Literature Analysis of Blastocystis sp. from 1990 to 2019 in China

Background Blastocystis sp. is ubiquitous presence in animals and humans worldwide and a broad genetically. The aim of this study was find and of in past Methods On the Chinese mainstream database China National Knowledge Infrastructure (CNKI), Wanfang database and PubMed database, the research literatures of Chinese Blastocystis sp. were searched and analyzed. Results 285 articles related to Blastocystis sp. were retrieved, including 244 published in Chinese and 41 English on pathogen morphology, epidemiological investigation, laboratory detection and diagnosis, clinical case reports, drug treatment, animal model establishment as well as pathogenicity studies. There were the most epidemiological investigations and researches on pathogenic morphology decreasing recently Surveys of Blastocystis sp. of human has been carried out among a total of 18 provinces, with infection rates ranging from 0.007–48.6%. Infants and young children, school students, hospitalized diarrhea patients, human immunodeficiency virus (HIV) patients, tuberculosis patients, and cancer patients as respondents had been included. ST1-ST7 and ST12 were the main subtypes in Chinese population. Moreover, surveys of Blastocystis sp. of animal were also conducted in 12 provinces, with infection rates ranging from 2.54–79.41%. A variety of animals were investigated including pigs, cattle, sheep, yak, giant panda, and Crested Ibis et al., with the main subtypes of ST1-ST8, ST10, ST12-ST14. In vitro culture and Polymerase chain reaction (PCR) were commonly used to detect Blastocystis sp. in research and less in clinical application. Traditional Chinese medicine and western medicine for clinical drug treatment appeared curative effects. However, there were few reports on the application of traditional Chinese medicine in clinical treatment. The pathogenicity of Blastocystis sp. remains controversial and needs further exploration.


Classification
In 1993, Jiang et al. proposed a new categorization standard for Blastocystis sp., and believed that Blastocystis sp. should be classified as protozoa, proposing a new subphylum Blastocysta, including class Blastocystea, order Blastocystida, family Blastocystidae and genus Blastocystis, this classification method has been widely recognized by domestic and foreign counterparts [7].

Morphology of Blastocystis sp.
There were 23 research articles on pathogenic morphology, 21 in Chinese and 2 in English. In 1990, He and Jiang et al. [6] published " Light Microscopy and Ultrastructure of Blastocystis Hominis" in Journal of Sun Yat-Sen University, which was the first record of Blastocystis sp. in China and the first observation of the structure of the Blastocystis sp. in vitro culture in China, found the original vacuolar, granular, and amoeboid forms and multiple fission form. Moreover, the granular form can reproduce by binary fission. The multiple fission form was also observed under a scanning electron microscope in 1992 [8]. Multiple fission form was also observed in 2001 [9].
In 1998, Qiao et al. [10] observed the morphology of Blastocystis sp. from patients with diarrhea and found that there were vacuolar, granular and amoeboid forms, of which the vacuolar form accounts for up to 84% and the granular form accounts for 12.8%, amoeboid form only accounts for 3.1%. In 2006, Qiao et al. [11] observed that the Blastocystis sp. have binary fission, endodyogeny, multiple fission and budding.
In 2000, cysts and their photogrammetry were reported in China firstly, which were relatively simple in form, both round or oval [12]. Cyst form was spherical, with two parts, an inner sphere and an outer cyst wall-like structure [13]. Cyst form contained more RNA-like substances under electron microscope [9]. Furthermore, Su et al. [14] observed the morphological characteristics of cysts in different environments and found that the cysts morphology was basically consistent under the light microscopy and the ultrastructure in different culture media, with different sizes and polymorphisms.
In 2007, cyst form in Roswell Park Memorial Institute (RPMI) 1640 medium firstly was observed [15]. In 2012, Xu Zhang et al. [16] observed multivacuolar, vacuolar, and amoeboid form in diarrhea, and found that the vacuolar form can be transformed into a granular form in the RPMI 1640 medium. Trophozoites were observed in different environments and found that the average diameter of vacuolar and granular trophozoites was larger in those with symptoms than in those without symptoms [17]. Moreover, asymptomatic trophozoites own little morphological changes after being cultured. However, after inoculation in the abdominal cavity of mice, the Blastocystis sp. enlarged, which was similar to that of symptomatic ones. The trophozoites were varying in shape and size depending on their living environment.
In China, research on the morphology of Blastocystis sp. has gradually decreased in recent years. 10 articles were published from 1990 to 1999, 10 articles were published from 2000 to 2009, and only 3 articles were published from 2010 to 2019.

Epidemiological investigations
There were 116 epidemiological investigations (88 in Chinese and 28 in English), which accounted for 41% of the research on Blastocystis sp. in China, including 98 population surveys and 18 animals. China has carried out three national human parasite investigations. In the first national human parasite survey from 1988 to 1992, it found that the nationally weighted average infection rate of Blastocystis sp. was 1.473(±0.075)%, with the highest infection rate in Sichuan 8.01% [18]. However, there was no data on Blastocystis sp. in the second national survey of important human parasites in 2001 2004. In the third national survey of key human parasites in 2014 2016, the highest weighted average infection rate was 5.69% in Guizhou, followed by Guangxi 4.52% [19].

Infection of Blastocystis sp. in humans
According to the current investigation of Blastocystis sp., the population infection rate was 0.007-48.6%. In 2000, surveyed the outpatient diarrhea patients in Guangzhou hospitals with the highest infection rate was 48.6% reported by Zhang et al. [20], and the lowest infection rate was 0.007% in the Xinjiang Uygur Autonomous Region, 2015 [21]. The general population, students, children, hospitalized and outpatients, patients with diarrhea, and people with other diseases were contained in the population epidemiological survey.
There were 44 epidemiological surveys of Blastocystis sp. infection in the general population, with 36 in Chinese, and 8 in English, of which 6 were genotyped. The percentage of Blastocystis sp. infection in the population in different regions of China was ranged from 0.007-43.26%. The infection status and subtype distribution of Blastocystis sp. in parts of China has been listed in Table 4. The highest infection rate of human Blastocystis sp. was 43.26% in Bama Yao Autonomous County [22]. The lowest infection rate was 0.007% [21]. With the development of molecular detection methods, PCR method was used to identify the subtype of Blastocystis sp. in China. Yan and others identified that the subtypes of Blastocystis sp. infected by humans was ST1 ST3. ST3 was the main subtype (40.0%), followed by ST1 (37.1%), mixed subtypes of ST1 and ST3 account for 14.3%. This study also detected unknown subtype [23].
The risk factors for Blastocystis sp. infection were diverse. A survey of children aged 7-12 years in the border areas of China and Myanmar found that not washing their hands after using the toilet may be a risk factor for Blastocystis sp. infection [24]. Helicobacter pylori infection and hepatitis B were also risk factors for Blastocystis sp. infection [25]. Drinking unboiled tap water, raising livestock and low immune function were the risk factors of Blastocystis sp. [26]. Female and body mass index < 19 were risk factors for intestinal parasite infection in patients with tuberculosis. Feeding poultry or livestock was important risk factor for Blastocystis sp. infection. Anemia and laboring barefoot in farmland were risk factors for intestinal helminth infection in patients with tuberculosis [27,28].
There were 8 investigations of Blastocystis sp. infections among students, all of which published in Chinese, with prevalence ranging from 1.1-16.61%, 3 of which were tested for subtype. The highest prevalence of students was 16.61% in 2010 grade freshmen admitted to Guilin Medical University [29]. In 1999, the infection rate of a primary school pupil in Nantang Town, Gan County was 1.1% [30], but the infection rate of Blastocystis sp. in Grade 2002 of Gannan Medical College reached 10.09% [31]. The prevalence of Blastocystis sp. was 14.93% among 2008-2010 grades students in Guangxi Medical University [32]. The comparison between these studies shows that it may be that under the management of parents and teachers, primary school students have developed good hygiene habits such as diligent hand washing. However, there are still regional differences, the prevalence of Blastocystis sp. was 1.41% (6/426) in college students in Dali, Yunnan Province, and the infection subtype was ST1 [33]. A study in Guangxi found that infection subtypes include ST1, ST3, ST4, ST6 and ST7, of which ST3 is the main genotype (32.08%) [34].
There were 8 investigations about infection in children of Blastocystis sp., including 7 published in Chinese and 1 in English. However, there was no DNA sequence analysis performed. According to these studies, the prevalence was 0.4-35.9%. The highest prevalence of children with diarrhea was 35.9% in Jiangxi Provincial Children's Hospital [35]. Moreover, the prevalence of children with repeated abdominal pain was 35.4% in the First Affiliated Hospital of Gannan Medical University [36]. The prevalence of children with diarrhea was 3.1% and 8.9% in four sentinel hospitals in Kunming, Yunnan Province and the First Affiliated Hospital of Fujian Medical University, respectively [37,38]. Cao et al performed fecal microscopy on children treated in the outpatient and inpatient departments of Shenzhen Children's Hospital, and found that the infection rate of Blastocystis sp. was 0.4% [39]. Children with diarrhea have a higher infection rate, but the infection rate in Jiangxi is the highest.
There were 11 investigations of Blastocystis sp. infection in inpatients or outpatients, all of which were published in Chinese with 1 sequence analysis. The prevalence was 3.7-36.6%. The highest prevalence in hospitalized patients was 36.6% [40]. The infection rate of patients in the First Affiliated Hospital of Guangxi Medical University is 22.78%, and the infection rate has a significant difference in gender [41]. The prevalence of some patients who attended the five hospitals were 16.27% and 16.77%, respectively in 2005 and 2013, in Nanning, Guangxi [42]. The infection rate of Blastocystis sp. in hospitals in Guangxi region was relatively high, and the infection rate has not changed much over time. It may be that the local population can see the doctor in time after experiencing symptoms such as diarrhea, resulting in a higher rate of infection of Blastocystis sp. in local hospitals. The subtypes of patients with Blastocystis sp. infection in two hospitals in Guangxi are ST1 ST4, ST6 and ST7, of which ST2 is the main genotype (35.09%) [43]. The prevalence of hospitalized patients was 9.47% in Tengchong City, Yunnan Province [44]. From 2014 to 2017, among the samples sent to the National Institute of Parasitic Diseases for testing, the infection rate of Blastocystis sp. was 3.7% [45].
13 (12 published in Chinese and 1 in English) surveys of Blastocystis sp. in patients with diarrhea, one of which did sequence analysis, and the prevalence was 0.42-48.6%. The infection rate of patients with diarrhea in outpatient clinics of hospitals in Guangzhou was 48.6% (84/138), which was the highest infection rate of diarrhea patients. Those diagnosed with Blastocystis sp. infection mainly complained of abdominal pain or diarrhea, 19% of them were associated with colitis, and some showed symptoms of fatigue and anorexia [20]. The lowest prevalence of Blastocystis sp. of patients with diarrhea was 0.42% in Fuzhou area. Blastocystis sp. was one of the main causes of diarrhea in local children. The infection rate of Blastocystis sp. in patients with diarrhea is significantly different in seasons, the highest in summer [46]. Blastocystis sp. was one of the common pathogens in patients with diarrhea. The prevalence was 4.8% in patients with diarrhea over 5 years old in Kunming, Yunnan, and it was highest in summer [47]. But Zhang et al. found that the infection rate of Blastocystis sp. in spring was the highest, followed by summer. In the four sentinel hospitals in Kunming, Yunnan Province the prevalence of diarrhea patients was 4.2%, and the subtypes were ST1 and ST2, of which ST1 occupied the predominate subtype (97.9%) [48]. The infection rate of patients with diarrhea was 18.54% in Guangxi. Most of the patients with Blastocystis sp. have a history of drinking raw water or eating outside for a long time, and some of them have or are raising animals. It was found that the mixed infection of Blastocystis sp. and other parasites accounted for 31.87%, of which 73.8% were co-infected with liver flukes. And patients with mixed liver fluke infection have eaten sashimi [49].
There were 14 investigations of Blastocystis sp. combined with other diseases, 9 published in Chinese and 5 in English. The prevalence was 3.7-20.7%, of which 3 did DNA sequence analysis. These investigations included HIV patients, tuberculosis patients, cancer patients and chronic disease patients co-infection with Blastocystis sp.. There were 8, 2, 2 and 2 surveys respectively. The average infection rate was 12.30% [26,[50][51][52][53][54][55][56], 6.1% [27,28], 13.1% [57,58], 17.8% [59,60], respectively. Among the co-infections, patients with chronic diseases had the highest rate of Blastocystis sp. infection, followed by cancer patients, and tuberculosis patients had the lowest. It has been found that the highest prevalence of Blastocystis sp. among HIV patients was 20.7% in Guangxi Province in 2015 [50]. The lowest prevalence was 3.7% among HIV seropositive individuals in Tengchong, Yunnan. The ST1, ST3, ST4, ST7, and ST12 subtypes were found, among which ST12 infection was first discovered in China. CD4 + cell count ≤ 500 cells/µl, and an HIV-RNA viral load 50 copies/ml were the influencing factors for Blastocystis sp. infection among HIV-seropositive individuals [26]. Tian et al suggested that HIV and Blastocystis sp. co-infection made IL-2 levels higher than those without HIV infection, altered the Th1/Th2 balance, and accelerated the transformation of HIV infection to AIDS [51]. The prevalence of HIV and Blastocystis sp. co-infection was 16.23% (49/302) in rural areas of China [52]. The prevalence of Blastocystis sp. among HIV positives was 19.57% in the suburbs of Fuyang City, Anhui Province [54]. The infection rate of Blastocystis sp. was 6.2% among tuberculosis patients without HIV infection. However, the infection rate was 7.6% in healthy group, there was no difference between the two groups [28]. The infection rate of Blastocystis sp. was 7.1% (27/381) in cancer patients. The subtypes ST1 and ST3 in cancer patients appeared the symptom of diarrhea, of which 66.7% (8/12) was ST1 and 40% (6/15) was ST3. Zhang and others detected Blastocystis sp. in cancer patients: lung, stomach, colorectal, liver, esophagus, breast and hematologic. The infection rate of Blastocystis sp. in the first three cancer patients was relatively high [57]. The infection rate of Blastocystis sp. was 18.29% in chronic disease patients. These patients have abdominal pain, diarrhea, vomiting and other symptoms. Low immune status and poor nutritional status of patients with chronic diseases are the factors that cause Blastocystis sp. infection [60].

Distribution of Blastocysts sp. in Animals
There were 18 investigations of Blastocystis sp. in animals, 5 published in Chinese and 13 in English, of which 13 did gene sequence analysis. Table 5 show the distribution and genetic diversity of Blastocystis sp. in different animals in some cities and province. According to references, nonhuman primates, birds, and mammals can be infected with Blastocystis sp. [61][62][63]. The subtypes of Blastocystis sp. infection animals were ST1 ST8, ST10, as well as ST12 ST14 in China. However, there was no ST9, ST11, ST15 ST17 subtypes found. ST10 was the main subtype on infection Blastocystis sp. in China [64][65][66][67], followed by ST5 [61,68]. Li et al. detected four unknown subtypes and the predominant subtype of sheep infection was ST10. However, only ST1 was observed in goats [66]. Song and others detected that the prevalence of Blastocystis sp. have significant difference in different study sites and age groups [61]. The infection rate of Blastocystis sp. in cats was relatively low. Among the 6 provinces in eastern China, only Blastocystis sp. was detected in Lu'an, Anhui Province [63]. Wang and others detected the infection rate was 9.5%, 8.8% and 5.5% in cattle, pig and sheep, respectively. They found 38.5% of Blastocystis sp. isolates were potential zoonotic subtypes [69]. Insects could also be infected. Body surface and digestive tract of Cockroach and Housefly can be infected with Blastocystis sp. The infection rate of Cockroach's and housefly's digestive tract was 24.76% and 4.35%, respectively [70].

Investigation on the distribution of Blastocystis sp. in different populations and animals
Surveys of Blastocystis sp. of human were conducted in 18 provinces, among which the survey in the general population was conducted in 15 provinces (Fig. 3). Calculate the average infection rate of each province, and the infection rate range is 0.82% 21.59%. The highest prevalence was 21.59% in Guangxi Zhuang Autonomous Region, followed by Hunan Provinces (18.4%), Sichuan Province (8.01%), Yunnan Province (7.82%), Fujian Province  (Fig. 4). The highest prevalence of animal was 79.41% in Guangzhou [71], and the lowest animal prevalence was 2.54% in Liaoning Province [62]. The prevalence of Blastocystis sp. of primates was 79.41% [71], 75.59% [61], and 19.33% [72] in Guangzhou, Shaanxi, and Guilin investigated, respectively. The infection of Blastocystis sp. in birds was investigated in three provinces, Sichuan, Heilongjiang and Shaanxi ( Table 5). Surveys of mammal Blastocystis sp. have conducted in 10 provinces/cities including Qinghai and Shaanxi ( Table 6). The lowest prevalence of the mammal was 2.54% in Liaoning and the highest was 61.74% in Shaanxi Province.  [73]. RPMI1640 medium was found superior to LES medium in the culture of Blastocystis sp.. Vacuolar, granular, multiple fission and cyst-like form can be seen in RPMI1640 medium [74]. Iscove's Modified Dulbecco's Medium (IMDM) was more suitable for the growth and reproduction of Blastocystis sp. than RPMI1640 and LES media. IMDM can be used for long-term culture in vitro [75]. Moreover, modified Locke-egg serum medium culturing (mLES culturing) has a higher detection rate of Blastocystis sp. than the iodine direct smear. Under the same culture conditions, the mLES was simpler, more sensitive than the LES. It was more convenient in the examination of Blastocystis sp. [76]. Xu Zhang and others observed the growth of Blastocystis sp. in three commercially available liquid media (RMRI1640 medium, 199 single-phase medium and Dulbecco's Modified Eagle Medium (DMEM)), and found that these three liquid media have the characteristics of environmental safety, convenient preparation and storage, high sensitivity, and strong specificity. This result was published in the International journal of infectious diseases, in 2012 [77].

Clinical detection of Blastocystis sp.
Direct wet-mount, iodine staining, improved centrifugal sedimentation with washing, and improved centrifugal sedimentation with hydrochloric acid and aether were applied to simultaneously stool samples detection. It was found that the detection rate of the improved centrifugal sedimentation with hydrochloric acid and aether was high and the rate of missed diagnosis was low. Although the detection rate of improved centrifugal sedimentation with washing was high, the operation was tedious and time-consuming. Direct wet-mount and iodine staining have lower detection rate and higher missed diagnosis rate [78]. In 2006, PCR was firstly applied to detect Blastocystis sp. in China. The molecular detection result was consistent with the results of microscopy [79]. After that, there were applications of PCR detection in research and clinical. In 2015, Yu et al. reported that PCR and in vitro culture can be used in the clinical diagnosis of Blastocystis sp. [80].

Clinical case reports and treatment of Blastocystis sp.
There were 19 clinical case reports, 18 published in Chinese and 1 in English. Blastocystis sp. patients have symptoms of abdominal pain, diarrhea, fatigue and abdominal distension [81,82]. 26 children cases for the first time were reported in 1994. Among the 26 cases, the youngest was only 3 months and the oldest was 10 years old [83]. In 1997, was the first to find a feeder infected with Blastocystis sp. in chicken farm was reported and believed that the feeder might be infected due to long-term close contact with poultry [84]. In 1999, Wang et al. reported a patient infection Blastocystis sp. with jam-like stool, which was treated orally and enema with traditional Chinese medicine Pulsatilla chinensis (Bunge) Regel soup. The symptoms of the patient disappeared, but Blastocystis sp. was still found in multiple stool tests [85]. In 2003, a case of amoeba complicated with Blastocystis sp. was also recorded with colon pathological manifestations of mucosal congestion, edema, ulcer and extensive erosion [86]. Furthermore, Liu et al. reported that a 4-month-old infant was infected in 2006. After 20 days of treatment with metronidazole, the infant was cured [87]. Wei-Ping Tai et al. found that 6 ulcerative colitis patients with refractory symptoms co-infective with Blastocystis sp., and showed the bloody purulent stool and some pathological manifestations including diffused edema, hyperemia and hemorrhage in the rectal and sigmoid flexure. After 3 weeks of metronidazole treatment, edema and hyperemia almost completely disappeared. After multiple stool examinations no Blastocystis sp. was found [88]. In 2017, Zhao et al. observed that a patient infection Blastocystis sp. but no abdominal pain, abdominal distension, abdominal distension, nausea and other symptoms [89].
There were 17 papers on drug therapy, 16 papers published in Chinese, and 1 paper in English. Among these articles, the drugs effect on killing Blastocystis sp. in vitro cultures has been recorded in 6 articles, 4 reports on clinical use of traditional Chinese medicine, 3 reports on western medicine and 4 reports on Chinese and western medicine combination therapy. Drugs that have effects on this protozoa include metronidazole, mefloquine, chloramphenicol and oxytetracycline [90], tinidazole and azithromycin [91], Coptis chinensis and Brucea javanica [92], Radix pulsatillae [93], Oregano oil [94], metronidazole plus cimetidine [95], metronidazole combined with streptomycin [96], erythromycin in combination with Radix pulsatillae [97]. Drugs that have a better effect on cultured blastocysts include: oregano oil [94], tinidazole [91]. Drugs that have good therapeutic effects on patients with Blastocystis sp. in clinical practice include: Metronidazole and streptomycin have a good combination effect, and the effective rate was 95.8% [96]. Erythromycin combined with Radix pulsatillae has better effect on Blastocystis sp. patient, the effective rate was 94% [97]. Therefore, it was considered that the combined use of two drugs appeared better effect clinical treatment, including the combination of two western medicines and the combination of Chinese and western medicines.

Animal model establishment and pathogenicity study
There were 25 studies to explore the pathogenicity of Blastocystis sp. by establishing animal models and animal infections, and 9 studies have introduced the relationship between intestinal mucosa and cytokines in patient infection Blastocystis sp..
In 1994, the infected female mice of Kunming species with oral Blastocystis sp. were firstly used to study its pathogenicity in China. After anatomy it was found that the terminal ileum and cecum had hyperemia and edema [98]. In 2003, Yao selected healthy female mice to infect with Blastocystis sp. by orally and rectum. The number of Blastocystis sp. in the jejunum was the largest, followed by the stomach, ileum, colon, rectum, and cecum. The small intestine and colon show pathological manifestations of epithelial cell shedding and inflammatory cell infiltration [99]. In 2005, it was found that the higher detection rate was in the ileocecal and cecum in Kunming female mice [100]. However, there were no obvious intestinal pathological changes in infected Sprague-Dawley (SD) rats. However, Blastocystis sp. was observed in the cecum [101].
Liu et al. infected SD male rats with different doses of Blastocystis sp. trophozoites, and found that the number of mice infected in different dose groups was different after 21 days of infection, of which the highest dose (10 8 ) group was all infected in 2019. The subtypes of the infected mice were all ST7. It was found the protozoa in the ileum, cecum, and colon, but not in the rest of the intestine [102]. In 2005, Yao et al. infected ICR mice by mouth and rectum, and found that by rectum was more susceptible to infection mice than by mouth, which is consistent with the results of their 2003 study [99]. When the immune function of mice was low, the pathogenicity of Blastocystis sp. can be more enhanced, which caused severe intestinal pathological changes [103]. This indicates that Blastocystis sp. infection was related to the immune status of the host. In 2011, it was found that cyst was more susceptible to infection ICR mice [104].
In the pathogenicity study, the intestine of ST1 infected SD rats showed pathological the mucus membrane slough and inflammatory cell infiltration [105]. The levels of interleukin-17 (IL-17) and IL-23 were higher in the intestinal mucosa of infected mice than those in the normal control group. The expression levels of IL-17 and IL-23 were higher in the duodenum and jejunum mucosa, and the expression level of IL-17 was also higher in the colon mucosa [106]. In 2003, Cui et al. observed T-cell subsets and membrane interleukin-2 receptor (mIL-2 receptor) in peripheral blood of patient infection Blastocystis sp. for the first time. T cell subsets and mIL-2 receptors were found to be closely related to the immune function of Blastocystis sp. infection [107]. Moreover, the intestinal epithelial cells of infected patient had a local immune response. Higher levels of IL-8, IL-18 and granulocyte-macrophage colony-stimulating factors were appeared in them. These three cytokines play a certain role in the pathogenesis and increase with the increase of the infection rate [108]. It was consistent with the results by Jin et al. in 2005 [109]. It has also been found that the intestine of patient had varying degrees of hyperemia, edema, erosion, and ulcers. Pathological sections revealed infiltration of eosinophils, and the number of eosinophils infiltration was positively correlated with the degree of Blastocystis sp. infection. IL-5 was one of the important cytokines that cause intestinal mucosal damage, and its level gradually increased with the increase of infiltrating eosinophils [110]. There were 116 articles on the epidemiological investigation in China and 98 articles on the population, of which14 of them were about molecular epidemiological investigations. According to the survey, the subtypes that can infect humans were ST1 ST7, ST12 at present in China [43,53,111], of which ST1 ST3 were the main subtypes [24,34,43,48], including mixed subtypes of ST1 and ST3, ST1 and ST2, and ST2 and ST3 [112]. Foreign studies have found that ST1 ST9 of Blastocystis sp. infect humans [113]. However, it has not been found ST8 and ST9 that can infect humans in China. There were 18 articles on animal infection. The subtypes of Blastocystis sp. infection animals were ST1 ST8, ST10, as well as ST12 ST14 in China. Some foreign studies have found that ST1 ST17 can infect animals [113]. However, there was no ST9, ST11, ST15 ST17 found. ST10 was the main subtype on infection Blastocystis sp. in China [64][65][66][67], followed by ST5 [61,68]. Dogs in the Italian rescue center were infected with ST3 [114]. The subtype infection in the United States was mainly ST8 (20.6%), followed by ST6 (17.3%) and ST5 (15.9%) [115]. Because ST1-ST9 subtypes can infect both humans and animals, there are studies abroad to explore the possibility of the transmission of Blastocystis sp. between people and animals. In Lebanon, the infection rate of Blastocystis sp. in cow was as high as 63.4% in 2019. Among the seven subtypes ST1, ST2, ST3, ST5, ST7, ST10 and ST14 of cow infection, ST10 and ST14 were the main subtypes. ST1, ST2, and ST3 were found in people who had been touched cows, with ST3 as the main subtype in feeders. ST1, ST2 and ST3 were subtypes that infection feeders and cows meanwhile, but only the ST3 sequence was completely identical between the two. It was believed that Blastocystis sp. has a potential risk of transmission from livestock to its contacts [116].

Discussion
There were fewer investigations of Blastocystis sp. infection among students, children, tuberculosis, cancer and patients with chronic diseases. Population and animal infections survey were merely carried out in some provinces. At present, there was no not investigated animals and its feeders at the same time in China, and has not carried out exploratory research on the possibility of zoonoses of Blastocystis sp..
Currently, the detection methods of Blastocystis sp. in China mainly include direct smear microscopy, iodinestained smear microscopy, improved acid ether precipitation method, in vitro culture method and PCR detection method. The microscopic diagnosis method is simple, fast and convenient, but its morphological identification is difficult [39]. The in vitro culture methods, such as RMRI1640 medium, 199 single-phase medium and DMEM medium should be considered for clinical diagnosis (except emergency) and field research application [77]. After the first application of the PCR method in China in 2006, the using frequency was increasing. The PCR detection method partially or completely amplifies The Small Subunit Ribosomal RNA (SSU-rRNA) gene, which was very sensitive to the identification and typing of Blastocystis sp. [117], and this method can also be considered for clinical detection and diagnosis. Until now, there are many applications of PCR methods in China, but few applications of other PCR methods. Foreign countries have used restriction fragment length polymorphism PCR (PCR-RFLP) to investigate animal Blastocystis sp. infections [118], and real-time fluorescence quantitative PCR (RT-PCR) was used to detect the infection status and subtype distribution of animals and their close contacts [116,119].
In China, the infection of Blastocystis sp. is treated with traditional Chinese medicine, western medicine as well as the combination of two drugs. However, the combination of two western medicines and a combination of Chinese and western medicines is more effective. It has been found that traditional Chinese medicines, such as Coptis chinensis, Brucea javanica, Oregano oil, Radix pulsatillae, all of which have effect on Blastocystis sp. in vitro culture media. However, there were few reports on the application of traditional Chinese medicine in clinical treatment, so the application of traditional Chinese medicine should be increased in clinical treatment.
The research on Blastocystis sp. in China includes pathogenic morphology, laboratory testing, epidemiology, pathogenicity, animal models, immunology, molecular biology, clinical case reports and treatment. Particularly, there were a large amount of data and literature accumulated in the field of epidemiological investigation and research. Although the research scope of Blastocystis sp. is extensive, many research filed have been ignored in China.

Conclusions
Although the research on Blastocystis sp in China covered a wide range of fields, the investigation and research on clinical therapeutic drugs, laboratory testing and diagnostic methods, and animal infections were still rare. Furthermore, there were fewer surveys of patients with colon cancer, tuberculosis, and chronic diseases infection Blastocystis sp. and fewer surveys on minority areas. Meanwhile, there were fewer reports on the investigation and research on the possibility and related relationship of the spread of Blastocystis sp. between humans and animals. Therefore, further studies are needed to reveal the possibility of Blastocystis sp. transmission between humans and animals. With the rapid development of the field of intestinal microecology recently, there have been many reports on related research, but there has been no report on the relationship between Blastocystis sp. and intestinal microecology in China. Thence, research in this area should be strengthened.

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Availability of data and materials
All datasets are presented in the main paper. Prevalence of Blastocystis sp. of human in different provinces in China. Note: The designations employed and the presentation of the material on this map do not imply the expression of any opinion whatsoever on the part of Research Square concerning the legal status of any country, territory, city or area or of its authorities, or concerning the delimitation of its frontiers or boundaries. This map has been provided by the authors.

Figure 4
Prevalence of Blastocystis sp. of animal in different provinces in China. Note: The designations employed and the presentation of the material on this map do not imply the expression of any opinion whatsoever on the part of Research Square concerning the legal status of any country, territory, city or area or of its authorities, or concerning the delimitation of its frontiers or boundaries. This map has been provided by the authors.