Viable Blastocystis Cysts in Scottish and Malaysian Sewage Samples

Abstract

Blastocystis cysts were detected in 38% (47/123) (37 Scottish, 17 Malaysian) of sewage treatment works. Fifty percent of influents (29% Scottish, 76% Malaysian) and 28% of effluents (9% Scottish, 60% Malaysian) contained viable cysts. Viable cysts, discharged in effluent, provide further evidence for the potential for waterborne transmission of Blastocystis.

Over the last decade, public health interest in the human intestinal protozoan parasite Blastocystis hominis has increased (17) as further evidence of its pathogenicity is identified (1, 3, 6, 20). Blastocystis is a polymorphic parasite and can present, under various conditions, a perplexing array of morphologies, including vacuolar, multivacuolar, avacuolar, granular, amoeboid, and cystic forms (14, 19). Only in the last decade has a detailed description of the small (6.65 to 12.65 μm) transmissive cystic stage been published (12, 23), and although avacuolar forms in recently voided feces have only been reported twice in the literature (13, 24), they are thought to be the form of B. hominis found in the human intestine (13). This variability of forms found in stools can also account for the unpredictability of detection in clinical parasitology laboratories.

Transmission is by the fecal-oral route, and evidence for experimental anthropozoonotic transmission using B. hominis cysts is available for a variety of nonhuman recipients (e.g., Wistar rats [15], mice [5], and gnotobiotic guinea pigs [9]). The size range of B. hominis cysts (12.65 μm, with their outer membranous coat; 6.65 μm, without their outer membranous coat [23]) is within the size range of both Cryptosporidium oocysts and Giardia cysts, which are etiological agents of waterborne disease. The description of two suspected waterborne outbreaks of blastocystosis (7, 8) together with the chlorine insensitivity of B. hominis cysts implicate chlorinated drinking water as a potentially significant transmission route (21).

Wastewater reuse is associated with parasitological health risks. Habbari et al. (4) demonstrated that children living in wastewater reuse regions of Morocco had an increased risk of parasite infections. One or more parasite infections were identified in 50.8% of children living in the wastewater reuse regions compared with only 8.2% of children living in regions with no wastewater reuse (n = 1,343 children). As for the enteric protozoan pathogens, Cryptosporidium and Giardia, the potential for environmental contamination with Blastocystis depends upon a variety of factors, including the number of infected human and nonhuman hosts, seasonal influences and duration of infection, the number of transmissive stages excreted, agricultural practices, host behavior and activity, socioeconomic and ethnic differences in human behavior, geographic distribution, sanitation, and climate and hydrogeology of the area (11).

An insight into the potential for contaminating surface waters used for abstraction of potable water can be provided by determining the presence of viable B. hominis cysts in sewage effluent. Apart from a report of the isolation of Blastocystis from sewage (22), no further studies have investigated its presence and viability in sewage.

The increasing reports of human blastocystosis and the lack of information on its waterborne occurrence together with our knowledge of the role that sewage effluent acts as a contributor to waterborne Cryptosporidium spp. oocysts and Giardia spp. cysts (2) indicate that the original finding of Zaman and Khan (22) should be confirmed. Furthermore, the occurrence of viable Blastocystis cysts in sewage treatment plants should be established so that the potential significance of the waterborne route of transmission can be determined.

Here, we analyzed sewage samples from Scotland, United Kingdom, and Kuala Lumpur, Malaysia, for the presence of Blastocystis cysts. A total of 73 1-liter samples from 37 Scottish sewage treatment plants (31 influent and 42 effluent samples) and 50 samples from 17 sewage treatment plants around Kuala Lumpur, Malaysia (25 influent and effluent samples, respectively), were collected. Influent and effluent samples were collected separately from different sewage treatment plants. The sewage samples were centrifuged (1,800 × g, 15 min at room temperature) and reduced in volume to ∼40 ml, ensuring that pellets were always retained. Each sample was further centrifuged (as above), the supernatant was discarded, and the pellet was resuspended to 100 μl, of which 10 μl of resuspended pellet was placed on a glass microscope slide to which a coverslip was added, and the wet film was examined by light microscopy at ×400 magnification for Blastocystis life cycle stages (vacuolar, multivacuolar, avacuolar, granular, amoeboid, and cystic). The remainder of the resuspended pellet (90 μl) was cultured in vitro to augment viable forms. The recovery efficiency of this method for concentrating Blastocystis cysts was 12.1 ± 2.9% (n = 3) when 1-liter sewage effluent samples were seeded with 10,000 cysts.

For in vitro culture, 30 μl of each resuspended pellet was dispensed into a sterile bijou bottle containing 3 ml of Jones' medium (16, 18, 22), incubated for 48 h at 37°C, and examined for the presence of various morphological forms of Blastocystis under the light microscope at ×400. Three replicates were performed for each sewage sample, and samples were regarded as positive when Blastocystis was observed in any of the three replicate cultures. The majority of forms seen were vacuolar and increased in density when reexamined after 5 days of incubation. Culture tubes negative for Blastocystis after 48 h remained negative at 5 days.

No morphological forms of Blastocystis were seen following direct microscopy of wet films prior to in vitro culture. In Scottish sewage samples cultured in vitro, 29% (9/31) and 9.2% (4/42) of influent and effluent samples, respectively, were positive for Blastocystis, while 76% (19/25) and 60% (15/25) of in vitro-cultured influent and effluent samples, respectively, were positive in the Malaysian sewage samples. Because influent and effluent samples were unmatched and collected separately from different sewage treatment plants, both in Scotland and in Malaysia, we are unable to determine the removal efficiencies of the sewage treatment process for Blastocystis cysts. However, being similar in size to Cryptosporidium spp. oocysts and Giardia spp. cysts, we would extrapolate from our previous work that the removal efficiencies of primary and secondary sewage treatments for Blastocystis cysts should range between 15 and 99% (10).

Zaman and Khan (22) stated that all four sewage influent and effluent samples in Pakistan were positive for Blastocystis, indicating that treatment does not completely remove Blastocystis cysts. In the present study, 9% of Scottish and 60% of Malaysian sewage effluent samples were Blastocystis positive, indicating that the sewage treatment works investigated neither completely remove cysts nor render the cysts discharged in effluent nonviable. Our data confirm the robustness of Blastocystis cysts to wastewater treatment processes.

Human contributors to the pool of Blastocystis cysts in sewage have been reported in both Scotland and Kula Lumpur. Blastocystis prevalence in humans with gastrointestinal symptoms is 3.9% in Scotland and 5.6% in Kuala Lumpur, Malaysia (18, 20). In the Scottish cases, 20.5% excreted cysts (18).

In vitro culture is useful for detecting Blastocystis in human stool samples and increases the percent positivity rate compared to direct microscopy (16, 18, 22), as viable organisms increase exponentially in culture medium, making them more easily identifiable by bright-field microscopy. The ability to culture Blastocystis cysts from sewage concentrates and to augment their numbers within 48 h to detectable levels confirms that this in vitro culture technique is also a useful adjunct for environmental samples. This is the first report of culturing viable Blastocystis from Scottish and Malaysian sewage samples. The study was designed to include sewage samples from geographically disparate and temperate and tropical countries using the same detection methods. It establishes that Blastocystis cysts can be found readily in sewage samples and that sewage treatment plants in Scotland and Malaysia are ineffective in removing or killing all Blastocystis cysts.

These findings have important implications and provide further evidence for the potential for waterborne transmission of Blastocystis, given the increasing reports of the parasite's pathogenicity. To determine the levels of contamination and the potential routes of transmission, Blastocystis should be included as a further parameter when investigating parasites in wastewater and other environmental samples.