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Article

Assessing Zoonotic Risks of Blastocystis Infection in Singapore

by
Thet Tun Aung
1,2,
Charlotte Kai Qi How
1,
Jean-Marc Chavatte
3,
Nazmi Bin Nazir
1,
Edgar Macabe Pena
4,
Bryan Ogden
4,
Grace Rou’en Lim
5,
Yasmina Arditi Paramastri
5,
Lois Anne Zitzow
5,
Hanrong Chen
6,
Niranjan Nagarajan
6,
Kevin Shyong Wei Tan
7 and
Benoit Malleret
1,*
1
Department of Microbiology and Immunology, Immunology Translational Research Programme, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117545, Singapore
2
Singapore Eye Research Institute, Singapore 169856, Singapore
3
National Public Health Laboratory, National Centre for Infectious Diseases, Singapore 308442, Singapore
4
SingHealth Experimental Medicine Centre and National Large Animal Research Facility, Singapore 169856, Singapore
5
Comparative Medicine, National University of Singapore, Singapore 119077, Singapore
6
Genome Institute of Singapore, A*Star, Singapore 138672, Singapore
7
Department of Microbiology and Immunology, Healthy Longevity Translational Research Programme, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117545, Singapore
*
Author to whom correspondence should be addressed.
Pathogens 2025, 14(8), 773; https://doi.org/10.3390/pathogens14080773
Submission received: 5 June 2025 / Revised: 3 July 2025 / Accepted: 18 July 2025 / Published: 5 August 2025
(This article belongs to the Section Parasitic Pathogens)
Browse Figures
Versions Notes

Abstract

Blastocystis spp. is an enteric protist that is present worldwide. Despite being discovered a century ago, there is still much to be learned about its pathogenicity and transmission. Different subtypes (ST) of Blastocystis spp. have been identified in various hosts, including humans, birds, and insects, and there is potential for zoonotic transmission through contact between humans and animals. The prevalence of Blastocystis spp. in humans and macaques in Singapore was understudied, and the findings revealed a significant prevalence of the parasite, with rates of 90% and 100% observed in each respective Macaca fascicularis population 1 and 2, with main subtypes (ST1, ST2, ST3, and ST5). Using metagenomics, the different subtypes of Blastocystis spp. (comprising ST2, ST3, and ST17) were identified in a healthy Singaporean cohort. Additionally, seven incidental findings of Blastocystis spp. were discovered in human patients with other gut parasites, including two ST1, two ST2, two ST3, and one ST8. Several factors such as diet or reverse zoonotic transmission are suggested to play a role in Blastocystis sp. subtype distribution.

1. Introduction

Blastocystis Alexeieff, 1911 is an anaerobic eukaryotic protist that inhabits the gastrointestinal tract of various hosts, including humans [1], mammals [2,3], birds [4], and insects [5]. Its prevalence in humans varies globally, with rates ranging from 22–56% in European countries to 37–100% in Asian and American countries [6]. Symptoms of Blastocystis spp. infections typically include diarrhea and irritable bowel syndrome [7]. However, instances of asymptomatic individuals have been reported [8]. Conversely, studies have indicated a positive correlation between a healthy gut microbiome and Blastocystis spp. colonization [9]. Thus, the parasite’s classification as either a commensal or a pathogen remains uncertain, making its clinical significance unclear.
While Blastocystis spp. naming used to be species-specific, no significant differences between isolates from different host species have been discovered [10]. This homogeneity between sequences is unexpected when one considers the extensive human–human or animal–human transmission that occurs, with animals forming a large reservoir for infection in humans [11]. Today, 18S SSU rRNA sequences are crucial in separating Blastocystis spp. isolates into subtypes (STs) for epidemiological studies.
At least 22 STs have been identified globally [12], with each ST possessing at least 4% divergence in sequences from other STs. ST occurrence depends on the species of the host and the geographical distribution. ST1–ST9 and ST12 have been isolated from humans, with ST4 being most common [13]. In Southeast Asia, the predominant Blastocystis spp. ST detected in humans was ST3 (Supplementary Figure S1).
ST5 is typically found in cows or pigs, ST6 in avian species, and ST8 in nonhuman primates [14]. The identification of ST5–ST8 in both animals and humans strongly supports transmission via zoonosis. As Blastocystis spp. subtypes have cryptic host specificity, more studies are required to fully elucidate the zoonotic potential of this parasite. The dominant subtypes of Blastocystis spp. vary across the different hosts ST1, ST2, and ST3 (humans); ST10 and ST5 (cows); ST1, ST2, and ST3 (monkeys); ST5 (pigs); ST7 and ST6 (hens); and ST3 and ST1 (rodents) [14].
Like many other gut parasites, Blastocystis spp. is primarily transmitted through the fecal–oral route, either by consuming contaminated food or water or by direct contact with infected feces [15]. In addition to human-to-human transmission, there have been documented cases of zoonotic transmission, with the parasite being transmitted from animals to humans. This is particularly evident in settings like zoos or research facilities, where close contact with animal reservoirs can occur [16].
Urbanization places humans much closer to wild animal habitats than before, resulting in humans now having more contact with wildlife than in the past, increasing the potential for zoonotic transmission of diseases. This can be seen in Singapore, where the long-tailed macaque (Macaca fascicularis Raffles, 1821), the most common nonhuman primate species, enters households and schools in search of food [17]. Previous studies have suggested that macaques are natural Blastocystis spp. hosts [2,18]. With increased interaction between humans and animals, these macaques become potential animal reservoirs for zoonotic pathogens. Moreover, Blastocystis spp. have been reported to have high prevalence rates of up to 50% in both livestock and captive animals [19]. In research, captive macaques are used as biomedical models due to similar biological and physiological functions with humans. Consequently, more investigation into Blastocystis spp. prevalence and effect on macaques is required.
This study is divided into two main areas. First, the prevalence and ST distribution of Blastocystis spp. in three populations of M. fascicularis from Vietnam or wild-caught (housed in Singapore research facilities) were examined, and several factors such as diet or reverse zoonotic transmission were analyzed to determine if they played a role in Blastocystis sp. subtype distribution. Second, metagenomic analysis was conducted on a human cohort from Singapore to determine Blastocystis sp. ST distribution, prevalence, and any potential association between physical characteristics and disease status. Finally, the STs of patients diagnosed with other gut parasites with an incidental finding of Blastocystis spp. from patients were determined.

2. Materials and Methods

2.1. Animals

2.1.1. Description of 3 Populations of M. fascicularis

Fecal samples from a total of 101 nonhuman primates were analyzed during the study. The NHPs were from different populations as described below.
a. Population 1
Although 42 M. fascicularis samples were received from the same captive-breeding primate facility in Vietnam, the macaques were housed in 2 different facilities as they were being used for various research purposes (Supplementary Figure S2). After a mandatory quarantine period (t = 0 days), fecal swabs were obtained from 42 M. fascicularis when they arrived at facility 1. Shortly after, 4 M. fascicularis were shipped to facility 2. At 48 days, 30 fecal swabs were obtained from the original batch of M. fascicularis at facility 1. These 30 macaques were then euthanized after completing an unrelated IACUC approved research protocol. Last, at 182 days, 8 fecal M. fascicularis samples from facility 1 and 4 from facility 2 were obtained, making up the last 12 samples (Supplementary Figure S3).
b. Population 2
After arrival in Singapore years ago (ranging from 2 to 8 years depending on individual macaques), the macaques underwent a compulsory quarantine period before moving to facility 3 and were isolated for their entire stay in Singapore. Nine M. fascicularis fecal samples were collected from facility 3 at 2–8 years after arrival in facility 3 (Supplementary Figure S4).
c. Population 3
An exploratory study of detecting Blastocystis spp. was conducted on 50 M. fascicularis from the research facility.

2.1.2. DNA Extraction from Fecal Samples

Three M. fascicularis populations (herein labeled population 1, population 2, and population 3) were selected from 2 different research facilities in Singapore. Fecal swabs (BD) were obtained from population 1 (n = 42), population 2 (n = 9), and population 3 (n = 42).
QiaAmp Fast DNA Stool Mini Kit (Qiagen, Venlo, The Netherlands) was utilized to extract DNA from fecal samples. The concentration, 260/230, and 260/280 values were determined using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA).

2.1.3. Real-Time Polymerase Chain Reaction (qPCR)

Amounts of 0.1 µL of F2 (5′-CCTACGGAAACCTTGTTACGACTTCA-3′) primer, 0.1 µL of PrimeTime Eco Probe and 0.1 µL of F5 (5′-GGTCCGGTGAACACTTTGGATTT-3′) primer, 5 µL of PrimeTime Gene Expression Master Mix (Integrated DNA Technologies (IDT), Coralville, IA, USA), 1 µL of DNA sample, and 3.7 µL of nuclease-free water (Cytiva, Marlborough, MA, USA) were added to 8-well PCR strips (Thermo Fisher Scientific). qPCR was run on an ABI 7500 Real-time PCR system (Thermo Fisher Scientific). The cycle was as follows: −50 °C for 2 min, 95 °C for 10 min, and 40 cycles of 95 °C for 15 s followed by 60 °C for 1 min. The cut-off Ct value was 30 for Blastocystis spp.-positive samples.

2.1.4. Polymerase Chain Reaction (PCR) Amplification

Amounts of 1.25 µL of 10 µM RD5 and BhRDr primers (Integrated DNA Technologies), 12.5 µL of HotStarTaq Master Mix (Qiagen), 1 µL of DNA sample, and 9 µL of nuclease-free water (Fisher Scientific) were added into 8-well PCR strips (Thermo Fisher Scientific). The samples were cycled through 95 °C for 15 min (for heat activation), then 30 cycles at 94 °C for 30 s, 60 °C for 30 s, and 72 °C for 1 min before 72 °C for 10 min.

2.1.5. Gel Electrophoresis of PCR Products

An amount of 0.5 g of agarose powder (1st Base) was dissolved in 50 mL of 1× TAE buffer (Vivantis, Hyderabad, India) before 5 µL of SYBR Safe (Invitrogen, Thermo Fisher Scientific) was added to the mixture. The 1% agarose solution was poured into a gel tray with a 15-well comb and allowed to cool until solidified. An amount of 5 µL of GeneRuler 1 kb DNA Ladder (Thermo Scientific Scientific) was added to one lane for reference. Amounts of 5 µL of the PCR products and 1 µL of 6× New England BioLabs Gel Loading Dye (Ipswich, MA, USA) were mixed before being loaded into the wells. The gel was run at 100 V for 60 min. Agarose gel was imaged using a ChemiDoc XRS+ Gel Imaging System (BioRad, Hercules, CA, USA) for the visualization of Blastocystis spp.-positive bands at ~600 bp.

2.1.6. Subtyping of Blastocystis spp.

qPCR positive Blastocystis spp. samples were sent to BioBasic Asia with forward (RD5) and reverse (BhRDr) primers for reference. The samples underwent Sanger sequencing. Sequences received in FASTA format were uploaded to PubMLST for subtype and allele identification.
For reference Blastocystis spp. sequence data used for phylogenetic analysis, at least 4 sequences of 18S SSU rRNA Blastocystis spp. isolates for ST1-ST8 (except ST6) were selected from humans, livestock, and nonhuman primates (NHP) in NCBI GenBank across various geographic regions. Sequence alignment was through ClustalW on MEGA X (Gap Opening Penalty (15.00) and Gap Extension Penalty (6.66)). Each alignment was trimmed at the beginning and end to improve the similarity scores. Similarity scores were used to plot phylogenetic trees using the Neighbour-Joining method in MEGA X. The best tree with the highest log likelihood was shown in each figure with bootstrap proportions at the nodes (100 replicates). Nucleotide substitution types were used with a maximum composite likelihood model. Trees were drawn to scale, with branch lengths measured as the number of substitutions per site.

2.2. Human

2.2.1. Metagenomic Datasets and Data Processing

One hundred nine stool metagenomic datasets from the multi-ethnic Singaporean population were obtained from the Singapore Platinum Metagenome Project [20]. Paired-end sequencing was performed on the Illumina HiSeq4K platform (San Diego, CA, USA), obtaining 18–190 million 150 bp reads per sample. Hybrid metagenomic assemblies were constructed from Illumina short reads and nanopore long reads using OPERA-MS [21].

2.2.2. Detection of Blastocystis spp. STs from Metagenomes

The short reads were mapped against 10 Blastocystis spp. genomes obtained from NCBI using BWA mem v0.7.17 [22]. The matches were filtered for 99% identity over 90% of the read length and ensured that reads are properly paired. Blastocystis spp. genome assemblies were divided into 10-kilobase (kb) windows, and the number of windows with at least one match were counted. Sample–genome pairs where >10% of genomic windows have nonzero coverage were flagged as indicating detection in the metagenome.
Separately, the short reads were also mapped against Blastocystis spp. rRNA full-length sequences obtained from PubMLST using bwa mem v0.7.17. The matches were filtered for 99% identity over 80% of the read length. Sample–gene pairs with mean BWA mapping quality > 5 were recorded. Blastocystis spp. 18S rRNA sequences were compared using BLAST+ 2.12.0 against metagenomic assemblies with an E-value threshold of 1 × 10−50 and >95% identity. The aligned contigs were extracted and utilized for phylogenetic analysis.

2.2.3. Statistical Analysis

Measurements from a list of characteristics from each of the 109 samples were measured and recorded [23]. Characteristics from the 109 humans were analyzed across 2 groups—Blastocystis spp.-positive and Blastocystis spp.-negative for any significant associations. Statistical tests were run on GraphPad PRISM 9.3.1 (https://www.graphpad.com/features, accessed on 17 July 2025). Sex was tested for statistical significance using Fisher’s exact test (to compare between 2 different groups), while ethnicity was tested using Pearson’s Chi-square test (to compare the difference for more than 2 different groups). For the rest of the characteristics, each group (Blastocystis spp.-positive and Blastocystis spp.-negative) was first tested for normality using the Shapiro–Wilk test to determine if the populations are normal across each characteristic. Subsequently, either an unpaired parametric t-test (if normal distribution) or an unpaired nonparametric Mann–Whitney t-test (if not normal distribution) was carried out to determine if the p-values were significant. The difference was considered statistically significant if the p-value was less than 0.05.

2.2.4. Blastocystis spp. Identification in Singapore Patients

Stool samples with morphological observations on fecal smears or positive detection by BioFire® FilmArray® GI Panel (BioFire Diagnostics, Salt Lake City, UT, USA) of enteritic Protozoa such as Cyclospora Schneider, 1881, Cryptosporidium Tyzzer, 1907, Entamoeba Casagrandi & Barbagallo, 1897, Enterocytozoon bieneusi Desportes, Lecharpentier, Galian, Bernard, Cochand-Priollet, Lavergne, Ravisse & Modigliani, 1985, or Giardia Künstler, 1882 were further tested in the Singapore National Public Health Laboratory (NPHL), by both microscopy and molecular methods. The presence of a suspected or detected primary parasite was confirmed and investigated for other enteritic parasites including Blastocystis spp. with subtyping as described in [24]; the ST was further confirmed by comparison with PubMLST.

3. Results

3.1. Subtyping and Phylogenetic Analysis of Blastocystis spp. in Population 1

After DNA extraction and analysis via qPCR, the Blastocystis spp. prevalence was determined. At 0 days, there was a Blastocystis spp. positive rate of 90.47% (38 out of 42). At 48 days, there was a Blastocystis spp. positive rate of 93.33% (28 out of 30). At 182 days, there was a Blastocystis spp. positive rate of 41.67% (5 out of 12).
At 0 days, the majority of Blastocystis spp.-positive samples were ST3 (42.1%), followed by ST1 (31.6%), ST2 (21.1%), ST5 (2.6%), and ST8 (2.6%). At 48 days, the majority of the samples were ST5 (78.5%), followed by ST3 (14.3%), then ST1 (3.6%) and ST2 (3.6%). Last, at 182 days, the majority of the samples were ST1 (80.0%) (Table 1).
From the phylogenetic tree at 0 days (Figure 1A), ST1, ST2, and ST3 sequences from the M. fascicularis were mostly clustered together with relatively high bootstrap values. These three subtypes formed monophyletic clades. ST2 (sample 7185; indicated with a black arrow) was interspersed among a ST1 cluster. ST5 (sample 0077; indicated with a black arrow) branched off from the majority of the samples, along with ST8 (sample 6337; indicated with a black arrow). In the phylogenetic tree from 48 days (Figure 1B), ST5 sequences were mostly clustered together, with ST1 (sample 0105; indicated by a black arrow) and ST3 (Sample 7187; indicated by a black arrow) sequences interspersed. The horizontal branch lengths were longer, indicating that the ST5 sequences were evolutionarily distant from each other. Sample 0077 (indicated with a black arrow) was the only sample that retained ST5 subtype. Last, for the phylogenetic tree from the 182 days batch (Figure 1C), the five sequences are evolutionarily close to each other, with short branch lengths. There is high confidence in the plotted phylogenetic tree due to high bootstrap values.

3.2. Subtyping and Phylogenetic Analysis of Blastocystis spp. in Population 2

After DNA extraction and analysis via qPCR, Blastocystis spp. prevalence was determined. The Blastocystis spp.-positive rate was 100% (nine out of nine) (Table 2).
Blastocystis spp.-positive samples from facility 3 were evenly distributed across ST1 (33.3%), ST2 (44.4%), and ST3 (22.2%) (Table 2).
For population 2 (Figure 2), ST1, ST2, and ST3 sequences were clustered together and were evolutionarily close to each other with short branch lengths. However, one of the ST2 sequences (sample 8168; indicated by a black arrow) branched off from the other ST2 sequences and was closer to the ST1 sequences.

3.3. Subtyping and Phylogenetic Analysis of Blastocystis spp. in Population 3

After DNA extraction and analysis via qPCR, the Blastocystis spp. prevalence was determined (42 out of 50 samples were Blastocystis spp. positive) (Table 3).
Blastocystis spp.-positive samples from population 3 were divided between ST1 (66.7%), ST2 (2.4%), ST3 (23.8%), ST5 (4.8%), and ST1 + ST3 (2.4%) (Table 3).
As seen from the phylogenetic tree (Figure 3), the majority of the branch lengths were 0.00–0.10, which indicated that the sequences were highly similar to each other. One hundred replicates were conducted with higher bootstrap values giving greater confidence in the observed branch. While some branches showed bootstrap values of more than 90, there were also some branches with values as low as 2. However, MEGA X computed the highest likelihood tree from the samples. With the exception of the co-infection sample, each subtype was clustered together and agreed well with subtyped sequences obtained from GenBank data.
Identification of Blastocystis spp. subtype distribution in two subgroups (wild caught and imported) from population 3 was performed. Hence, we reported that ST1 was the major subtype in both subgroups, followed by ST3 (Supplementary Figure S5).

3.4. Incidental Cases of Blastocystis spp.

Seven human cases with an incidental finding of Blastocystis spp. were identified from positive stool samples suspected or confirmed positive for other enteritic parasites such as Cyclospora, Cryptosporidium, Entamoeba, Enterocytozoon, or Giardia. They were discovered by microscopy on fecal smear, confirmed by molecular methods, and then subtyped. The different STs identified were as follows: 2 ST1, 2 ST2, 2 ST3, and 1 ST8 (Table 4).

3.5. Blastocystis spp. in Human Metagenomic Data

Prevalence of Blastocystis spp. and subtype distribution detected in metagenomic data.
Out of 109 samples taken from the multi-ethnic cohort in Singapore [20], there was a prevalence of 11.0% (12 out of 109) when compared against whole-genome sequences of Blastocystis spp. The majority of the samples were ST6 (58.3%; 7 out of 12), followed by ST3 (33.3%; 4 out of 12) and then ST2 (8.33%; 1 out of 12) (Table 5). However, 41.6% (5 out of 12) samples had a genomic window coverage of less than 80%. Additionally, there were a few samples with ≤30% match with ST6 sequences.
A low genomic window percentage coverage could be due to the presence of a different Blastocystis spp. ST whose genome was not in NCBI when we performed the analysis (Table 5). Hence, it was decided to match the reads against 18S SSU rRNA full-length sequences obtained from PubMLST.
There was a prevalence of 7.34% (8 out of 109) of Blastocystis spp. when comparing with 18S SSU rRNA full-length sequences from the same multi-ethnic cohort in Singapore [20] (Table 6). The majority of the samples were ST17 (50%), followed by ST3 (37.5%) and then ST2 (12.5%). All eight samples showed a high percentage identity with the query sequence (at least 95%) (Table 6). Apart from ST3 (sample ID-5), which branched off before the rest, the other sequences were quite clustered together according to subtype (Figure 4).
This is unusual as ST17 has mainly been identified in rodents (such as gundi, squirrel, and chinchilla) thus far [13,25,26,27], the probable natural hosts for ST17. Since the ST17 genome sequence is not available in NCBI, we could not corroborate the rRNA matches for these four samples of ST17-positive cases with the genomic analysis above. This dataset showing the identification of four samples that were positive for ST17 in human samples is unprecedented and could be an indicator of zoonotic transmission from rodents to human samples (Table 6).

3.6. Population Characteristics and Links to Blastocystis spp.

The dataset spanned diverse characteristics—from BMI and waist circumference to blood pressure and cholesterol levels (Table 7) [20]. Both Blastocystis spp.-positive and Blastocystis spp.-negative groups had similar distributions of sex and race (p-value > 0.05). The average and standard deviation across both populations were calculated, and the p-values were determined. The results were considered significant if the p-value was ≤0.05. From this table, the right apex neurothesiometer value was considered significant across both groups. Additionally, the total cholesterol had a low p-value of 0.072 compared with the rest of the metabolites.

4. Discussion

Blastocystis spp. is an enteric protist with widespread distribution throughout the world. In today’s global context, zoonotic diseases are gaining attention, especially with increased exposure to animal reservoirs. Hence, it becomes imperative that reservoirs of infection are identified from the community. In the local context of Singapore, one study determined the prevalence (3.3%; 9 out of 276) and subtype distribution (78% ST3 and 22% ST1) of Blastocystis spp. in patients from one of the hospitals in Singapore [28]. Additionally, another study determined the prevalence (10.1%; 25 out of 248) and subtypes (64% ST7; 16 out of 25) of Blastocystis spp. in patients with Clostridioides difficile (Hall & O’Toole, 1935) Lawson & Rainey, 2016 infection in Singapore [29]. However, no sampling of macaques or the healthy human population for Blastocystis spp. in Singapore had been conducted thus far.
In this study, five subtypes were identified across 51 M. fascicularis stool samples (population 1 and population 2)—ST1, ST2, ST3, ST5, and ST8. Additionally, many samples were not only of the same subtype but also the same allele. There was also a chronological comparison performed across six months for population 1, which enabled the investigation of how time affected the subtype distribution.
Macaca fascicularis is a nonhuman primate that is native to Southeast Asia and is one of the most popular choices as a nonhuman primate for research purposes due to suitability in reproductive and endocrine physiology [18,30]. There was a high prevalence of Blastocystis spp. present initially (90.47% (38 out of 42) (t = 0 days)) (Table 1). This result is comparable to other studies conducted in M. fascicularis in Asia [18].
High prevalence rates were expected as M. fascicularis are natural hosts of Blastocystis spp. and remain asymptomatic despite infection. Additionally, these macaques were held captive in an enclosed area, which could lead to an increase in Blastocystis spp. transmission. However, there was a notable drop in prevalence in the batch of 12 M. fascicularis at t = 182 days when the prevalence went from ≥90% to 41.67% (5 out of 12).
Although 47 M. fascicularis were housed individually, there were 4 that were socially housed at facility 2. The 51 M. fascicularis shared the same diet (i.e., commercial monkey chow and enrichment fruits from suppliers); however, enrichment (i.e., toys) was not shared across macaques.
To explain the loss in infections after a long time point (Table 1), one should first observe what was seen in the prospective study on Blastocystis spp. subtypes pre- and post-travel [31]. It reported that 27.6% changed subtype or lost infection after travelling and concluded that Blastocystis spp. carriage and transmission was highly dynamic—depending on the location of travel and various host factors. There are three other explanations for why 75% of the t = 182 days batch experienced a loss in infections. First, after a long period of time, there could be an alteration in the composition of the gut microbiota that causes disturbances in Blastocystis spp. growth conditions in the gut [32]. Second, it is possible that the natural Blastocystis spp. infection had run its course and been removed by the macaque immune system; however, currently, there is no consensus on how long Blastocystis spp. infection remains in the gut. Last, the socially housed macaques were younger in age, and all four of these macaques either lost the infection or did not acquire one due to their young age [33].
Sample 0077 was the only sample that was ST5 in population 1 (t = 0 days). Additionally, it maintained its ST5 subtype in population 1 (t = 48 days), which could have indicated a potential point source for contamination, (genome sequence percentage identity of 87.65% and e-value of 1 × 10−113 between t = 0 and t = 48 days). The majority of the macaques that changed subtype were living in separate cages with no intermingling (including sample 0077), hence warranting a deeper look into other commonalities.
Our results showed that 80% (20 of 25) of macaques that changed ST were changed to ST5.
First, food could have promoted ST5 proliferation, and enrichment fruits obtained from the suppliers may have been obtained from the wet market, which present a source for contamination [34,35]. Additionally, not only were they of the same ST5 subtype, but they also had the same allele (i.e., 115), which indicates a possible common environmental source of infection. Second, there could have been a low sensitivity for the method of detection. In this study, the utilized barcoding method that was suggested by Scicluna et al. (2006) (forward primer-generic and amplifying other eukaryotes) was more suitable for screening, rather than in-depth analysis of subtypes [36]. In fact, one study found that mixed-subtype infections are more common than detected when comparing next-generation sequencing methods with the conventional PCR method [37]. Although there is one study that found that stable Blastocystis spp. colonization is subtype-independent, it is possible that ST5 could have been in low abundance at the t = 0 day time point, and afterward, it proliferated by t = 48 days to stable gut colonization and outgrew other subtypes present in the gut [9].
Third, ST5 is typically considered a livestock subtype due to its high prevalence in pigs [38]. There were other animals held at facilities 1 and 2, including pigs and horses, which were located at another end of the facility.
Fourth, human subtypes obtained (ST1, ST2, and ST3) from M. fascicularis could be indicative of reverse zoonosis. This was supported by the higher prevalence of Blastocystis spp. infections in zookeepers, as well as similarity in subtype sequences obtained from both the animals and the zookeepers [2,39]. However, this is also rather unlikely as in a research facility, there are workflows in place to reduce the transmission of zoonotic organisms, and personal protective equipment (PPE) is not likely to be breached. Last, as macaques are social animals, living in isolation for a prolonged period of time could have increased their stress levels and exacerbated the pathogenicity of ST5 Blastocystis spp. [40].
For the nine M. fascicularis samples held in facility 3, there were only three subtypes identified: ST1 (33.3% (three of nine)), ST2 (44.4% (four of nine)), and ST3 (22.2% (two of nine)). It is worth noting that these macaques have been living at the facility for 2 to 8 years, and this proximity could have led to evolutionary closeness, as evidenced by short branch lengths (Figure 3). ST1, ST2, and ST3 are also typically considered as human subtypes and atypical of macaques, which provides further evidence of reverse zoonosis from the caretakers. Although contracting Blastocystis spp. from the caretakers in Singapore is unlikely due to extensive PPE and protective procedures, the level of compliance with PPE procedures at the macaque vendor’s breeding facility prior to import is unknown, and it is conceivable that Blastocystis spp. was contracted from the caretakers in Vietnam instead. It is possible that in the future, the subtype distribution of Blastocystis spp. in population 1 will continue to evolve to look more like population 2 (extrapolation of population 1).
All 51 M. fascicularis in this study (population 1 and population 2) were bred from the same breeding facility in Vietnam. Although the expectation is that wild-caught macaques would have higher subtype and allele variability in Blastocystis spp., this was not seen in population 1 when compared with population 3 (Supplementary Figure S5). Compared with population 3, there was a similar number of subtypes detected in population 1 but also a larger number of alleles, indicating allelic variability. Additionally, there was a mix of imported (n = 25) and wild-caught (n = 17) animals in population 3 (n = 42). As seen from Supplementary Figure S5, a significant proportion of macaques that were imported from Vietnam were ST1, followed by ST3. Wild-caught macaques from Singapore (this study) also displayed the same trend but at lower proportions. However, this research has low power due to the small sample size. For future studies, a comparison between imported and wild-caught macaques can be performed to determine if wild-caught would have a more diversified distribution of Blastocystis spp.
The Singapore Platinum Metagenomics Project [20], a multi-ethnic cohort analysis of 109 fecal samples, was the first metagenomic analysis of Blastocystis spp. for Singapore. Initially, the Illumina Short Reads were matched against Blastocystis spp. genomes found on NCBI and produced a prevalence of 11.0% (12 out of 109). However, many of the sequence matches had low percentage identity (Table 5), and whole-genome sequences were not available for every subtype on NCBI.
The decision was made to match the reads against 18S SSU rRNA full-length sequences obtained from PubMLST. All of the matches had ≥95% similarity (Table 6). This provided a final prevalence of 7.33% (8 out of 109), which was comparable to other studies conducted in Singapore. We note, however, that the metagenomics approach was less sensitive than the qPCR-based detection method in macaques. Since only 10 genomes were used for the genomic analysis, subtypes not in the reference database may have been missed (and may have caused the low % window coverage for 8 of the 12 samples). Blastocystis spp. present at low abundance may also cause a low % window coverage and/or lack of mapping to 18S rRNA.
The incidental detection of Blastocystis spp. among human stool samples suspected and/or confirmed positive for other enteritic parasites highlighted the regular occurrence of mixed pathogens infection as previously reported [24,30]. It is worth noting that since Blastocystis spp. is not included within the commercial syndromic gastro-intestinal molecular testing panels, its detection relies solely on microscopy, likely leading to a widely underestimated prevalence.
Interestingly, the subtyping of the incidental human cases confirmed the presence of ST1, ST2, and ST3, which are three dominant subtypes in human populations that have also been reported in animals, such as NHPs and rodents [6,14,41]. In addition, ST8 was also identified. This ST has been found in animals such as NHPs, rodents, birds, and arthropods [6]; is rarely found in humans [6]; and has not previously been reported from Singapore. Altogether, these results and a recent global report of widespread presence of Blastocystis spp. in rodents [41] support the idea of zoonotic transmission. Given this, more testing, subtyping, and implementation of a comprehensive monitoring of Blastocystis spp. to try to identify infections routes and reservoirs should be conducted. This could also shed light on the still controversial pathogenicity of the different Blastocystis spp. STs.
Based on the data collected from the human cohort (Table 7), only the right apex neurothesiometer value was significant (p-value = 0.043) when comparing Blastocystis spp.-positive and Blastocystis spp.-negative groups. While numerous studies have reported that Blastocystis spp.-positive individuals tend to have a lower BMI [42], the difference in BMI between the two groups in this study was not statistically significant (p-value = 0.186). Notably, Mirjalali et al.’s study was conducted in Iran, whereas Beghini et al.’s data were primarily from European or American populations [9,42]. Asians generally have lower BMIs compared with other populations worldwide, which may have contributed to the unexpected trend observed in this study.
To gain a more comprehensive understanding of the potential pathogenic role of Blastocystis spp., there are several studies that could be performed in the future. Gut microbiome analysis of Blastocystis spp. positive and negative patients can be compared for the presence of healthy gut microbiota differences to investigate whether Blastocystis spp. are part of the healthy gut or pathogens. Regular checking (monthly) of Blastocystis spp. subtype distribution from M. fascicularis is encouraged to determine the effects of zoonotic transmission over time. Finally, environmental swabs of the macaque’s living area, food and water provided, cages, and fecal swabs from the caretakers would elucidate the contamination pathways or potential reverse zoonosis transmission.

5. Conclusions

This study employed a comprehensive approach to analyze Blastocystis spp., incorporating molecular techniques such as subtype and phylogenetic analyses. This approach facilitated the determination of the prevalence and subtype distribution of Blastocystis spp. in M. fascicularis populations, indicating evidence of zoonosis. Additionally, metagenomics was also employed to determine the prevalence of Blastocystis spp. in the Singaporean population, revealing potential risk factors. Moreover, the incidental findings of Blastocystis spp. in humans with other parasites suggest that inclusion of Blastocystis spp. comprehensive testing could help investigate the controversial pathogenic role of the parasites by different STs, identify the possible infection routes, and possibly break the transmission cycle. By elucidating the zoonotic transmission pathways and capabilities of Blastocystis spp., tailored strategies for screening and controlling transmission can be developed for local contexts including animal facilities, providing valuable insights for public health initiatives. Through these findings, the study helps to bridge gaps in current Blastocystis spp. research, particularly concerning epidemiology and pathogenicity.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/pathogens14080773/s1, Figure S1. Diagram depicting the subtype distribution of Blastocystis spp. infections in humans in Southeast Asia. Figure S2. Phylogenetic trees of 18S SSU rRNA Blastocystis spp. sequences. (A) Complete sequences between Blastocystis spp. subtypes 1–9 obtained from NCBI [5]. (B) Partial Blastocystis spp. sequences ranging from 986 to 1983 bp obtained from PubMLST. (C) Partial Blastocystis spp. sequences ranging from 337 to 600 bp obtained from PubMLST. Best trees from the Neighbour-Joining method are displayed with bootstrap proportions (BPs) at each branch node. The branch length is proportional to the number of substitutions. Figure S3. Schematic describing the distribution of M. fascicularis (population 1) obtained from Vietnam by location and time. Figure S4. Schematic of timeline of arrival of M. fascicularis from Vietnam of population 1 and when fecal swabs were collected. Orange circles indicate time points at which fecal swabs were collected and subsequently analyzed. Figure S5. Timeline depicting the arrival of M. fascicularis from Vietnam of population 2 and when fecal swabs were collected. Orange circles indicate time points at which fecal swabs were collected and subsequently analyzed. References [43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59] are cited in the Supplementary Materials.

Author Contributions

T.T.A. and C.K.Q.H., investigation, methodology, validation, and writing—original draft. J.-M.C., N.B.N., E.M.P., B.O., G.R.L., Y.A.P., L.A.Z., H.C., N.N., and K.S.W.T., validation, data curation, formal analysis, review, and editing. B.M., conceptualization, project administration, supervision, funding, acquisition, and writing—review and editing. All authors have read and agreed to the published version of the manuscript.

Funding

The study was supported by NUS start-up grant NUHSRO/2018/006/SU/01 and NUSMed Post-Doctoral Fellowship NUHSRO/2019/046/PDF/19. K.S.W.T. acknowledges funding support from the Yong Loo Lin School of Medicine, National University of Singapore, grant numbers A-8000685-00-00 and A-8000629-00-00, and the European Cooperation in Science & Technology (EU-COST) Grant OC-2021-1-25116 “Blastocystis under One Health” from the European Union.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Incidental case samples were obtained for surveillance purposes for the Ministry of Health Singapore under the Infectious Diseases Act.

Data Availability Statement

The data that support the findings of this study are available on reasonable request from the corresponding author. Genetic sequences obtained from the incidental cases are deposited in GenBank.

Acknowledgments

The authors are grateful to Maiwald M. and Loo L.H. from KK Women’s and Children Hospital; to Tan K.X. and the clinical laboratory staff from the National University Hospital, for sharing the samples suspected or confirmed positive for enteritic Protozoa; and to Lin R.T.P., who agree on their additional investigations for parasites.

Conflicts of Interest

The authors declare that they have no conflicts of interest.

Abbreviations

The following abbreviations are used in this manuscript:
STSubtypes
M. fascicularisMacaca fascicularis
18S SSU rRNA18S small sub-unit ribosomal RNA
PCRPolymerase chain reaction
qPCRReal-time polymerase chain reaction
NPHLSingapore National Public Health Laboratory
NCBINational Center for Biotechnology Information
BMIBody mass index
NHPNonhuman primates

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Figure 1. Phylogenetic relationships of the 18S small subunit (SSU) rRNA gene of Blastocystis spp. sequences inferred by Neighbour-Joining tree based on the 18S rRNA gene. The numbers on the branches are percentage bootstrap values of 100 replicates. Evolutionary distances between sequences were computed using the maximum composite likelihood method, and the horizontal branch is proportional to the estimated number of substitutions. (A) Phylogenetic tree from Blastocystis spp. sequences at 0 days. (B) Phylogenetic tree from Blastocystis spp. sequences at 48 days. (C) Phylogenetic tree from Blastocystis spp. sequences at t = 182 days. The black arrows indicate the samples that were either branched off or interspersed from their respective ST groups.
Figure 1. Phylogenetic relationships of the 18S small subunit (SSU) rRNA gene of Blastocystis spp. sequences inferred by Neighbour-Joining tree based on the 18S rRNA gene. The numbers on the branches are percentage bootstrap values of 100 replicates. Evolutionary distances between sequences were computed using the maximum composite likelihood method, and the horizontal branch is proportional to the estimated number of substitutions. (A) Phylogenetic tree from Blastocystis spp. sequences at 0 days. (B) Phylogenetic tree from Blastocystis spp. sequences at 48 days. (C) Phylogenetic tree from Blastocystis spp. sequences at t = 182 days. The black arrows indicate the samples that were either branched off or interspersed from their respective ST groups.
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Figure 2. Phylogenetic relationships of one small subunit (SSU) rRNA gene of Blastocystis spp. sequences inferred by the Neighbour-Joining tree based on the 18S rRNA gene. The numbers on the branches are the percentage bootstrap values of 100 replicates. Evolutionary distances between sequences were computed using the maximum composite likelihood method, and the horizontal branch is proportional to the estimated number of substitutions. The phylogenetic tree is from Blastocystis spp. sequences from population 2. The black arrow indicates the branched off sample (sample 8168) from other ST2 samples.
Figure 2. Phylogenetic relationships of one small subunit (SSU) rRNA gene of Blastocystis spp. sequences inferred by the Neighbour-Joining tree based on the 18S rRNA gene. The numbers on the branches are the percentage bootstrap values of 100 replicates. Evolutionary distances between sequences were computed using the maximum composite likelihood method, and the horizontal branch is proportional to the estimated number of substitutions. The phylogenetic tree is from Blastocystis spp. sequences from population 2. The black arrow indicates the branched off sample (sample 8168) from other ST2 samples.
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Figure 3. Subtype analysis and allele distribution of Blastocystis spp. Phylogenetic tree constructed using the maximum likelihood method, with a log likelihood of −9609.78. Bootstrap values are provided above the node, whereas the branch lengths are provided below the nodes. * represents the sample with co-infection (ST1 6093 and ST3 6093).
Figure 3. Subtype analysis and allele distribution of Blastocystis spp. Phylogenetic tree constructed using the maximum likelihood method, with a log likelihood of −9609.78. Bootstrap values are provided above the node, whereas the branch lengths are provided below the nodes. * represents the sample with co-infection (ST1 6093 and ST3 6093).
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Figure 4. Phylogenetic tree of 18S small subunit (SSU) rRNA gene of Blastocystis spp. sequences pieced together from contigs from human gut samples from the Singapore Platinum Metagenome Project and incidental cases of Blastocystis spp. The tree was inferred by Neighbour-Joining based on the 18S rRNA gene. The numbers on the branches are percentage bootstrap values of 100 replicates. Evolutionary distances between sequences were computed using the maximum composite likelihood method, and the horizontal branch is proportional to the estimated number of substitutions.
Figure 4. Phylogenetic tree of 18S small subunit (SSU) rRNA gene of Blastocystis spp. sequences pieced together from contigs from human gut samples from the Singapore Platinum Metagenome Project and incidental cases of Blastocystis spp. The tree was inferred by Neighbour-Joining based on the 18S rRNA gene. The numbers on the branches are percentage bootstrap values of 100 replicates. Evolutionary distances between sequences were computed using the maximum composite likelihood method, and the horizontal branch is proportional to the estimated number of substitutions.
Pathogens 14 00773 g004
Table 1. Blastocystis spp.-positive subtype distribution across Macaca fascicularis population obtained from population 1.
Table 1. Blastocystis spp.-positive subtype distribution across Macaca fascicularis population obtained from population 1.
0 Days (n = 38)48 Days (n = 28)182 Days (n = 5)
NumberPercentageNumberPercentageNumberPercentage
ST11231.6%13.6%480%
ST2821.1%13.6%--
ST31642.1%414.3%--
ST512.6%2278.6%120.0%
ST812.6%----
Table 2. Table describing the Blastocystis spp.-positive subtype distribution across the Macaca fascicularis population obtained from population 2 (n = 9).
Table 2. Table describing the Blastocystis spp.-positive subtype distribution across the Macaca fascicularis population obtained from population 2 (n = 9).
NumberPercentage
ST1333.3%
ST2444.4%
ST3222.2%
Table 3. Table describing the Blastocystis spp.-positive subtype distribution across the Macaca fascicularis population obtained from population 3 (n = 42).
Table 3. Table describing the Blastocystis spp.-positive subtype distribution across the Macaca fascicularis population obtained from population 3 (n = 42).
NumberPercentage
ST12866.7%
ST212.4%
ST31023.8%
ST524.8%
ST1 + ST312.4%
Table 4. Incidental cases of Blastocystis spp. detected among stool samples positive for other enteritic parasites.
Table 4. Incidental cases of Blastocystis spp. detected among stool samples positive for other enteritic parasites.
Sample YearProviderPrimary Diagnosis *
(M/FA)		Microscopy ResultsMolecular ResultsBlastocystis spp. STGenBank Acc. No.
2016 ‡NUHCyclospora cayetanensis Ortega, Gilman & Sterling, 1984 (M)C. cayetanensis
Entamoeba sp.
Blastocystis spp.
Cryptosporidium sp.		C. cayetanensis
Entamoeba hartmanni Prowazek, 1912
Blastocystis spp.
Cryptosporidium hominis/parvum		ST3KX618192
2018MOHGiardia lamblia Lambl, 1859 (FA)Negative
Blastocystis spp.		G. lamblia
Blastocystis spp.		ST3PV335991
2018MOHEntamoeba histolytica Schaudinn, 1903 (FA)E. histolytica
Blastocystis spp.		E. histolytica
Blastocystis spp.		ST2PV335994
2020TTSHParagonimus sp. Braun, 1899? (M)
Blastocystis spp.? (M)		Negative
Blastocystis spp.		Not confirmed
Blastocystis spp.		ST1PV335992
2020NUHMicrosporidia (M)
Cryptosporidium sp. (M)		Microsporidia
Cryptosporidium sp. G. lamblia		E. bieneusi
Cryptosporidium hominis/parvum
G. lamblia
Blastocystis spp.		ST1PV335993
2023KKHSalmonella Lignieres, 1900 (FA)
G. lamblia (FA)		n.a.
G. lamblia
Blastocystis spp.		n.a.
G. lamblia
Blastocystis spp.		ST2PV335995
2024KKHCryptosporidium sp. (FA) G. lamblia (FA)Cryptosporidium sp.
Negative
Blastocystis spp.		Cryptosporidium sp.
Not confirmed
Blastocystis spp.		ST8PV335996
* Primary diagnosis provided by clinical laboratories with M for microscopy and FA for BioFire® FilmArray® GI Panel; ‡ Case previously reported in [24].
Table 5. Table describing the corresponding subtype that matched to each sample with Blastocystis spp. whole-genome reference sequences from NCBI.
Table 5. Table describing the corresponding subtype that matched to each sample with Blastocystis spp. whole-genome reference sequences from NCBI.
SampleGenBank Accession NumberSubtype% of Windows with Reads Mapped
5GCA_000963385399.75
13GCA_000963415626.97
18GCA_000963415632.83
27GCA_000963415626.28
28GCA_0009633853100
44GCA_000963385390.11
46GCA_000963415612.41
48GCA_000963385399.84
52GCA_000963415626.59
83GCA_000963415627.10
88GCA_000963415650.03
102GCA_000963365295.46
Table 6. Table describing the corresponding subtype that matched to each sample with Blastocystis spp. 18S SSU rRNA full-length sequences obtained from PubMLST.
Table 6. Table describing the corresponding subtype that matched to each sample with Blastocystis spp. 18S SSU rRNA full-length sequences obtained from PubMLST.
SampleQuery Sequence ID (PubMLST)SubtypePercentage Identity (%)
218S_rRNA_full_length_1601795.60
518S_rRNA_full_length_34399.45
918S_rRNA_full_length_1601795.37
2018S_rRNA_full_length_1601796.92
2818S_rRNA_full_length_34399.94
4818S_rRNA_full_length_34399.89
6518S_rRNA_full_length_1601795.37
10218S_rRNA_full_length_9298.02
Table 7. The average (±standard deviation) of each characteristic measured in Blastocystis spp.-positive and Blastocystis spp.-negative populations along with the corresponding p-value from various statistical tests.
Table 7. The average (±standard deviation) of each characteristic measured in Blastocystis spp.-positive and Blastocystis spp.-negative populations along with the corresponding p-value from various statistical tests.
Blastocystis spp.-PositiveBlastocystis spp.-Negativep-Value
Number8 (7.33%)101 (92.67%)-
Sex
 Female5 (62.5%)60 (59.4%)>0.999
 Male3 (37.5%)41 (40.6%)
Race
 Chinese3 (37.5%)50 (49.5%)0.673
 Indian2 (25.0%)27 (26.7%)
 Malay3 (37.5%)24 (23.8%)
Characteristics
 Age53.4 ± 6.352.7 ± 5.80.769
 Height (cm)162.06 ± 9.58161.49 ± 8.250.853
 Weight (kg)61.14 ± 15.0865.9 ± 13.40.355
 BMI (kg/m2)23.22 ± 5.1725.2 ± 4.260.186
 Waist circumference (cm)77.45 ± 10.6582.72 ± 10.620.180
 Hip circumference (cm)94.48 ± 9.3498.62 ± 8.510.157
 Systolic blood pressure (mmHg)115.31 ± 21.39124.49 ± 27.30.367
 Diastolic blood pressure (mmHg)71.88 ± 10.3779.36 ± 22.010.278
Neurothesiometer (Mv)
 Right apex8.38 ± 4.096.06 ± 3.210.043
 Left apex7.31 ± 2.846.19 ± 3.330.166
 Right medial malleolus8.81 ± 3.817.82 ± 3.780.391
 Left medial malleolus7.94 ± 2.277.63 ± 3.820.347
Metabolites
 Fasting blood glucose (mmol/L)5.06 ± 0.585.19 ± 0.670.951
 Serum creatinine61.38 ± 20.0463.78 ± 18.070.619
 Total cholesterol (mmol/L)5.21 ± 0.675.8 ± 0.970.072
 Triglycerides (mmol/L)0.91 ± 0.571.23 ± 0.870.217
 HDL (mmol/L)1.37 ± 0.381.38 ± 0.360.970
 Ratio of total blood cholesterol: HDL4.02 ± 0.974.46 ± 1.320.590
 LDL (mmol/L)3.41 ± 0.443.77 ± 0.840.236
 Glycosylated hemoglobin (DCCT %)5.55 ± 0.465.68 ± 0.430.518
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Aung, T.T.; How, C.K.Q.; Chavatte, J.-M.; Nazir, N.B.; Pena, E.M.; Ogden, B.; Lim, G.R.; Paramastri, Y.A.; Zitzow, L.A.; Chen, H.; et al. Assessing Zoonotic Risks of Blastocystis Infection in Singapore. Pathogens 2025, 14, 773. https://doi.org/10.3390/pathogens14080773

AMA Style

Aung TT, How CKQ, Chavatte J-M, Nazir NB, Pena EM, Ogden B, Lim GR, Paramastri YA, Zitzow LA, Chen H, et al. Assessing Zoonotic Risks of Blastocystis Infection in Singapore. Pathogens. 2025; 14(8):773. https://doi.org/10.3390/pathogens14080773

Chicago/Turabian Style

Aung, Thet Tun, Charlotte Kai Qi How, Jean-Marc Chavatte, Nazmi Bin Nazir, Edgar Macabe Pena, Bryan Ogden, Grace Rou’en Lim, Yasmina Arditi Paramastri, Lois Anne Zitzow, Hanrong Chen, and et al. 2025. "Assessing Zoonotic Risks of Blastocystis Infection in Singapore" Pathogens 14, no. 8: 773. https://doi.org/10.3390/pathogens14080773

APA Style

Aung, T. T., How, C. K. Q., Chavatte, J.-M., Nazir, N. B., Pena, E. M., Ogden, B., Lim, G. R., Paramastri, Y. A., Zitzow, L. A., Chen, H., Nagarajan, N., Tan, K. S. W., & Malleret, B. (2025). Assessing Zoonotic Risks of Blastocystis Infection in Singapore. Pathogens, 14(8), 773. https://doi.org/10.3390/pathogens14080773

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