The impact of chlorine, ultraviolet-C, and microwave treatment on the survivability of Blastocystis sp. cysts

Blastocystis is the most common and widespread unicellular intestinal eukaryote in humans and many non-human hosts (Jinatham et al. 2021). Over a billion people are thought to be colonized by Blastocystis sp. worldwide, while the numbers are lower in wealthy nations than in developing ones (Parija & Jeremiah 2013). Blastocystis sp. is one of the top protists reported in 19 African countries, with the highest estimated prevalence range of >50% (Ahmed et al. 2023).

At least 28 subtypes (ST) of the genus Blastocystis have been identified based on SSU-rRNA sequence variants, ST1–ST17, ST21, and ST23–ST32 (Higuera et al. 2021). Fourteen of these ST have been identified in humans (ST1–ST10, ST12, ST14, ST16, and ST23), with ST1–ST4 being the most commonly found globally (Jinatham et al. 2021). Except for ST9, which has only been isolated from humans, all other ST have been detected in a variety of avian and non-human mammalian hosts, indicating low host specificity and potential zoonotic transmission of the parasite (Higuera et al. 2021; Jinatham et al. 2021).

The debate about Blastocystis sp. pathogenicity has grown due to the frequent isolation of this organism from healthy and symptomatic subjects who lacked any other detectable pathogen. However, frequent substantial parasite detection in patients with acute and chronic gastrointestinal illnesses, such as irritable bowel syndrome and inflammatory bowel disease, as well as in those with allergic skin diseases, has been documented (Abdel Hameed et al. 2011; Yakoob et al. 2010).

All species of the genus Blastocystis exhibit morphological homogeneity, and four distinct forms, vacuolar, granular, amoeboid, and cysts, are recognized (Ahmed & Karanis 2019). The cyst form was determined to be the only transmissible stage of Blastocystis sp. via the faecal–oral pathway, where as few as 10 cysts were sufficient to establish infection in experimental animals (Yoshikawa et al. 2004). In contrast to other parasite forms, cysts were resistant to water, temperature change, and desiccation, suggesting that cyst-contaminated food and drink may contribute to human and animal infection (Moe et al. 1996; Yoshikawa et al. 2004). Indeed, several epidemiological studies worldwide have reported the detection of the parasite in different water resources, soil samples, fresh fruits and vegetables (Leelayoova et al. 2004; Jinatham et al. 2021). These investigations demonstrated that cysts may tolerate various environmental conditions and traditional water treatment methods.

Chlorine has been widely employed in water treatment processes to disinfect against various infections. Chlorine concentrations in drinking water up to 4 parts per million (ppm) are considered safe and unlikely to harm human and animal health (CDC 2020). In a prior investigation, cysts of Blastocystis sp. were found to be resistant to different chlorine doses (0.2–2.2 ppm) used to disinfect drinking water (Zaki et al. 1996).

In comparison to chemical methods, physical technologies for microbial disinfection, such as microwave (MW) and ultraviolet (UV) irradiations, have been used to destroy microorganisms on surfaces, in food, water, and even in the air (Wang et al. 2019; Hazell et al. 2021).

MW radiation is non-ionizing electromagnetic radiation with a high frequency (0.3–300 GHz) (Michaelson 1974). MW-based technologies have much promise as a new approach to water and wastewater treatment disinfection. It has been demonstrated to have microbicidal effects on many pathogens, including bacteria (Woo et al. 2000), viruses (Wang et al. 2022), fungi (Wu & Yao 2010), and parasites (Hussein et al. 2021).

The use of UV irradiation in water treatment has also been expanding significantly. It has long been employed as an efficient disinfectant against protozoans recognized for their chlorine resistance (Clancy et al. 2004; Adeyemo et al. 2019). According to wavelengths, UV radiation can be divided into UV-A (400–315 nm), UV-B (315–280 nm), UV-C (280–200 nm), and vacuum UV (200–100 nm). The germicidal range of wavelengths (200–300 nm), which is most efficient for microbial disinfection in water, is included in the UV-C type (Hazell et al. 2021). The UV dose applied, which is the product of exposure time and UV light intensity, and is typically expressed in micro watt seconds per square centimetre (mWs/cm²) or, equivalently, in micro joules per square centimetre (mJ/cm²), determines the extent of a microorganism's inactivation by UV radiation (Clancy et al. 2004).

To the author's knowledge, the current study is the first of its kind using disinfection methods on Blastocystis cysts. Due to the paucity of studies on the efficacy of water treatment strategies to inactivate cysts of Blastocystis sp. despite the potential risk it poses (Zaki et al. 1996), the current study investigated the effects of chlorine, MW, and UV-C treatments on the survivability of two different isolates of Blastocystis sp. cysts in vitro.

Patients referred to the Suez Canal University Hospital's Parasitology Lab for stool analysis provided 51 samples. Each sample was examined with direct smear examination (saline and iodine mounts) and formalin-ethyl acetate concentration. Fourteen samples tested positive for Blastocystis sp., while only 2/14 had a significant number of cysts, and therefore they were included in the current study. The number of cysts per high power field (HPF) was calculated at the high dry objective lens.

Using a light microscopy with an oil immersion lens and a calibrated micrometer, the mean diameter of 20 cysts from each sample was determined. The fresh faecal samples underwent further processing to purify cysts. For further DNA extraction and subtyping of Blastocystis sp., around 200 mg of fresh faecal samples were stored at −20 °C.

Following the manufacturer's instructions, genomic DNA from Blastocystis was isolated from faecal samples using a Qiagen DNA extraction kit (QIAamp; Qiagen Inc., Hilden, Germany). The two isolates were molecularly identified with seven sequenced-tagged sites (STS) primers. Seven distinct ST of Blastocystis sp., ST1 (351 bp), ST2 (704 bp), ST3 (526 bp), ST4 (487 bp), ST5 (317 bp), ST6 (338 bp), and ST7 (650 bp) have been molecularly targeted (Yoshikawa et al. 2004).

For each ST, a 25-μL PCR reaction mixture was prepared, containing 1 μL template DNA, 25 pmol of each primer pair, 12.5 μL Master Mix (Applied Biotechnology Co., Ltd, Egypt) and 9.5 μL nuclease-free water. The reaction contained positive and negative control tubes. The reaction consisted of initial denaturing at 94 °C (3 min), 30 cycles of denaturing at 94 °C, annealing at 57 °C (30 s each), and extension at 72 °C (1 min), followed by a chain elongation cycle at 72 °C (10 min). PCR products were electrophoresed on an agarose gel (1.5% concentration) stained with ethidium bromide and visualized with a UV trans-illuminator.

The PCR product and the identified subtype primers were sent to Department of Basic and Clinical Sciences, University of Nicosia Medical School, Nicosia, Cyprus, for purification and sequencing by the Macrogen company. The PCR product samples were mixed with GelRed^® Prestain Plus 6X DNA Loading Dye (Biotium, US) and run on a 1% agarose gel with a 1 kb Plus DNA ladder (Invitrogen, US). The DNA bands were then extracted and purified using the Blirt ExtractMe DNA kit (Blirt, Gdansk, Poland). The purified PCR products were sent to Macrogen Ltd Europe, Amsterdam, for sequencing. The sequences were subjected to BLAST searches (https://blast.ncbi.nlm.nih.gov/Blast) at NCBI GenBank to determine Blastocystis species. Using the MUSCLE algorithm (https://www.megasoftware.net/web_help_10/Part_II_Assembling_Data_For_Analysis/Building_Sequence_Alignments/MUSCLE/About_Muscle.htm) of the MEGA software (https://www.megasoftware.net/), multiple nucleotide sequence alignments were performed.

Cysts of Blastocystis sp. were collected using the Ficoll density gradient centrifugation method (Zaman & Khan 1994). This technique effectively concentrated Blastocystis cysts, eliminating most bacteria and faecal debris while maintaining the cysts' viability (Zaman 1996). About 10–15 g of faeces were emulsified in 50 mL distilled water (DW) and sieved through three layers of gauze to eliminate coarse particles. The faecal suspension was dispersed into 15 mL Falcon tubes with screw-on caps and rinsed thrice with DW at 300 g for 20 min. About 1 mL of sediment was placed on 5 mL of Ficoll-Paque solution (Lymphoflot; Biotest, Germany) and centrifuged at 2,000 g for 20 min. The cystic stages banded about 1 cm below the surface were transferred to a clean tube and washed three times by DW at 300 g for 20 min. The final cyst pellet was suspended in DW at 4 °C.

Cysts were counted under light microscopy (×400) using a Neubauer haemocytometer and trypan blue (TB) dye 0.4%. Viable cysts appeared unstained, whereas non-viable cysts were stained blue. One week before disinfection experiments, purified cysts were subjected to in vitro cultivation at 37 °C into Jones' medium (2 × 10⁴ cysts/mL) supplemented with 10% horse serum to assess viability before disinfection testing (Yoshikawa et al. 2004). On the second to the third day of culture, the emergence of vacuolar and granular forms of Blastocystis sp. proved viability. The concentrated cysts were used consequently in the disinfection experiments.

Before use in disinfection experiments, all glassware used in the current study was soaked overnight in DW containing 50 mg of free chlorine per litre and thoroughly washed with chlorine demand-free (CDF) water. According to the procedure outlined by Korich et al. (1990), CDF water was prepared (Korich et al. 1990). Briefly, 6 L of DW were mixed in a closed container with 3 mL of commercial bleach (Clorox©) containing 5% active chlorine. To altogether remove free chlorine, which was confirmed by DPD (N, N-dimethyl-p-phenylenediamine) colorimetric method (WinLab Photometer LF2400, Germany), the water was kept at room temperature for 24 h before being exposed to direct sunlight for at least 6 h (Korich et al. 1990).

Purified cysts were used for disinfection experiments within 2 weeks of sample collection. For the disinfection assays, 50-mL Pyrex beakers (height: 56 mm, inner diameter: 40 mm) were used as reaction vessels. Each disinfection and control experiment was replicated three times. All experiments were prepared and conducted at room temperature (25 °C ± 0.3).

Sodium hypochlorite (Clorox©, 5% NaoCl) was used to create solutions containing 1 ppm, 2 ppm, and 4 ppm of free chlorine. Cysts were treated with chlorine using the technique outlined by Johnson et al. (2003) with a few modifications. In CDF 0.05 M potassium dihydrogen phosphate buffer, purified cysts were suspended after being washed three times at 3, 220 g for 10 min (pH 7.0).

At time zero, an inoculum containing 2.5 × 10⁶ viable cysts in 1 mL CDF buffer was added to each reaction vessel. Each reaction vessel contained the chlorine concentration (1, 2, 4 ppm), achieving a total volume of 50 mL (each 10 mL contains 5 × 10⁵ cysts). At room temperature, the contents of the reaction vessels were continuously stirred with a magnetic stirrer using a 10 mm stir bar. At 30, 60, 120, and 180 min, 10-mL samples were taken out of the reaction vessels and put into sterile 15-mL centrifuge tubes. Adding 0.1 mL of 10% (wt/vol) sodium thiosulphate (Na₂S₂O₃) immediately neutralized the free chlorine in the withdrawn samples. With potassium iodide starch paper, the total neutralization of chlorine was tested (ADVANTEC, Japan).

The DPD colorimetric method measured the free chlorine levels at time zero and at each exposure time. Sodium hypochlorite was added to bring the free chlorine level to the appropriate concentration if a drop of more than 5% in the free chlorine concentration was found at any exposure time points.

Control vessels were handled like samples exposed to chlorine and contained 2.5 × 10⁶ cysts suspended in 50 mL CDF buffer.

The UV-C source was a low-pressure mercury vapour lamp emitting 254 nm of monochromatic UV-C radiation (LEYBOLD DIDACTIC GMBH, 665635, Germany). The intensity of the UV light emitted was measured using a radiometer calibrated at a wavelength of 254 nm. The lamp was housed on a solenoid-operated shutter that was timed and spaced to the UV-C light source approximately 150 mm from the surface of the cyst suspension.

A suspension of 5 × 10⁵ viable cysts in 10 mL DW was placed in a 50-mL Pyrex beaker. The test suspension was placed on a magnetic stirrer and stirred continuously with a 10 mm stir bar throughout each exposure time. Before radiation, the lamp was given at least 30 min to warm up. The UV lamp shutter was opened, and the cyst suspension was irradiated following the predetermined exposure times necessary to achieve the target UV-C doses of 5.13, 10.26, 20.52, and 40.47mJ/cm².

Concurrently, a controlled dose (0 mJ/cm²) was conducted with the highest dose level irradiation experiment. Without UV-C irradiation, controls consisted of 50-mL Pyrex beakers containing 5 × 10⁵ viable cysts suspended and stirred in 10 mL DW (Clancy et al. 2004). The suspensions of control and test cysts were transferred into 15 mL sterile centrifuge tubes.

Cyst suspensions of 5 × 10⁵ viable cysts in 10 mL of DW were placed in 50-mL Pyrex beakers. Each beaker was covered with a stretch plastic wrap. The samples were placed in the centre of the MW oven (45 L; dimensions: 60 × 44 × 35 cm³), operating at 2,450 MHz frequency, and 900 W maximum output power (Samsung, model MC455TBRCSR, Malaysia). Cyst suspensions were exposed to MW irradiation at 100% power for varying periods (10, 15, 30, and 45 s) (one sample at a time and a total of three experiments/each exposure time). Using a digital thermocouple (MR, China), the temperature of the parasitic suspension was recorded immediately after each MW irradiation. The samples were rapidly cooled in water at 4 °C to stop the heating process until the suspension reached 23 °C. The MW oven was left to cool for 30 min between MW irradiation experiments. The control samples consisted of 5 × 10⁵ cysts suspended in 10 mL DW and were treated in the same manner as MW-irradiated samples but without MW irradiation. Then, suspensions were poured into 15-mL centrifuge tubes.

After each disinfection experiment, the control and test tubes were spun at 3,220 g for 10 min. The pellets containing cysts were resuspended in sterile DW, and the volume was adjusted to 1 mL after the supernatants were removed. Pellets were examined microscopically for counting viable cysts with a Neubauer haemocytometer and TB dye.

We analysed each treatment's effects on the cysts' morphology using wet mount, TB, and Lugol's iodine staining. The tubes were centrifuged at 3,220 g for 10 min, and the sediments (about 0.5 mL each) were inoculated into 2 mL tubes of Jones' media and incubated at 37 °C. For up to 7 days, the culture tubes were checked every day. When trophic (vacuolar, granular, or amoeboid) forms of Blastocystis sp. were found in the 7-day cultures, the samples were deemed to contain viable cysts; however, if these forms were not seen, the samples were considered to contain no viable cysts (Yoshikawa et al. 2004).

The inactivation data were represented as viability reduction percentage (VR%) which was calculated using the formula (N₀ − N/N₀) × 100, and as log₁₀ inactivation using the formula log₁₀ (N/N₀) where N₀ is the mean number of viable cyst forms in the control tube, while N is the mean number of viable cyst forms in each tube of the disinfection experiments after treatment.

When no viable cysts were detected, the minimum log₁₀ inactivation was calculated by assuming that one viable cyst was seen in the experiment.

Data were analysed using analysis of variance (ANOVA) to test significance among the studied groups. Means separation and pairwise comparisons were performed using Duncan's Multiple Range test. SPSS conducted statistical analyses for Windows (SPSS version 25). Results are considered significant at a probability level of 0.05 for each (P ≤ 0.05).

Adding cyst suspension to each used chlorine concentration (1, 2, 4 ppm) exerted a slight initial chlorine demand with a decline of ≤0.05 mg/L ± 0.02 of the initially used chlorine concentrations.

Compared to the control group, a statistically significant decline in the mean cyst counts was observed in both isolates for all free chlorine concentrations examined. This decline was proportionate to the fraction of viable cysts lost over varied exposure durations and concentrations (Table 2).

When the mean cyst counts for the two isolates were compared, a statistically significant drop was seen for isolate II compared to isolate I, indicating that isolate II is more vulnerable to chlorination.

The UV exposure of 40 mJ/cm² thoroughly disinfected isolate II compared to >l log₁₀ inactivation in isolate I at the same dose. After exposure to UV dosages of 20 and 40 mJ/cm² for isolate II and 40 mJ/cm² for isolate I, cysts had no in vitro culture growth.

TB was an exceptional predictor of cyst viability, with viable cysts appearing refractile and non-viable cysts appearing gradually non-refractile with mild greyness, then absorbing the TB colour. The iodine staining of Lugol's revealed vacuolation, cyst contour distortion, and content expulsion, clearly distinguishing the morphological alterations of disinfected cysts. Even with a trained eye, it was difficult to differentiate between viable and non-viable disinfected cysts during wet mount examination.

Chlorine and UV-C disinfected cysts underwent comparable morphological alterations. Internal morphological characteristics of cysts displayed faint staining with TB and Lugol's iodine, and some cysts even appeared as featureless hollow shells (author's observation). Iodine staining revealed numerous vacuoles, or sometimes black streaks inside some cysts, giving them a honeycombed appearance. These cysts lacked intracytoplasmic circular structures and had a less distinct cyst wall. Both disinfected isolates had the same morphological alterations. The typical size of chlorine and UV-C disinfected cysts did not alter (Figure 6, photos a–d).

MW disinfected cysts displayed TB staining in both isolates due to loss of viability. Iodine staining revealed a wrinkled cyst wall shape, a shrivelled appearance, and a reduction in the size of the intracytoplasmic circular bodies, which now appeared as dark, shrunk granules. Some cysts began to exhibit lacerated cyst walls with the ejection of intracellular material after 15 s of MW exposure. The size of MW disinfected cysts was smaller on average (4.7 mm ±0.5 for isolate I and 6.2 mm ± 1.2 for isolate II) (Figure 6, photos e–h).

To disinfect water contaminated with Blastocystis sp. cysts, it is required to consider several criteria that influence the decision of which of the three investigated disinfectants to use (Table 5). The two isolates exhibited dissimilar responses to chlorine and UV-C disinfection. On the other hand, MW is highlighted as the most effective disinfectant due to its uniform influence on the isolates used.

Blastocystis remains one of the prevailing intestinal protozoans worldwide that has been previously implicated in waterborne outbreaks (de la Cruz & Stensvold 2017). The parasite is presently recognized as one of the possible waterborne pathogens in the WHO guidelines for controlling drinking water quality (WHO 2011). Nevertheless, there is a lack of information on the Blastocystis species disinfection during water treatment procedures. Therefore, the current study aimed to determine the efficacy of chlorine, UV-C, and MW treatments as disinfectants against cysts of Blastocystis sp.

The two samples used in the present study were subtyped as ST3. Globally, Blastocystis ST3 is one of the most frequently found ST in water sources (Jinatham et al. 2022) and the most commonly recognized subtype in humans (Ahmed et al. 2022). Previously, ST3 with genetically identical sequences was identified as concomitantly infecting humans and contaminating their drinking water (Jinatham et al. 2021). This fact strengthened the potential importance of waterborne ST3 transmission. Therefore, isolates from the current study were utilized to represent ST3.

In the current investigation, although the two isolates reacted differently, the cysts of Blastocystis sp. were resistant to chlorine disinfection at all concentrations, up to 4 ppm. The existence of viable cysts in in vitro cultivation validated the results. Zaki et al. (1996) previously demonstrated cyst resistance to chlorine disinfection without identifying the ST of the utilized isolates. The robust structure of the parasite's cyst stage may account for its probable resistance to chlorine. Giardia duodenalis (G. duodenalis) (Adeyemo et al. 2019) and Encephalitozoon sp. have been effectively controlled by chlorination of water within the recommended dosage range (<4 ppm) (Johnson et al. 2003). However, Cryptosporidium sp. (Adeyemo et al. 2019), Toxoplasma gondii (Dumètre et al. 2021), and Entamoeba histolytica (Chowdhury et al. 2022) can resist high concentrations of chlorine. The structure of the protozoan (oo) cysts' wall exposed to chlorine disinfection plays an essential role in determining its resistance or vulnerability, as seen in the walls of Toxoplasma oocysts and sporocysts. It has been reported that the bilayer feature forms a robust hermetic barrier protecting the enclosed sporozoites from the toxic effects of chemical disinfectants, particularly chlorinated-based disinfectants (Dumètre et al. 2021).

UV-C was efficient to disinfect cysts of Blastocystis at 40 mJ/cm² even though the two isolates also responded differently. In agreement with our findings, Leelayoova et al. (2004) observed a decreased risk of Blastocystis infection among individuals who consume UV-treated water. Following the present investigation, UV has been reported efficient in eradicating other waterborne protozoan parasites such as T. gondii, G. duodenalis, and Cryptosporidium sp., according to previous research (Clancy et al. 2004; Ware et al. 2010; Adeyemo et al. 2019). This UV-disinfection effect on cysts of Blastocystis and other protozoan (oo) cysts may be due to the large size of protozoan parasites and their genomes, which have a more significant number of nucleic acid targets for UV photons compared to various health-related waterborne enteric viruses and some enteric bacteria (Linden et al. 2002).

The current study demonstrates that the sensitivity of the two ST3 isolates to chlorine and UV radiation differs, even though both isolates lacked genetic diversity at the level of sequencing. Vassalos et al. (2010) reported intra-ST3 differences in the clinical outcome of infection, responsiveness to metronidazole in treated individuals, and in vitro morphotypes. In addition, a highly significant difference between symptomatic and asymptomatic ST3 isolates in protease activity at 32 kDa has been reported (Abdel-Hameed & Hassanin 2011).

Regarding chlorine as a chemical disinfectant, Li et al. (2004) hypothesized that overcoming the cyst wall barrier is essential for the effective chemical inactivation of encysted protozoan parasites. Thin-walled and thick-walled Blastocystis cyst populations were previously identified using transmission electron microscopy (TEM) in faecal samples. It was suggested that thick-walled cysts were responsible for external transmission of the parasite, whereas thin-walled cysts were responsible for autoinfection (Moe et al. 1996). According to King et al. (2005), Cryptosporidium's thick-walled oocysts are exceedingly resistant to chlorine.

In the current study, thick-walled cysts may account for their resistance. Whereas the variable excretion of thin- and thick-walled cysts in faeces may account for the variable sensitivity of the two isolates to chlorine.

It has been reported that UV-C irradiation induces the formation of dimers between adjacent pyrimidine bases in DNA. The biological effect was contingent on both the induced lesions' genomic location and the afflicted cells' developmental state (Einarsson et al. 2015). These authors demonstrated that G. duodenalis trophozoites and developing cysts are more UV-resistant than mature cysts (Einarsson et al. 2015). The author hypothesized that the active replication machinery might assist the cell in avoiding cell death caused by UV irradiation. This might occur as a result of the post-replication repair process and proteins shared by both DNA replication and UV damage repair. Different degrees of Blastocystis sp. cyst maturity may account for the variable UV-C sensitivity of the two isolates used.

Other elements that can contribute to this intra-subtype variation in sensitivity to chlorine and UV-C include the varying amounts of debris particles in the two isolates, which might hinder cysts from absorbing chlorine and might absorb, scatter, or otherwise obstruct cyst-targeting UV radiation (Christensen & Linden 2003; Shields et al. 2008). Other protozoa, including two C. parvum (Iowa and Maine isolates) oocyst lines, have also been found to vary in susceptibility to chlorine inactivation (Shields et al. 2008). In the Glasgow and Maine C. parvum strains, nearly 6 log₁₀ inactivation was attained at 5 and 20 mJ/cm², respectively, in response to UV irradiation (Clancy et al. 2004). It remains unknown why various strains of the same Blastocystis subtype respond differently to the same treatment, a topic worthy of future inquiry.

In the present work, the morphology of Blastocystis cysts subjected to chlorine and UV-C has been altered. Cysts of Blastocystis sp. are small (2–5 μm), mostly spherical or ovoid but sometimes irregular, with a multi-layered cyst wall which may or may not be surrounded with a loose surface coat (Moe et al. 1996). It was observed that both disinfectants generated identical morphological changes in the form of cysts vacuolation and inner structure abnormalities while leaving the cysts' outline and size unchanged. In G. duodenalis chlorine-treated cysts, light microscopy revealed degradation or loss of interior morphological characteristics following exposure to chlorine (Sauch & Berman 1991). In another work, TEM of chlorine-treated G. duodenalis cysts demonstrated a series of events commencing with the loosening of cyst wall filaments and plasma membrane disruption, allowing chlorine to enter cysts and destroy intracytoplasmic organelles and nuclei. The cysts reached a point of no return in the death cascade when intracytoplasmic vacuole development increased, indicating granulation and aggregation of cytoplasmic material (Li et al. 2004). Recent investigations have shown that membrane permeability alterations and interference with specific enzyme activities after chlorine entrance into the cytoplasm damage nucleic acid and leak critical cellular biomolecules (Adefisoye & Olaniran 2022).

UV-induced protozoan cysts' morphological changes are sparsely described. In previous work on the effect of UV light on Trichomonas vaginalis trophozoites, abnormalities in the parasite morphology were reported, including vacuolization of the cytoplasm and cytolysis (Karanis et al. 1991). TEM revealed cell wall alterations, enhanced membrane permeability, cytoplasmic gelatinization, and DNA aggregation/loss in UV-C-irradiated pathogenic bacteria Acinetobacter baumannii (Li et al. 2022). UV-C irradiation is known to interfere with essential biological processes such as DNA replication and transcription (Einarsson et al. 2015). It can also cause the creation of reactive oxygen species, which can affect the structure of cell membranes and proteins (Fulgentini et al. 2015). However, the biocidal mechanism of chlorine and UV-C against Blastocystis cysts has yet to be studied.

In the current study, both ST3 isolates were disinfected after 15 s of MW exposure at 56 °C (the cysts' suspension temperature), indicating that MW has a uniform effect on both isolates. According to Moe et al. (1996), temperatures between 40 and 50 °C have a lethal effect on Blastocystis sp. cysts. MW irradiation inactivated other protozoan (oo) cysts to varying degrees. Cryptosporidium oocysts were inactivated after 20 s, G. duodenalis cysts were 90% inactivated after 30 s, and Cyclospora cayetanensis sporulation was reduced to 1.9% after 45 s (Ortega & Liao 2006; Hussein et al. 2021). In contrast, MW irradiation for 7.5 min failed to inactivate microsporidian spores of Enterocytozoon bieneusi and Encephalitozoon intestinalis in sewage sludge (Graczyk et al. 2007). Such variances could be attributable to various ultrastructural features of different protozoans or variability in MW oven temperatures, where processing time mostly depends on MW heating power and sample volume (Ortega & Liao 2006; Vialkova et al. 2021).

In the current study, MW-treated cysts of Blastocystis shrank, lacerated, and ejected intracellular material. Similarly, MW-irradiated Eimeria magna oocysts showed timely progressive destruction of wall layers and intracellular organelles (Bouchet & Boulard 1991). Hussein et al. (2021) found intracellular contents of MW-irradiated G. duodenalis cysts shrinking and disappearing.

The microbicidal effect of MW is generally attributed to its thermal effects resulting from cell molecules' absorption of MW energy, which makes them vibrate faster, producing general cell heating (Michaelson 1974). The MW thermal effects dominate the inactivation of waterborne organisms since water molecules absorb MW photons quickly, causing a rise in water temperature (Wang et al. 2019). MW non-thermal effects have been elucidated (Woo et al. 2000). These non-thermal effects also play a role in the inactivation of waterborne microorganisms by producing hydrogen peroxide and chemical bond cleavage in small molecules (Kim et al. 2008). Thermal and non-thermal MW impacts can denature nucleic acids and proteins of irradiated cells, change membrane permeability, leak intracellular contents, and disturb critical biological processes (Woo et al. 2000; Wang et al. 2019). Uncertainty remains regarding the precise mechanism through which MW irradiation inactivates cysts of Blastocystis.

Three factors typically determine the effectiveness of a disinfection method: (i) the concentration or dosage of the disinfectant, (ii) the contact duration, and (iii) the particular organism involved (Lin et al. 2020). A chlorine dosage of up to 4 ppm was used in the current experiment since it is deemed safe and unlikely to harm human or animal health (CDC 2020). While both Blastocystis isolates resisted the employed chlorine concentrations, increasing the contact time from 120 to 180 min did not affect the parasite's viability. This may be due to the chlorine's capacity to disinfect the population of thin-walled cysts, while the thick-walled cysts provide the actual chlorine resistance.

The range of UV-C doses required for typical potable water and wastewater applications is 16–40 mJ/cm² (Linden et al. 2002). In the present study, UV-C at 40 mJ/cm² efficiently disinfected Blastocystis cysts from both isolates. However, this approach risks UV disinfection hampered by particulate matter in water. This might occur through decreasing UV transmittance and particles associating with the parasite and shielding it from UV radiation. Furthermore, certain microorganisms, like bacteria, can repair and reverse UV-induced damage (Einarsson et al. 2015). Prior research demonstrated that C. parvum oocysts undergo DNA photo and dark repair following UV irradiation, despite the parasite's infectivity not being restored (Oguma et al. 2001). Concerning the Blastocystis parasite, this is a subject that merits further research.

In contrast, MW irradiation in water treatment has the benefit of being a rapid, instantaneous procedure that heats water precisely and uniformly, causing dipolar oscillations and ionic conductivity in the water medium (Vialkova et al. 2021). MW was more effective than chlorine and UV-C at disinfecting cysts of Blastocystis in terms of reducing their numbers, changing their morphology, and having a uniform effect on both isolates; it was also safer and easier to use and had the highest impact on viability after processing. However, the high-energy cost required for MW water treatment may limit its use in developing countries.

Disinfection experiments with few samples have limited the current investigation. However, the opportunity to have an abundance of cysts in the samples proved to be advantageous to our investigation of the most effective disinfection methods. Previous research found it challenging to harvest large quantities of Blastocystis sp. cysts from human faeces due to their rarity or low numbers (Iguchi et al. 2007). Although in vitro encystation was documented (Chen et al. 1999), we used faeces-separated cysts to simulate the natural situation. Moreover, maintaining the parasite in laboratory culture could have affected how the cysts behaved in the disinfection experiments.

Blastocystis sp. cysts were resistant to chlorine at all tested concentrations and exposure durations. UV-C entirely disinfected the cysts at a dose of 40 mJ/cm². Nevertheless, the two ST3 isolates exhibited dissimilar chlorine and UV-C disinfection responses.

However, MW is deemed the most effective disinfectant due to its uniform effect on both isolates. Disinfected cysts exhibited morphological alterations, morphological defects, and a loss of viability, making them incapable of proliferating in culture.

The findings of the present study may prove helpful in water disinfection technologies. These findings might also have household and food business ramifications for the safety of ready-to-eat or minimally processed fruits and vegetables, thereby contributing to controlling parasite transmission. Even though these results only pertain to Blastocystis sp. ST3, further research is required to characterize the response of different Blastocystis sp. subtypes to MW irradiation.

The authors thank Dr Abdelghafar Mohamed Abu-Elsaoud, Department of Botany and Microbiology, Faculty of Sciences, Suez Canal University, for providing the UV-C instruments and directing the instructions for preparation. The authors would also like to thank Dr Ahmed Mohamed Osman Ali, Department of Soil and Water, Faculty of Agriculture, Suez Canal University, for preparing the free chlorine concentrations.

Ethical approval of the study protocol was given by the Research Ethics Committee of the Faculty of Medicine, Suez Canal University (Approval number 5150; Dec. 2022). Free and informed consent of the participants or their legal representatives was obtained.

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

A. M. conceptualized the whole article. A. M., C. S. and S. A. developed the methodology, rendered support in data curation and formal analysis, and validated the data. A. M., C. S., S. A., and P. K., investigated the data, visualized the process, and validated the article. A. M. wrote the original draft. A. M., C. S., S. A., P. K. reviewed and edited the final manuscript. P. K. rendered support in language proof and overall mentoring.

All relevant data are included in the paper or its Supplementary Information.

The authors declare there is no conflict.