The Coexistence of Blastocystis spp. in Humans, Animals and Environmental Sources from 2010–2021 in Asia

Simple Summary Blastocystis spp. are unicellular parasites that infect the gastrointestinal tract of humans and animals. Their occurrence in the environment had been detected in water sources, thus causing contamination. The presence of the parasites in humans, animals and environmental sources in Asia were reviewed according to countries in Asia, different categories of human and animal populations, and environmental sources including water samples, food and ambient air. The coexistence of the parasites poses a public health concern as the parasites are commonly found in most studies. Hence, there is a growing interest in the study of Blastocystis spp. Due to the isolation of Blastocystis spp. from living and non-living sources, a collaborative, multisectoral and transdisciplinary approach known as One Health is proposed for future study of Blastocystis spp. in order to achieve optimal health outcomes through the recognition of interconnection between people, animals and their shared environment. Abstract Blastocystis spp. are controversial unicellular protists that inhabit the gastrointestinal tract of humans and a wide range of animals worldwide. This review provides an overview of the prevalence and distribution of Blastocystis spp. and their subtypes throughout Asia. Research articles reporting on the presence of Blastocystis spp. in locations within Asia, between 1 January 2010, and 10 May 2021, were obtained from Scopus, PubMed, and Google Scholar. In 427 articles, the prevalence of Blastocystis spp. in 31 countries within the last decade was revealed. Isolates were found in humans, various mammals, birds, reptiles, insects, water sources, vegetables, and ambient air. Prevalence of Blastocystis spp. varied widely across host categories. Subtypes identified throughout Asia were STs 1–14, and ST18–22 (novel subtypes). ST1, ST2, ST3, ST4 were the most frequently isolated in humans; ST5 in pigs; ST10 and ST14 in goats, sheep, and cattle; and ST6 and ST7 in chickens. ST1 and ST3 were most common in water samples. ST1, ST2, ST3, ST4, ST5 and ST6 were shared by humans, animals, and water sources. There is a growing interest in the study of Blastocystis spp. and their subtypes in Asia. Due to the isolation of Blastocystis spp. from biotic and abiotic sources in Asia, the application of the One Health (OH) approach to the study of Blastocystis spp. is proposed for improved perception of this organism.


Introduction
Blastocystis spp. are anaerobic unicellular eukaryotes that are widespread among humans and animals around the world [1][2][3]. They reside in the gastrointestinal tract wherein their role in gut health and disease is unresolved [4]. Several attempts were made at the classification of Blastocystis spp. by means of physiological and morphological characteristics [5], however, its place as a member of the phylum stramenopiles was revealed by Silberman et al. [6] based on phylogenetic analysis of the small subunit ribosomal rRNA (SSU rRNA) gene. Prevalence and subtypes of Blastocystis spp. in immunocompromised individuals in Asia are summarized in Table 2. This category comprised mostly cancer, HIV/AIDS, and pulmonary tuberculosis patients. Reported prevalence rates were generally not above 30% except 54.8% in immunocompromised children with diarrhea in Indonesia, and 42.2% and 53.6% prevalence in HIV/AIDS cases and pulmonary tuberculosis patients respectively in Uzbekistan. Blastocystis spp. subtypes 1, 2, 3, 4, 5, and 7 were identified.  It is noted that hematologic and non-hematologic (cranial) cancers with Blastocystis spp. infections are most commonly reported in children [82][83][84][85]. Whereas, colorectal, stomach, esophagus and non-gastrointestinal cancer such as lung, liver, breast, ovarian, hematologic and other cancers were detected in adults. Among the 10 studies focused on cancer patients, six studies clearly stated that cancer patients were receiving chemotherapy treatment [82][83][84][85]87,88]. One study recruited cancer patients who have not received any chemotherapy [89]. While the remaining two were classified as follow-up cases [90] and in-or out-patient cases [91], respectively. It is noted that the highest prevalence of Blastocystis spp. infection in cancer patients is detected in those who have not received chemotherapy [89] as compared to the other six studies. This could be due to the existing immunocompromised condition of the cancer patients that allowed an opportunistic infection to occur.
Patients with different gastrointestinal complaints and disorders such as constipation, abdominal pain, diarrhea, irritable bowel syndrome (IBS) and inflammatory bowel disease (IBD) have been examined for Blastocystis spp. infection with positive results recorded as shown in Table 3. The prevalence rate was as low as 0.5%, with the highest being 67.1% and all isolates belonged to Blastocystis spp. subtypes 1, 2, 3, 4, 5, 6, and 7.  The occurrence of Blastocystis spp. in mental rehabilitation centers was documented by several authors from Iran only ( Table 4). Prevalence ranged from 4% to 55.2%; and out of all nine of these studies, only one reported the use of molecular methods wherein ST1, ST3 and ST9 were identified. Studies on the status of Blastocystis spp. infection in hospital in-and out-patients are shown in Table 5. The diseases/illnesses of these patients were, however, not stated in the reports. Nonetheless, they did not show any gastrointestinal-related symptoms and volunteered as healthy participants in the gastrointestinal studies. As a result of their involvement, though asymptomatic, they were detected positive for Blastocystis spp. infection. Infection rate as low as 0.02% was recorded in 23,278 Saudi Arabian patients, while all (100%) of 15 hospital patients without any gastrointestinal complaints were found positive for Blastocystis spp. Asides Blastocystis spp. subtypes 1, 2, and 3 which were the most commonly observed, STs 6 and 7 were also commonly identified while STs 4 and 5 were few.  Table 6 is a summary of Blastocystis spp. infection in students and working populations in Asia between 2010 and 2021. Food handlers and immigrant workers were commonly screened in Iran and Qatar, respectively. In addition to Blastocystis spp. subtypes 1, 2, and 3; ST6 was isolated from chicken slaughterhouse staff in Lebanon [199], and ST5 in pig handlers in Thailand [214].
The majority of the studies on Blastocystis spp. infections in humans in Asia within 2010 and 2021 were on general populations of apparently healthy status; such participants comprised urban dwellers, rural dwellers, and healthy control for immunocompromised persons. As depicted in Table 7, low prevalence rates of less than 5% and rates as high as 50% were reported from the different countries where these studies were undertaken, and various techniques were used for the detection of this protist. Blastocystis spp. subtypes reported were STs1-7 and ST10, whose only record was from Lebanon.   The presence of Blastocystis spp. in various other human categories that do not quite fit into those discussed above is summarized in Table 8.

Blastocystis spp. Infection in Animals
In Asia, Blastocystis spp. infection have been documented in hoofed mammals (Table 9), carnivores (Table 10), non-human primates (NHPs) ( Table 11), birds (Table 12), rodents (Table 13), reptiles (Table 14), insects and some other mammalian groups (Table 15).          The prevalence of Blastocystis spp., reported in the last ten years, varied widely among the ungulates. Infection was mostly reported in livestock animals such as cattle, goats, sheep and pigs. Blastocystis spp. ST10 and ST14 were the most frequently isolated from deer, alpacas, cattle, yaks, sheep and goats, while ST1 and ST5 were the most common in pigs.
Blastocystis spp. has been isolated from carnivores, both domestic and wild, in Asia. Prevalence ranged from 0.6% to 100%, with STs 1-8 and ST10 being identified. NHPs have been commonly described to harbor Blastocystis spp., with a reported prevalence reaching a 100%. Genetic analyses have recognized ST1, ST2, and ST3 as being the most common in this group of mammals. Interestingly, Blastocystis spp. ST9 was isolated from ring-tailed lemur from China [250].
Blastocystis spp. infections in birds have been reported. Prevalence varied widely, however, subtype identification revealed ST6, S7, ST8 as the most frequently isolated. The isolation of Blastocystis spp. ST9 in chicken in Malaysia [15] is peculiar. Diverse genera of rodents have been found as hosts to Blastocystis spp. Although STs 1, 3, 5, 7 and 13 have been reported, ST4 and ST17 were the most frequently identified.
A few studies have reported on the infection of reptiles with Blastocystis spp. with the highest sample size being 19. Prevalence ranged from 26.3% to 100%, no subtype has yet been mentioned. Although studies are still few, cockroaches have been found as hosts to Blastocystis spp. Two out of six studies have described infection to the subtype level, ST2 was identified in China [418] while ST3 was identified in Malaysia [438].
Other animals found as hosts to Blastocystis spp. are the gray kangaroo, red-necked wallaby, sugar glider, rabbit, and hedgehog.

Blastocystis spp. in Food and Environmental Sources
In the past decade, the presence of Blastocystis spp. has been reported in tap water, river water, seawater, wells, fishponds, wastewater, food and even ambient air in Asia. The prevalence rate ranged from 2.1% to 100% in the various water sources, and 2.8% to 10.2% in leafy vegetables (Table 16). The only study on Blastocystis spp. in ambient air reported a prevalence of 1.4%. Blastocystis spp. subtype identification is only available for water sources. STs 1, 2, 3, 4, 6, 8, 10 have so far been recorded from water samples; and although the prevalence of ST3 was highest, ST1 was the most widespread subtype.

Distribution of Blastocystis spp. by Country
From 2010 till now, the identification of Blastocystis spp. has been described for a total of 31 Asian countries. Out of these 31, genetic characterization and Blastocystis spp. subtype identification was available for 22 countries. Figure 1 reveals the distribution of the subtypes of Blastocystis spp. in these countries with a glimpse of subtypes shared by humans, animals, and water sources. Blastocystis spp. ST1 was the most widespread subtype, found in all of the 22 countries.

Discussion
Blastocystis spp. have been reported in over 50% of the countries in the continent of Asia. Although the most documented hosts to infection were humans and several animal species, this organism has also been detected in water sources, vegetables, and ambient air.
Variation of prevalence rates was seen within and between the various human host categories. Although authors have described both significant and insignificant differences between Blastocystis spp. infection in patients with and without known disease conditions, this variation could be a result of the methods employed in the detection of Blastocystis spp. Blastocystis spp. STs 1-7 have been identified in humans in Asia. ST1, ST2, ST3 and ST4 were more widespread and more frequently isolated than ST5, ST6 and ST7. This finding is in agreement with studies from other parts of the world [2,13,452,453].
The isolation of Blastocystis spp. STs 1-14, and ST18-22 (novel subtypes) were reported in animal hosts. ST1, ST2, ST3, ST4, ST5, ST6 and ST7 were found common to humans and animals. ST9 was observed in ring-tailed lemurs and chickens in China [250] and Malaysia [15] respectively; however, no article included in this review reported on the identification of ST9 in humans in these countries. The characteristic presence of ST5 in pigs, ST10 and ST14 in goats, sheep and cattle, and ST6 and ST7 in chickens underscore suggestions that these STs are specific to the respective animal hosts. Also, reports of isolation of ST5 in pig handlers [214] and ST6 in chicken slaughterhouse staff [199] are pointers to possible zoonotic transmission.
Where stated, cysts were the Blastocystis spp. forms observed in vegetables and water samples. The presence of cysts in the life cycle of Blastocystis spp. enable their existence outside of human and animal hosts; also, the chloroform-resistant nature of these cysts probably explains the presence of Blastocystis spp. even in treated water.

Conclusions
The growing interest in the study of Blastocystis spp. as an area of research is very obvious and fundamental to unraveling the much that is hitherto unknown of the epidemiology, biology and pathogenicity of this protist. Blastocystis spp. have been isolated from biotic and abiotic sources in Asia. Considering that humans and animals are in constant interactions with their environment, epidemiological studies of Blastocystis spp. from an ecological perspective are essential. In essence, continuous surveillance of human and animal hosts alongside their food and water sources and other possible sources of infection such as soil across different geographical locations and climatic conditions is needed. The use of molecular detection methods in epidemiological studies are recommended to provide information on Blastocystis spp. STs in as many regions as possible. Incorporating the One Health (OH) method into epidemiological studies will equip researchers and other stakeholders with information on the possible influence of ecosystems on Blastocystis spp., it will further elucidate transmission routes and provide clues required to break the transmission of this protist successfully. Morphological studies of Blastocystis spp. in various host species and environmental sources are insufficient but essential; electron microscopy could help to accentuate structural details of isolates from various hosts and the differences or similarities between them, and contribute to the understanding of a proper, more detailed Blastocystis spp. lifecycle.  Data Availability Statement: All data generated or analyzed during this study are included in this published article.

Conflicts of Interest:
The authors declare no conflict of interest.