Introduction

Blastocystis hominis has been recognized as an enigma among the intestinal parasites. Since the first description of the organism in the early 1900s (Alexeieff 1911; Brumpt 1912), only its morphology had been extensively studied by light and electron microscopy (Stenzel and Boreham 1996). Many other aspects of Blastocystis biology, such as pathogenicity, culture characteristics, life cycle, mode of transmission, taxonomy, biochemistry, and molecular biology, remain minimally understood (Tan et al. 2002).

Blastocystis hominis is a highly polymorphic organism with various morphological forms being reported in the literature including vacuolar, granular, amoeboid, cyst, avacuolar, and multivacuolar forms (Stenzel and Boreham 1996). The vacuolar form is the most common morphotype seen in in vitro cultures and stools. The granular form is rarely seen in stools, but is found in in vitro cultures. The amoeboid form is rarely found in culture, but has been seen in diarrheic stools. The morphological descriptions of the amoeboid form are conflicting (Tan et al. 2002). The avacuolar form is seen in colonoscopy samples while the multivacuolar form is occasionally found in stools. The purpose of the current study is to compare symptomatic and asymptomatic isolates of B. hominis in terms of their morphology in in vitro cultures.

Materials and methods

Source of Blastocystis hominis isolates

A total of 20 isolates of B. hominis, comprised of ten asymptomatic (A1–A10) and ten symptomatic (S1–S10) isolates, were included in the present study. Details of the subjects and their clinical history are given in Table 1. Parasites were isolated from stool samples received at the Department of Parasitology, Faculty of Medicine, University of Malaya. All samples were sent from local clinics, hospitals and communities except those indicated as coming from other countries.

Table 1 Source of Blastocystis hominis isolates
Full size table

Selection criteria

Stool samples from patients were examined for pathogenic bacteria (Campylobacter, Shigella, Salmonella and Vibrio) and other parasites including helminths ova, Entamoeba histolytica, Cryptosporidium parvum, Giardia intestinalis and microsporidia. Only isolates from patients presenting with gastrointestinal symptoms with B. hominis appearing as the single causative agent were included as symptomatic isolates in the present study.

Culture of Blastocystis hominis isolates

The parasites were isolated from patients and healthy individuals by in vitro cultivation using Jones’ medium (Jones 1946) supplemented with 10% horse serum at 37°C (Suresh et al. 1994a; Rajah et al. 1997; Zaman 1997). Subsequently, after isolation, the parasites were maintained in Jones’ medium by consecutive subcultures every 3 to 4 days for at least 1 month prior to the phenotypic analysis.

Morphological study

A drop of the sediment from parasite cultures was placed on a glass slide daily until day 10 of culture and viewed under ×400 magnification using light microscopy. The percentages of the vacuolar, granular and amoeboid forms were determined in 100 parasites randomly selected from each isolate for morphological examination.

TEM study

The contents from day 5 culture from asymptomatic and symptomatic isolates were centrifuged at 500×g for 5 min. The pelleted cells were resuspended overnight in 4% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.3 at 4°C, washed thoroughly with cacodylate buffer, and postfixed for 30 min in 1% osmium tetroxide in cacodylate buffer. The fixed cells were dehydrated in ascending series of ethanols and embedded in epoxy resin. Semithin sections were stained with toluidine blue. Ultrathin sections were cut using an ultramicrotome, contrasted with uranyl acetate and lead citrate, and viewed using a transmission electron microscope (LEO Libra 120).

Acridine orange staining

Parasites from days 2 to 10 culture of each isolate were stained with acridine orange solution according to the method previously described by Suresh et al. (1994a). Briefly, 5 ml of 0.1% acridine orange stock solution was diluted with 45 ml of phosphate buffered saline pH 7.4 before use. A drop of culture sediment containing parasites was mixed thoroughly on a clean glass slide with a drop of diluted acridine orange. The preparation was viewed with a fluorescence microscope (Leitz Wetzlar, Germany) using incident light transmission at ×400 magnification.

Results

The amoeboid forms of B. hominis (Fig. 1) were present in all symptomatic isolates (S1–S10), and none of the asymptomatic isolates (A1–A10) showed the presence of the amoeboid form during the 10-day cultivation period. The amoeboid forms were irregular in shape with a prominent nucleus at the central zone and multiple extended pseudopodia at the periphery. No movement of either the pseudopodia or the whole parasite was observed on the amoeboid forms. The kinetics of the presence of the amoeboid form in symptomatic isolates is shown in Fig. 2. The amoeboid forms appeared on day 2 (isolate S8), day 3 (isolates S3, S5, S6, S7, S9 and S10), and day 4 (isolates S1, S2 and S4). By day 4 parasite culture, the amoeboid forms were seen in all symptomatic isolates with varying percentages ranging from 2% (isolate S2 and S4) to 13.7% (isolate S1). The maximum percentage (28%) of the amoeboid form in culture was seen in isolate S1 on day 6. Generally, the percentages of the amoeboid form in symptomatic isolates peaked on days 3 to 6. After day 6, the percentages of the amoeboid form declined.

Fig. 1
figure 1

Phase contrast images of Blastocystis hominis. The amoeboid form appears irregular in shape and possesses multiple extended pseudopodia (arrows). A prominent nucleus is clearly visible at the central zone. A rounded vacuolar form is visible beside the amoeboid form (×400)

Full size image
Fig. 2
figure 2

Percentages of the presence of amoeboid forms in symptomatic isolates (S1–S10) over a 10-day cultivation period in Jones’ medium. Parasites in isolate S7 died off on day 9 while no viable parasite was detected in isolates S8 and S10 on day 10

Full size image

Granular forms were more frequently seen in asymptomatic isolates except isolate A8. No granular form was observed in isolate A8 throughout the 10-day cultivation period. The granular form appeared similar to the vacuolar form except for the presence of inclusion bodies or granules in the central vacuole. The granular form started to appear on day 2 (isolates A3, A5, A6, A9 and A10), day 3 (isolates A1 and A2), day 4 (isolate A7), and day 5 (isolate A4). On day 6 cultures, the percentages of the granular form ranged from 2.7% (isolate A7) to 38% (isolate A1). Generally the percentages of the granular form in asymptomatic isolates peaked on days 2 to 6. After day 6, the percentages of the granular form decreased. Granular forms were also seen occasionally in several symptomatic isolates, but with relatively lower percentages (0.3–2.3%). In initial cultures (day 2 to day 6) the inclusion bodies in the granular forms appeared small and solid, but the granular forms became full of tiny granules with Brownian movement after 7 days of cultivation prior to death.

Transmission electron microscopy (TEM) studies showed two types of amoeboid forms, one contained a large central vacuole completely filled up with tiny electron-dense granules (Fig. 3a) and the other did not have a large central vacuole, but instead showed multiple small vacuoles in the cytoplasm (Fig. 3b). The cytoplasm contained strands of ribosome structure resembling rough endoplasmatic reticulum. Bacteria were frequently seen in close contact with the pseudopodia (data not shown). The thickness of the surface coat of the amoeboid form was uneven within the same parasite cell. Some parts of the surface coat of the amoeboid form were about five times thicker than the other parts of the same cell.

Fig. 3
figure 3

a A large central vacuole (cv) containing electron-dense granules is visible (isolate S1). A thin surface coat surrounds the parasite cell. A prominent nucleus (n) is visible. Note: The peripheral cytoplasm is highly vacuolated. Strands of ribosome structure resembling rough endoplasmatic reticulum are visible (arrow). b The parasite cell (isolate S8) (at the center) appears irregular in shape and contains multiple small vacuoles (v). The surface coat (sc) tends to be thicker at some parts surrounding the parasite

Full size image

Acridine orange stained the nuclei and the central body of vacuolar forms of B. hominis bright and dull green, respectively. The granular forms were stained yellow to orange, with the central zone of the amoeboid form stained yellow, and the peripheral region stained orange (Fig. 4). Multiple projected pseudopodia were clearly seen at the periphery.

Fig. 4
figure 4

The peripheral region of the amoeboid form is stained an intense red-orange while the central zone is stained yellow. Note: Projected pseudopodia are visible (arrow)

Full size image

Discussion

Previous studies have shown the existence of at least two demes of B. hominis with distinct protein content, DNA profiles, and isoenzyme patterns which may be related to their varying pathogenic potential (Kukoschke and Müller 1991; Mansour et al. 1995; Clark 1997; Gericke et al. 1997; Lanuza et al. 1999). The present study is a comprehensive, comparative study of the morphology of both symptomatic and asymptomatic isolates of B. hominis, with detailed descriptions of the amoeboid form.

Major morphological differences were noted between symptomatic and asymptomatic isolates of B. hominis. The vacuolar form was the most common morphotype found in both groups. Granular forms were more frequently seen in asymptomatic isolates (up to 38%) than in symptomatic isolates (0.3 to 2.3%). The amoeboid form was seen in all symptomatic isolates (up to 28%). No amoeboid forms were seen in cultures of asymptomatic isolates throughout the entire 10-day period of in vitro growth. These findings are in agreement with previous reports by Dunn et al. (1989) and Rajah et al. (1997). Dunn et al. (1989) found the amoeboid form in one of the symptomatic isolates. The amoeboid form was not observed in the other seven symptomatic isolates in that particular study. Because no information on the age of the culture was available, it is possible that the authors might not have noticed some of the amoeboid forms that appeared in older cultures. Results in the present study have shown that in some symptomatic isolates, the amoeboid form started to appear on day 4 (isolates S1, S2 and S4) with percentages as low as 1.7%. In a separate study, Rajah et al. (1997) observed that granular forms were present in all three asymptomatic isolates from Malaysia, Indonesia, and Bangladesh. The percentage of granular form at peak parasite counts on day 8 for the Malaysian, Indonesian, and Bangladeshi isolates were 17.5%, 6.8%, and 47.3%, respectively. Rajah et al. (1997) did not find any amoeboid form of B. hominis in asymptomatic isolates throughout their cultivation of the parasites in a period of more than 12 days.

In the present study, B. hominis isolates were studied in the xenic condition, mainly because the xenic condition is a better mimic of the natural environment in the human intestine than the axenic condition. The high prevalence of the amoeboid form (10 out of 20 isolates) suggests that the amoeboid form is not an accidental occurrence and should be considered a stage of the life cycle of the parasite. Close association with bacteria may be an important factor for the formation of the amoeboid form. The engulfment of bacteria by B. hominis has been reported (Dunn et al. 1989). It has also been suggested that the amoeboid form is an intermediate form between the vacuolar and cyst forms and that it ingests bacteria to provide nutrition for the encystation (Singh et al. 1995), as has previously been shown for Entamoeba (McConnachie 1969). Isolates of B. hominis may differ in their ability to produce the amoeboid form, which could be a factor contributing to pathogenicity. This hypothesis is partly supported by Lanuza et al. (1997), who reported that the amoeboid form of B. hominis was more frequently seen in primary cultures (xenic cultures) than in axenic cultures.

The amoeboid form of B. hominis has been reported infrequently, and its morphological descriptions have yielded conflicting and confusing reports particularly on the presence of cell membrane, surface coat, central vacuole, and organelles (Zierdt 1973, 1991; Zierdt and Tan 1976; McClure et al. 1980; Dunn et al. 1989; Suresh et al. 1994b; Tan et al. 2001). In the present study, two types of amoeboid forms were found, one contained a large central vacuole completely filled up with tiny electron-dense granules, and the other did not have a large central vacuole, but instead showed multiple small vacuoles in the cytoplasm. This explains the controversy on the presence of the central vacuole in amoeboid forms in previous reports. Most of the vacuoles were surrounded by strands of ribosome structure resembling rough endoplasmatic reticulum, and TEM analysis showed that the amoeboid form was surrounded by a surface coat of varying thickness. The cytoplasmatic regions showed moderately dense concentrations of ribosomal particles, suggesting active protein synthesis. This ultrastructural appearance of the amoeboid form of B. hominis is similar to the previous descriptions by Suresh et al. (1994b) and Tan et al. (2001), except for the sizes, which were bigger and ranged from 5 to 65 μm.

Acridine orange is known to stain the DNA of the nucleus, mucus, and RNA as bright green, dull green, and intense red-orange, respectively (Humason 1972). Suresh et al. (1994a) were the first to use acridine orange in differentiating the various life cycle stages of B. hominis. Subsequently, acridine orange was used to measure the parasite cell activity during in vitro encystation of B. hominis (Villar et al. 1998). The present study represents the first report on the acridine orange staining of the amoeboid form of B. hominis. The amoeboid form showed intensive red-orange coloration with acridine orange staining indicating that it was in the active state and had high levels of RNA which could be related to active protein synthesis.

The amoeboid form was previously detected in colonoscopy samples (Stenzel et al. 1991) and only in patients with acute diarrheal syndrome (Lanuza et al. 1997). Trophozoite of E. histolytica is known to invade the intestinal mucosa and engulf red blood cells, resulting in amoebic colitis (Stanley 2003). Furthermore a phylogenetic analysis of the elongation factor-1α (EF-1α) gene suggests a close relationship between B. hominis and E. histolytica (Ho et al. 2000). Hence, it is postulated that the amoeboid form seen in the present study could be the life cycle stage that contributes to the pathogenicity of B. hominis.

It is currently unknown whether these amoeboid forms reflect different genetic make up and biology. Because the in vitro culture technique using Jones’ medium has been proven to be more sensitive than the formol-ether concentration technique (Suresh and Smith 2004), it is recommended that the culture technique be used for the diagnosis of Blastocytis. This should be followed by careful examination for amoeboid forms up to day 4 of culture to predict the pathogenic potential of the parasite isolate.