by rRNA sequence comparison. Exp. Parasitol. 68, 283–8. Johnson, P.J., Bradley, P.J., Lahti, C.J., 1995. Boothroyd, J.C., Komuniecki, R. (Eds.), Cell Biology of Trichomonads: Protein Targeting to the HydrogenosomeWiley-Liss, New York, NY, pp. 399–411. Jones, W.R., 1946. The experimental infection of rats with Entamoeba histolytica. Ann. Trop. Med. Parasitol. 40, 130. Junod, C., 1995. Blastocystis hominis: a common commensal in the colon. Study of prevalence in different populations of Paris. Presse Med. 24, 1684–8 (in French). Kaneda, Y., Horiki, N., Cheng, X.J., Tachibana, H., Tsutsumi, Y., 2000. Serologic response to Blastocystis hominis infection in asymptomatic individuals. Tokai J. Exp. Clin. Med. 25 (2), 51–56. Kukoschke, K.G., Mu¨ ller, H.E., 1991. SDS-PAGE and immunological analysis of different axenic Blastocystis hominis strains. J. Med. Microbiol. 35, 35–39. Lanuza, M.D., Carbajal, J.A., Borra´ s, R., 1996. Identification of surface coat carbohydrates in Blastocystis hominis by lectin probes. Int. J. Parasitol. 26, 527–32. Lanuza, M.D., Carbajal, J.A., Villar, J., Borra´ s, R., 1997. Description of an improved method for Blastocystis hominis culture and axenization. Parasitol. Res. 83, 60–63. Lanuza, M.D., Carbajal, J.A., Villar, J., Mir, A., Borra´ s, R., 1999. Solubleprotein and antigenic heterogeneity in axenic Blastocystis hominis isolates: pathogenic implications. Parasitol. Res. 85, 93–97. Lewis, K., 2000. Programmed death in bacteria. Microbiol. Mol. Biol. Rev. 64, 503–14. Lindmark, D.G., Mu¨ ller, M., 1973. Hydrogenosome, a cytoplasmic organelle of the anaerobic flagellate Tritrichomonas foetus, and its role in pyruvate metabolism. J. Biol. Chem. 248, 7724–8. Lindmark, D.G., Mu¨ ller, M., Shio, H., 1975. Hydrogenosomes in Trichomonas vaginalis. J. Parasitol. 61, 552–4. Lloyd, D., Hillman, K., Yarlett, N., Williams, A.G., 1989. Hydrogen production by rumen holotrich protozoa: effects of oxygen and implications for metabolic control by in situ conditions. J. Protozool. 36, 205– 13. Maercker, C., Kortwig, H., Nikiforov, M.A., Allis, C.D., Lipps, H.J., 1999. A nuclear protein involved in apoptotic-like DNA degradation in Stylonychia: implications for similar mechanisms in differentiating and starved cells. Mol. Biol. Cell 10, 3003–14. Mansour, N.S., Mikhail, E.M., El Masry, E.A., Sabry, A.G., Mohareb, E.W., 1995. Biochemical characterization of human isolates of Blastocystis hominis. J. Med. Microbiol. 42, 304–7. Matsumoto, Y., Yamada, M., Yoshida, Y., 1987. Light microscopical appearance and ultrastructure of Blastocystis hominis, an intestinal parasite of man. Zentralbl. Bakteriol. Microbiol. Hyg. A 264, 379–85. McClure, H.M., Strobert, E.A., Healy, G.R., 1980. Blastocystis in a pigtailed macaque: a potential enteric pathogen for non-human primates. Lab. Anim. Sci. 30, 890–9. Mehlhorn, H., 1988. Blastocystis hominis, Brumpt, 1912: are there different stages or species? Parasitol. Res. 74, 393–5. Moe, K.T., Singh, M., Howe, J., Ho, L.C., Tan, S.W., Ng, G.C., Chen, X.Q., Yap, E.H., 1996. Observations on the ultrastructure and viability of the cystic stage of Blastocystis hominis from human feces. Parasitol. Res. 82, 439–44. Moe, K.T., Singh, M., Howe, J., Ho, L.C., Tan, S.W., Ng, G.C., Chen, X.Q., Yap, E.H., 1997. Experimental Blastocystis hominis infection in laboratory mice. Parasitol. Res. 83, 319–25. Moe, K.T., Singh, M., Gopalakrishnakone, P., Ho, L.C., Tan, S.W., Chen, X.Q., Yap, E.H., 1998. Cytopathic effect of Blastocystis hominis after intramuscular inoculation into laboratory mice. Parasitol. Res. 84, 450– 4. Moe, K.T., Singh, M., Howe, J., Ho, L.C., Tan, S.W., Chen, X.Q., Yap, E.H., 1999. Development of Blastocystis hominis cysts into vacuolar forms in vitro. Parasitol. Res. 85, 103–8. Mu¨ ller, H., 1994. Four serologically different groups within the species Blastocystis hominis. Zentralbl. Bakteriol. 280, 403–8.

Nakamura, Y., Hashimoto, T., Yoshikawa, H., Kamaishi, T., Nakamura, F., Okamoto, K., Hasegawa, M., 1996. Phylogenetic position of Blastocystis hominis that contains cytochrome-free mitochondria, inferred from the protein phylogeny of elongation factor 1 alpha. Mol. Biochem. Parasitol. 77, 241–5. Nasirudeen, A.M., Tan, K.S., Singh, M., Yap, E.H., 2001a. Programmed cell death in a human intestinal parasite, Blastocystis hominis. Parasitology 123, 235–46. Nasirudeen, A.M., Singh, M., Yap, E.H., Tan, K.S., 2001b. Blastocystis hominis: evidence for caspase-3-like activity in cells undergoing programmed cell death. Parasitol. Res. 87, 559–65. Ng, G.C., Tan, K.S.W., 1999. Colony growth as a step towards axenization of Blastocystis isolates. Parasitol. Res. 85, 678–9. O’Fallon, J.V., Wright, R.W., Calza, R.E., 1991. Glucose metabolic pathways in the anaerobic rumen fungus Neocallimastix frontalis EB188. Biochem. J. 274, 595–9. Ok, U.Z., Cirit, M., Uner, A., Ok, E., Akcicek, F., Basci, A., Ozcel, M.A., 1997. Cryptosporidiosis and blastocystosis in renal transplant recipients. Nephron 75, 171–4. Palmer, J.D., 1997. Organelle genomes: going, going, going, gone!. Science 275, 790–1. Pakandl, M., 1999. Blastocystis sp. from pigs: ultrastructural changes occurring during polyxenic cultivation in Iscove’s modified Dulbecco’s medium. Parasitol. Res. 85, 743–8. Paul, R.G., Williams, A.G., Butler, R.D., 1990. Hydrogenosomes in the rumen entodiniomorphid ciliate Polyplastron multivesiculatum. J. Gen. Microbiol. 136, 388–96. Philippe, H., Laurent, J., 1998. How good are deep phylogenetic trees? Curr. Opin. Genet. Dev. 8, 616–23. Prasad, K.N., Nag, V.L., Dhole, T.N., Ayyagari, A., 2000. Identification of enteric pathogens in HIV-positive patients with diarrhoea in northern India. J. Health Popul. Nutr. 18, 23–26. Rajah Salim, H., Suresh Kumar, G., Vellayan, S., Mak, J.W., Khairul Anuar, A., Init, I., Vennila, G.D., Saminathan, R., Ramakrishnan, K., 1999. Blastocystis in animal handlers. Parasitol. Res. 85, 1032–3. Roberts-Thomson, I.C., Stevens, D.P., Mahmoud, A.A.F., Warren, K.S., 1976. Giardiasis in the mouse: an animal model. Gastroenterology 71, 57–61. Roger, A.J., Sandblom, O., Doolittle, W.F., Philippe, H., 1999. An evaluation of elongation factor 1 alpha as a phylogenetic marker for eukaryotes. Mol. Biol. Evol. 16, 218–33. Scott, D.A., North, M.J., Coombs, G.H., 1995. Trichomonas vaginalis: amoeboid and flagellated forms synthesize similar proteinases. Exp. Parasitol. 80, 345–8. Shlim, D.R., Hoge, C.W., Rajah, R., Rabold, J.G., Echeverria, P., 1995. Is Blastocystis hominis a cause of diarrhea in travelers? A prospective controlled study in Nepal. Clin. Infect. Dis. 21, 97–101. Silard, R., Burghelea, B., 1985. Ultrastructural aspects of Blastocystis hominis strain resistant to antiprotozoal drugs. Arch. Roum. Pathol. Exp. Microbiol. 44, 73–85. Silberman, J.D., Sogin, M.L., Leipe, D.D., Clark, C.G., 1996. Human parasite finds taxonomic home. Nature 380, 398. Singh, M., Suresh, K., Ho, L.C., Ng, G.C., Yap, E.H., 1995. Elucidation of the life cycle of the intestinal protozoan Blastocystis hominis. Parasitol. Res. 81, 446–50. Singh, M., Ho, L.C., Yap, A.L.L., Ng, G.C., Tan, S.W., Moe, K.T., Yap, E.H., 1996. Axenic culture of reptilian Blastocystis isolates in monophasic medium and speciation by karyotypic typing. Parasitol. Res. 82, 165–9. Snowden, K., Logan, K., Blozinski, C., Hoevers, J., Holman, P., 2000. Restriction-fragment-length polymorphism analysis of small-subunit rRNA genes of Blastocystis isolates from animal hosts. Parasitol. Res. 86, 62–66. Stenzel, D.J., Boreham, P.F.L., 1991. A cyst-like stage of Blastocystis hominis. Int. J. Parasitol. 21, 613–5. Stenzel, D.J., Boreham, P.F.L., 1996. Blastocystis hominis revisited. Clin. Microbiol. Rev. 9 (4), 563–84.

K.S.W. Tan et al. / International Journal for Parasitology 32 (2002) 789–804 Stenzel, D.J., Dunn, L.A., Boreham, P.F.L., 1989. Endocytosis in cultures of Blastocystis hominis. Int. J. Parasitol. 19, 787–91. Stenzel, D.J., Boreham, P.F.L., McDougall, R., 1991. Ultrastructure of Blastocystis hominis in human stool samples. Int. J. Parasitol. 21, 807–12. Stenzel, D.J., Lee, M.G., Boreham, P.F.L., 1997. Morphological differences in Blastocystis cysts – an indication of different species? Parasitol. Res. 83, 452–7. Suresh, K., Howe, J., Ng, G.C., Ho, L.C., Ramachandran, N.P., Loh, A.K., Yap, E.H., Singh, M., 1994. A multiple fission-like mode of asexual reproduction in Blastocystis hominis. Parasitol. Res. 80, 523–7. Suresh, K., Chong, S.Y., Howe, J., Ho, L.C., Ng, G.C., Yap, E.H., Singh, M., 1995. Tubulovesicular elements in Blastocystis hominis from the caecum of experimentally-infected rats. Int. J. Parasitol. 25, 123–6. Suresh, K., Khairul Anuar, A., Saminathan, R., Ng, K.P., Init, I., 1997. In vitro culture technique: a better diagnostic tool for Blastocystis hominis. Int. Med. Res. J. 1, 5–7. Suresh, K., Init, I., Reuel, P.A., Rajah, S., Lokman, H., Khairul Anuar, A., 1998. Glycerol with fetal calf serum – a better cryoprotectant for Blastocystis hominis. Parasitol. Res. 84, 321–2. Taamasri, P., Mungthin, M., Rangsin, R., Tongupprakarn, B., Areekul, W., Leelayoova, S., 2000. Transmission of intestinal blastocystosis related to the quality of drinking water. Southeast Asian J. Trop. Med. Public Health 31, 112–7. Tan, H.K., Zierdt, C.H., 1973. Ultrastructure of Blastocystis hominis. Z. Parasitenkd. 42, 315–24. Tan, S.W., Ho, L.C., Moe, K.T., Chen, X.Q., Ng, G.C., Yap, E.H., Singh, M., 1996a. Production and characterization of murine monoclonal antibodies to Blastocystis hominis. Int. J. Parasitol. 26, 375–81. Tan, S.W., Singh, M., Yap, E.H., Ho, L.C., Moe, K.T., Howe, J., Ng, G.C., 1996b. Colony formation of Blastocystis hominis in soft agar. Parasitol. Res. 82, 375–7. Tan, S.W., Singh, M., Thong, K.T., Ho, L.C., Moe, K.T., Chen, X.Q., Ng, G.C., Yap, E.H., 1996c. Clonal growth of Blastocystis hominis in soft agar with sodium thioglycollate. Parasitol. Res. 82, 737–9. Tan, S.W., Singh, M., Ho, L.C., Howe, J., Moe, K.T., Chen, X.Q., Ng, G.C., Yap, E.H., 1997. Survival of Blastocystis hominis clones after exposure to a cytotoxic monoclonal antibody. Int. J. Parasitol. 27, 947–54. Tan, K.S.W., Ng, G.C., Quek, E., Howe, J., Ramachandran, N.P., Yap, E.H., Singh, M., 2000. Blastocystis hominis: a simplified, high-efficiency method for clonal growth on solid agar. Exp. Parasitol. 96, 9–15. Tan, K.S.W., Howe, J., Yap, E.H., Singh, M., 2001a. Do Blastocystis hominis colony forms undergo programmed cell death? Parasitol. Res. 87, 362–7. Tan, K.S.W., Ibrahim, M., Ng, G.C., Nasirudeen, A.M.A., Ho, L.C., Yap, E.H., Singh, M., 2001b. Exposure of Blastocystis species to a cytotoxic monoclonal antibody. Parasitol. Res. 87, 534–8. Tanuri, A., Andrade, P.P., Almeida, D.F.D., 1981. A simple, highly efficient plating method for trypanosomatids. J. Protozool. 28, 360–2. Tasova, Y., Sahin, B., Koltas, S., Paydas, S., 2000. Clinical significance and frequency of Blastocystis hominis in Turkish patients with hematological malignancy. Acta Med. Okayama 54, 133–6. Teow, W.L., Zaman, V., Ng, G.C., Chan, Y.C., Yap, E.H., Howe, J., Gopalakrishnakone, P., Singh, M., 1991. A Blastocystis species from the seasnake, Lapemis hardwickii (Serpentes: hydrophiidae). Int. J. Parasitol. 21, 723–6. Teow, W.L., Ng, G.C., Chan, P.P., Chan, Y.C., Yap, E.H., Zaman, V., Singh, M., 1992. A survey of Blastocystis in reptiles. Parasitol. Res. 78, 453–5. Upcroft, J.A., Dunn, L.A., Dommett, L.S., Healy, A., Upcroft, P., Boreham, P.F.L., 1989. Chromosomes of Blastocystis hominis. Int. J. Parasitol. 19, 879–83. Vardi, A., Berman-Frank, I., Rozenberg, T., Hadas, O., Kaplan, A., Levine, A., 1999. Programmed cell death of the dinoflagellate Peridinium gatunense is mediated by CO(2) limitation and oxidative stress. Curr. Biol. 9, 1061–4. Vesey, G., Hutton, P., Champion, A., Ashbolt, N., Williams, K.L., Warton,

803

A., Veal, D., 1994. Application of flow cytometric methods for the routine detection of Cryptosporidium and Giardia in water. Cytometry 16, 1–6. Villar, J., Carbajal, J.A., Lanuza, M.D., Munˇ oz, C., Borra´ s, R., 1998. In vitro encystation of Blastocystis hominis: a kinetics and cytochemistry study. Parasitol. Res. 84, 54–58. Welburn, S.C., Dale, C., Ellis, D., Beecroft, R., Pearson, T.W., 1996. Apoptosis in procyclic Trypanosoma brucei rhodesiense in vitro. Cell Death Differ. 3, 229–36. Welburn, S.C., Barcinski, M.A., Williams, G.T., 1997. Programmed cell death in Trypanosomatids. Parasitol. Today 13, 22–26. Wilairatana, P., Radomyos, P., Radomyos, B., Phraevanich, R., Plooksawasdi, W., Chanthavanich, P., Viravan, C., Looareesuwan, S., 1996. Intestinal sarcocystosis in Thai laborers. Southeast Asian J. Trop. Med. Public Health 27, 43–46. Wirtz, E., Leal, S., Ochatt, C., Cross, G.A., 1999. A tightly regulated inducible expression system for conditional gene knock-outs and dominant-negative genetics in Trypanosoma brucei. Mol. Biochem. Parasitol. 99, 89–101. Yarlett, N., Hann, A.C., Lloyd, D., Williams, A., 1981. Hydrogenosomes in the rumen protozoon Dasytricha ruminantium Schuberg. Biochem. J. 200, 365–72. Yarlett, N., Orpin, C.G., Munn, E.A., Yarlett, N.C., Greenwood, C.A., 1986. Hydrogenosomes in the rumen fungus Neocallimastix patriciarum. Biochem. J. 236, 729–39. Yee, J., Mowatt, M.R., Dennis, P.P., Nash, T.E., 2000. Transcriptional analysis of the glutamate dehydrogenase gene in the primitive eukaryote, Giardia lamblia. Identification of a primordial gene promoter. J. Biol. Chem. 275, 11432–9. Yoshikawa, H., Kuwayama, N., Enose, Y., 1995a. Histochemical detection of carbohydrates of Blastocystis hominis. J. Eukaryot. Microbiol. 42, 70–74. Yoshikawa, H., Katakiri, I., Li, X., Becker-Hapak, M., Graves, D.C., 1995b. Antigenic differences between Blastocystis hominis and Blastocystis sp. revealed by polyclonal and monoclonal antibodies. J. Protozool. Res. 5, 118–28. Yoshikawa, H., Nagono, I., Yap, E.H., Singh, M., Takahashi, Y., 1996. DNA polymorphism revealed by arbitrary primers polymerase chain reaction among Blastocystis strains isolated from humans, a chicken and a reptile. J. Eukaryot. Microbiol. 43, 127–30. Yoshikawa, H., Nagano, I., Wu, Z., Yap, E.H., Singh, M., Takahashi, Y., 1998. Genomic polymorphism among Blastocystis hominis strains and development of subtype-specific diagnostic primers. Mol. Cell. Probes 12, 153–9. Yoshikawa, H., Abe, N., Iwasawa, M., Kitano, S., Nagano, I., Wu, Z., Takahashi, Y., 2000. Genomic analysis of Blastocystis hominis strains isolated from two long-term health care facilities. J. Clin. Microbiol. 38, 1324–30. Zaman, V., 1996. The diagnosis of Blastocystis hominis cysts in human faeces. J. Infect. 33, 15–16. Zaman, V., Khan, K.Z., 1994. A concentration technique for obtaining viable cysts of Blastocystis hominis from faeces. J. Pak. Med. Assoc. 44, 220–1. Zaman, V., Howe, J., Ng, M.L., 1995. Ultrastructure of Blastocystis hominis cyst. Parasitol. Res. 81, 465–9. Zaman, V., Howe, J., Ng, M.L., 1997a. Variation in the cyst morphology of Blastocystis hominis. Parasitol. Res. 83, 306–8. Zaman, V., Howe, J., Ng, M.L., 1997b. Observations on the surface of Blastocystis hominis. Parasitol. Res. 83, 731–3. Zaman, V., Howe, J., Ng, M.L., 1998. Scanning electron microscopy of Blastocystis hominis cysts. Parasitol. Res. 84, 476–7. Zaman, V., Howe, J., Ng, M., Goh, T.K., 1999a. Scanning electron microscopy of the surface coat of Blastocystis hominis. Parasitol. Res. 85, 974–6. Zaman, V., Zaki, M., Manzoor, M., Howe, J., Ng, M., 1999b. Postcystic development of Blastocystis hominis. Parasitol. Res. 85, 437–40.

804

K.S.W. Tan et al. / International Journal for Parasitology 32 (2002) 789–804

Zierdt, C.H., 1973. Studies of Blastocystis hominis. J. Protozool. 20, 114– 21. Zierdt, C.H., 1986. Cytochrome-free mitochondria of an anaerobic protozoan-Blastocystis hominis. J. Protozool. 33, 67–69. Zierdt, C.H., 1988. Blastocystis hominis, a long-misunderstood intestinal parasite. Parasitol. Today 4, 15–17. Zierdt, C.H., 1991. Blastocystis hominis-past and future. Clin. Microbiol. Rev. 4, 61–79. Zierdt, C.H., Tan, H.K., 1976. Ultrastructure and light microscope appearance of Blastocystis hominis in a patient with enteric disease. Z. Parasitenkd. 50, 277–83.

Zierdt, C.H., Williams, R.L., 1974. Blastocystis hominis: axenic cultivation. Exp. Parasitol. 36, 233–43. Zierdt, C.H., Rude, W.S., Bull, B.S., 1967. Protozoan characteristics of Blastocystis hominis. Am. J. Clin. Pathol. 48, 495–501. Zierdt, C.H., Zierdt, W.S., Nagy, B., 1995. Enzyme-linked immunosorbent assay for detection of serum antibody to Blastocystis hominis in symptomatic infections. J. Parasitol. 81, 127–9. Zwart, K.B., Goosen, N.K., Schijndel, M.W.V., Broers, C.A.M., Stumm, C.K., Vogels, G.D., 1988. Cytochemical localization of hydrogenase activity in the anaerobic protozoan Trichomonas vaginalis, Plagiopyla nasuta and Trimyema compressum. J. Gen. Microbiol. 134, 2165–70.