Blastocystis is an intestinal parasite commonly found in humans and is estimated to have infected over one billion people worldwide (Kühnelová et al., 2025). Although the association between Blastocystis sp. and gastrointestinal disease is uncertain, infection with Blastocystis can cause severe illness, including diarrhea, in both immunocompromised and immunocompetent individuals (Pawelec-Pęciak et al., 2025; Popruk et al., 2021). Nonetheless, recent research indicates that Blastocystis is not a pathogen but rather a gut inhabitant that may even be beneficial (Deng and Tan, 2025). Evidence supports its links to improved cardiometabolic markers and suggests potential protective effects against intestinal inflammation (Piperni et al., 2024). Surveillance of diverse hosts and populations, particularly those displaying symptoms of diarrhea, offers critical insights into the pathogenesis of Blastocystis and its potential therapeutic applications. Meanwhile, research on Blastocystis ought to be specifically focused on genetic characterization, with the objective of discerning host specificity and promoting the discovery of potentially pathogenic variants.

PCR with Sanger sequencing has been extensively used for epidemiological surveillance; this method can identify subtypes of Blastocystis (Stensvold and Clark, 2016). To date, at least 44 subtypes have been acknowledged through sequence and phylogenetic analysis of the small ribosomal subunit RNA (SSU rRNA) (Santin et al., 2024). Although ST1 to ST4 represent the most prevalent subtypes detected in humans, ST5 to ST9, ST12 to ST14, ST16, ST35, and ST41 have also been identified within the human population (Hernández-Castro et al., 2023; Zhao et al., 2024). The extensive genetic diversity of Blastocystis may be the primary reason for the discrepancy in pathogenicity, as different lineages exhibit varied ecological roles within the host gut (Deng and Tan, 2025). Research findings have indicated that ST4 serves as a beneficial commensal microorganism, and its colonization can confer protection against colitis. In contrast, infection with ST7 might represent a potential risk factor for the onset of experimentally induced colitis (Deng et al., 2022, 2023). Therefore, conducting research on the subtype distribution of Blastocystis in symptomatic patients is of utmost significance for achieving an in - depth comprehension of its genetic variation and functional pathogenicity.

In China, the initial report of Blastocystis infection involved two children suffering from chronic diarrhea in Guangdong Province. Subsequently, over 12 provinces and municipalities in China reported cases of Blastocystis infection, primarily affecting children, diarrhea patients, immunocompromised individuals, and cancer patients (Table 1). Despite this data, the majority of studies conducted before 2019 utilized morphological or cultural methods, lacking molecular typing data (Deng et al., 2019). Consequently, our understanding of the pathogenicity and transmission patterns, as well as the routes of transmission, of Blastocystis is limited or insufficient.

Zhuhai, a city in Guangdong Province, China (Figure 1), is distinguished by a dense network of rivers and lakes. As the city with the largest marine area, the greatest number of islands, and the longest coastline in the Pearl River Delta region, it boasts a thriving tourism industry and high population mobility. The warm and humid climate further facilitates the dissemination of intestinal protozoa. However, molecular data on Blastocystis infection in the Zhuhai population are still lacking. Therefore, this study employs genotype typing technology to detect Blastocystis in diarrhea patients in Zhuhai, with the aim of comprehending the prevalence and genetic subtypes of Blastocystis in the region. This provides fundamental data for the in - depth exploration of Blastocystis’s epidemiology and prevention, as well as its pathogenic mechanisms and their association with public health.

In the present study, the mean infection rate of Blastocystis in surveyed diarrhea patients was 4.9%, consistent with previously reported rates in diarrhea patients from Yunnan, China (4.8%) and in Qatari children with diarrhea (4.7%) (Zhang et al., 2016; Boughattas et al., 2017). However, it was lower than the rates in patients with diarrhea in Indonesia (5.7%), Korea (11.6%), Iran (67.1%), and Turkey (5.9%), and higher than in patients with diarrhea in Singapore (1.0%) and children with diarrhea in Nepal (0.9%) (Maçin et al., 2016; Kwon et al., 2024; Oyofo et al., 2002; Koltas et al., 2017; Dahal et al., 2017; Feurle et al., 2021). Of the 24 molecular survey studies conducted in China, only six reported infection rates in a specific region that were lower than those found in this study, while the other 18 studies indicated infection rates higher than those in this study (Table 1). Certainly, when comparing these datasets, it is essential to account for variations among the survey subjects, including those from HIV-positive populations, cancer patients, children, healthy individuals, and those with other medical conditions. For instance, our previous survey found that the prevalence rate of Blastocystis spp. in diarrheic children in Zhejiang (8.8%) was significantly higher than that in asymptomatic children (2.0%) (Zhao et al., 2024). Overall, the population in China exhibits a low prevalence of Blastocystis, which may be associated with drinking habits. Individuals in China frequently consume boiled water and seldom drink untreated water, potentially decreasing the likelihood of ingesting infectious-stage Blastocystis and thus lowering the infection rate. Previous studies have indicated that in a mountain village in Yunnan Province, China, Blastocystis infection was associated with drinking unboiled water (Deng et al., 2020). Meanwhile, outbreaks of human Blastocystis infection were observed in China, resulting in over 1,122 cases of diarrhea confirmed after consuming contaminated drinking water (Wu et al., 2000). Given the high prevalence of Blastocystis in China’s animal populations, its frequent detection in aquatic environments, and its presence on the surfaces of vegetables and fruits, the population remains at significant risk (Deng et al., 2019; Li et al., 2020). Continuous monitoring of Blastocystis prevalence across different demographics is essential to better understand its pathogenicity and its relationship with public health.

Similar to some opportunistic pathogens, Blastocystis prevalence may be influenced by factors like host immunity, age, occupation, and climate. Detection methods are crucial for detection rates. Molecular detection techniques are more sensitive and specific than morphological examination and culture methods (Tan, 2008). In North Cyprus, Blastocystis prevalence in humans was 10.5%, 10.5%, and 27.8% by direct microscopy, trichrome method, and PCR respectively (Seyer et al., 2017). In a study of school - aged children in Colombia, it was 25.19% by microscopy and 39.22% by PCR (Villamizar et al., 2019). However, molecular techniques such as PCR can be inhibited by various factors; for instance, PCR inhibition in fecal DNA samples can lead to false-negative results. Additionally, primer pairs may produce nonspecific amplicons, particularly when screening directly extracted fecal DNA (Clark et al., 2013). For example, PCR using low-specificity eukaryotic primers may preferentially amplify shorter and more abundant SSU rDNA from co-infecting or co-colonizing organisms present in the intestine. Therefore, it is necessary to develop standard epidemiological methods for detecting Blastocystis (Tan, 2008). It is recommended to use Real-Time PCR in investigations, as it not only screens samples for positivity but also assays to estimate Blastocystis infection intensity in healthy carriers and patients with gastrointestinal issues, which would clarify the potential link between parasite intensity and symptoms (Stensvold et al., 2012).

Blastocystis infection is commonly observed in children. For instance, Gong et al.’s survey on the population at the China-Myanmar border revealed that the prevalence of Blastocystis in children (12.37%) is significantly higher than in adults (4.95%) (Gong et al., 2019). However, contrary research results have also been described, indicating that the colonization of Blastocystis is linearly related to age, meaning that the infection rate of Blastocystis increases with age. It could be hypothesised that Blastocystis is dependent on a more mature (diverse) microbiota in order to be able to establish (Stensvold, 2025). The current study indicates that the infection rate among adults exceeds that among children, although the difference is not statistically significant. However, this research discovered that females have a significantly higher infection rate (8.1%) compared to males (2.9%). In numerous households, women predominantly care for pets, which increases their exposure to potentially pathogen-carrying animals. Additionally, they may be more engaged in household chores that expose them to infection sources, such as cleaning areas frequented by animals or handling animal waste. These activities may result in females being more susceptible to infections than males. At the same time, due to the relatively small sample size and the number of positive cases, a definitive conclusion regarding the correlation with age or gender cannot yet be drawn. Consequently, further research is required to elucidate the relationship between age, gender, and Blastocystis infection.

It is generally believed that the infection rate in rural populations is higher than in urban populations because the prevalence of Blastocystis may be closely related to living environment and sanitation conditions, and the sanitary environment in cities is undoubtedly superior to that in rural areas (Deng et al., 2020). However, in some parts of China, the sanitary environment in rural areas has been widely improved, which may directly affect the prevalence of some parasitic diseases. In any case, the detection of Blastocystis prevalence in the population with diarrhea in this study suggests that these patients should be taken seriously. On the one hand, these patients require improvement in their clinical symptoms; on the other hand, they may become sources of infection, leading to infections in other populations and animals, thereby facilitating the spread of Blastocystis.

The increasing utilization of PCR-based methods has, likewise, expanded the comprehension of the genetic diversity and transmission of Blastocystis spp. Over the past decade, research on Blastocystis spp. at the subtype level has conspicuously intensified (Stensvold, 2025). Among the 44 subtypes identified, ST1 to ST4 are commonly found in humans (Zhao et al., 2024). In the current study, three known STs (ST1, ST2, and ST3) were detected, with ST3 being the most prevalent, followed by ST1. This finding aligns with studies conducted in other parts of the world, where subtype ST3 accounts for at least 44% of all human cases, and ST1 is the second-most predominant, causing over 27% of human cases (Popruk et al., 2021). In the present study, the proportion of ST2 was relatively low, with only one patient testing positive for ST2. This subtype has a relatively high proportion in populations of African, American, and European countries, while it has a relatively small proportion in Asian populations (Popruk et al., 2021). In China, except for the Hebei region, where ST2 was reported to be in absolute dominance, the proportion of the ST2 subtype was small in other regions (Ma et al., 2020). For example, in Hainan Province, among 144 reported human cases, only one case was ST2, and ST2 was not found in Chongqing, Guangxi, Guizhou, etc (Table 1). This indicates that the transmission of Blastocystis subtypes may exhibit regional prevalence. Additionally, in China, an increasing number of rare ST subtypes of Blastocystis have been identified in humans. For instance, two studies in Chongqing reported a high proportion of ST6 and ST7, and a study in Heilongjiang showed that only ST5 was present in the population; ST14 was also identified in HIV - infected individuals from Heilongjiang (Table 1). This may suggest that the mode of human infection may be shifting from human - to - human transmission to animal - to - human transmission, or that the animal - to - human transmission mode is increasing.

In Guangdong Province, the study area, documented cases of Blastocystis infection have been reported in various animals, including black goats, chickens, pigs, farmed bamboo rats, and dogs (Yu et al., 2023; Liu et al., 2022; Song et al., 2021; Zou et al., 2021; Liao et al., 2020). Subtype analysis indicates that farm animals predominantly carry ST1 to ST4 subtypes, which are less likely to infect humans. Conversely, the ST3 subtype is predominant among companion animals like pet dogs (Liao et al., 2020). While there is no direct evidence indicating that human infection with Blastocystis comes from pet dogs, the discovery of the ST3 subtype in both humans and dogs within the same region underscores the potential for transmission between the two. Therefore, this study suggests that the prevalent Blastocystis in surveyed diarrhea patients may not only spread through human-to-human transmission but also through pet-to-human and human-to-pet transmission routes.

In fact, an increasing number of infection studies indicate that contaminated water serves as a significant source of Blastocystis infections (Attah et al., 2023). Previous research in Yunnan Province of China found that consuming raw aquatic plants is associated with ST1 infections, while drinking unboiled water correlates with ST3 infections (Li et al., 2007b). Although specific data on Blastocystis contamination in Guangdong Province remains unavailable, other studies have confirmed the presence of Blastocystis in tap water. For instance, Blastocystis ST3 has been detected in tap water in northern Thailand (Jinatham et al., 2022). Therefore, the next step involves conducting a detection of Blastocystis in the water bodies of Guangdong Province to better infer and assess the possible sources of human Blastocystis infection.

This study presents a preliminary exploration of the prevalence and genetic characteristics of Blastocystis in patients with diarrhea in Zhuhai, Guangdong Province, China. The results show an average infection rate of 4.9% in the investigated population. The infection rate is significantly higher in females than males, but there are no significant differences among age groups and residential areas. The identification of zoonotic ST1, ST2, and ST3 suggests the potential role of diarrhea patients in transmitting Blastocystis to both humans and animals. Although there is a lack of local farm animal infection data for ST1, ST2, and ST3, the high prevalence of ST3 in pet dogs indicates potential cross-transmission between humans and pets. Meanwhile, these patients experiencing diarrhea should be given attention to identify and address the underlying causes of the disease, thereby improving their health.