Current Views on the Clinical Relevance of Blastocystis spp.
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- Tan, K.S.W., Mirza, H., Teo, J.D.W. et al. Curr Infect Dis Rep (2010) 12: 28. doi:10.1007/s11908-009-0073-8
Blastocystis is an enteric protistan parasite of uncertain clinical relevance. Recent studies indicate that the parasite is a species complex and humans are potentially hosts to nine Blastocystis subtypes, most of which are zoonotic. Subtype 3 is the most common in prevalence studies, followed by subtype 1. Laboratory diagnosis is challenging; the currently recommended diagnostic approach is trichrome staining of direct smears coupled with stool culture. Polymerase chain reaction testing from stools or culture is useful for determining Blastocystis subtype information. The controversial pathogenesis of Blastocystis is attributed to subtype variations in virulence; although current studies seem to support this idea, evidence suggests other factors also contribute to the clinical outcome of the infection. Clinical signs and symptoms of blastocystosis include abdominal pain, diarrhea, bloating, and flatulence. Extraintestinal manifestations, predominantly cutaneous, also were reported. In vitro and animal studies shed new light on the pathobiology of Blastocystis.
KeywordsBlastocystis Pathogenesis Subtypes Diagnosis Prevalence
Pathogenicity of Blastocystis subtypes: a tentative list
Primates, pigs, cattle, birds
Associated with symptomatic infections; pathologic outcomes in experimentally-infected rats
Eroglu et al. 
Mainly primates, occasionally birds and pigs
Generally associated with asymptomatic infections
Primates, pigs, cattle
Generally associated with asymptomatic infections
Associated with symptomatic infections; exhibits cytopathic effects in parasite-exposed tissue cultures; inflammatory response in parasite-exposed tissue cultures and experimentally infected rats
Associated with symptomatic infections; proteases are putative virulence factors
Human infections with Blastocystis are called “blastocystosis.” A variety of signs and symptoms are associated with blastocystosis, ranging from nonspecific intestinal symptoms to cutaneous disorders.
Blastocystosis most commonly presents with diarrhea and abdominal pain [1••, 2]. Other nonspecific gastrointestinal symptoms (eg, flatulence, bloating, anorexia, weight loss, nausea, vomiting, constipation, and dysentery) may be associated with the infection . Severity is reported to range from mild and chronic diarrhea to acute gastroenteritis [1••]. Two recent reports were suggestive of invasive Blastocystis infections [7, 8]. However, a coinfection with Entamoeba histolytica in the first case, and an invasive adenocarcinoma in the second, probably contributed to the perforation of the intestinal lining, allowing extraintestinal dissemination of Blastocystis.
Several studies associate the severity of disease with parasite density. The presence of greater than five parasites per high-power field (× 400) for wet mounts or under oil immersion (× 1,000) in permanent-stained smears is frequently associated with an acute presentation of gastrointestinal symptoms [9, 10]. Recent studies focus on the question of whether pathology is subtype-dependent, without information on infection density [2, 3, 4, 5]. Because limited experimental data are available to rule out the association of parasite density with symptoms, it is important that future studies investigate if Blastocystis-induced pathology may be both parasite density- and subtype-dependent.
Accumulating data suggest a correlation between Blastocystis and cutaneous lesions, particularly urticaria . Case reports suggest a causal link between Blastocystis and acute or chronic urticaria, delayed-pressure urticaria, angioedema, and palmoplantar pruritus [1••]. Treatment of the parasite leads to resolution of both the infection and the cutaneous lesions. These cutaneous manifestations are likely immune-mediated, although the mechanism is unclear.
The formol ether concentration technique (FECT), which is commonly used to detect parasite ova and cysts, is not recommended for laboratory identification of Blastocystis because of extremely poor sensitivity [12•, 13]. This shortcoming is attributed to the inability of FECT to isolate Blastocystis spp subtype 3 [12•, 14], the most common subtype in humans. The use of FECT as the sole method of parasite isolation probably contributes to the underestimation of parasite burden in epidemiologic studies. For parasite detection, trichrome-stained fecal smears or short-term xenic in vitro culture (XIVC) offer the best sensitivity. When performing XIVC, it should be noted that slower-growing subtypes (eg, subtype 7) [15•] and mixed infections containing slower- and faster-growing subtypes may be missed. Emerging data suggest that pathogenesis is subtype-dependent; therefore, reference laboratories particularly should also determine which subtype is present in the specimen. This determination may be achieved by sequence analysis of Blastocystis-specific polymerase chain reaction (PCR) products  or with the use of subtype-specific diagnostic PCR primers . Multiple stool specimens should be examined because the parasite may exhibit irregular shedding . Recent surveys indicate that 20% to 30% of stool samples contain the fecal cyst form of the parasite [13, 14]. Hence, despite challenges in identification because of their small size and morphology distinct from vacuolar forms, laboratory staff should also be trained to identify this parasite stage. Blastocystis continues to be one of the more difficult enteric parasites to identify in clinical samples. In a recent study comparing the diagnostic performance of various European reference laboratories in diagnosing intestinal parasites, laboratories revealed poorest agreement in reporting Blastocystis-positive specimens [19•].
Serologic approaches provide rapid, sensitive, and quantitative detection of microbe-specific antibodies. Only a handful of reports have used this approach on Blastocystis. Some studies using the enzyme-linked immunosorbent assay report a correlation between antibody titers and symptoms [20, 21]. If a serologic method of diagnosis were to be established, the genetic and hence antigenic diversity of the parasite must be taken into consideration. Additionally, a panel of monoclonal antibodies against specific subtypes would be an enormously powerful tool to detect the parasite from stool specimens.
Epidemiologic Studies and Laboratory Detection Methods
A major hindrance in determining the pathogenic potential of Blastocystis is the ambiguity in reports of its prevalence. Given the variations in sensitivity of different diagnostic methods, the choice of technique used to identify Blastocystis in stool samples plays a pivotal role in the overall outcome of a particular study. Despite these observations, authors often fail to provide details on the diagnostic methods used in such studies. The popular use of inefficient techniques (eg, FECT) results in underestimation of Blastocystis-positive samples and an incorrect assignment of individuals to case and control groups. Future epidemiologic studies should incorporate data for which Blastocystis was detected using optimal methods. In addition, subtype information should be included.
An often overlooked aspect of Blastocystis biology is the differences in doubling time between different subtypes [15•]. Subtype 7 parasites have dramatically longer doubling times, about 50 to 80 h when cultured in vitro at 37°C, whereas subtype 4 parasites double every 20 to 30 h [15•]. Laboratory culture (XIVC) of stool samples might under-represent such subtypes in epidemiologic surveys compared to faster growing subtypes. Additionally, XIVC of mixed infections was shown to preferentially amplify the growth of one subtype over the other, resulting in underestimation of mixed infections in prevalence studies [22, 23]. Therefore, accurate identification of subtypes and precise estimation of mixed infections requires that Blastocystis DNA genotyping be performed directly from stool samples.
A lack of prevalence data in the past seriously hampered our understanding of Blastocystis infection. In recent years, and despite the discrepancy in diagnostic methods used, interest in Blastocystis epidemiology has been renewed [1••]. The recent prevalence studies of Blastocystis in recent years have significantly increased our understanding of the parasite’s geographic and demographic distribution, mode of transmission, risk factors, and pathobiology.
Genotyping studies have highlighted the diversity in Blastocystis in humans and other animal hosts [22, 24•]. Yoshikawa et al.  investigated the Blastocystis subtype distribution among isolates from Bangladesh, Germany, Japan, Pakistan, and Thailand and found that, apart from Thailand, where subtype 1 was the most common subtype, subtype 3 is the most frequently isolated subtype from humans, followed by either subtype 1 or 6. Other prevalence studies in a wide range of geographic locations support the observation that subtype 3, followed by subtype 1, predominates in human infections [1••]. This finding suggests that subtype distribution does not vary much between human populations from different geographic locations. Other genotypes have also been isolated from humans in surveys but at lesser frequencies. In decreasing frequency, these are subtypes 2, 4, 6, 7, 8, and 9 [24•]. Most surveys suggest that each infected individual harbors a particular Blastocystis subtype, but mixed infections—generally involving subtype 1/3 and subtype 1/2 combinations—are reported occasionally [1••]. Accumulating studies are shedding light on the possibility that pathogenesis is subtype-dependent [2, 3, 4, 5]. Collectively, the data suggest that pathogenic strains belong to subtypes 1, 4, and 7 [2, 3, 4, 5], whereas subtypes 2 and 3 [3, 5] are likely to be nonpathogenic (Table 1).
Mode of Transmission
Numerous studies have shown Blastocystis infections are associated with poor hygiene practices, exposure to animals, and consumption of contaminated food or water [1••]. These studies indicate that, like other enteric parasites, the mode of transmission is via the fecal-oral route. Studies implicating water as a source of infection support the idea that the water-resistant fecal cyst is the transmissible form of the parasite .
Irritable Bowel Syndrome
Irritable bowel syndrome (IBS) is a functional gastrointestinal disorder in which abdominal pain is associated with a defect or a change in bowel habits. Accumulating studies report higher incidence of Blastocystis infections in IBS patients compared with healthy controls [2, 27, 28], although others have shown a lack of association between blastocystosis and IBS . Stark et al.  proposed that low-grade inflammation through persistent antigenic exposure in a chronic Blastocystis infection might be a possible mechanism. Current studies do not provide a clear role for Blastocystis as an etiologic agent of IBS, because the altered intestinal environment of IBS patients may provide conditions favorable for Blastocystis growth. Studies showing eradication of infection concomitant with resolution of symptoms in Blastocystis-infected IBS patients are therefore necessary to clarify this uncertainty. Because persistent, low-grade inflammation was suggested to play a role in IBS, animal infection and in vitro immunologic studies may also provide clues about the role of Blastocystis in IBS.
Several case reports provide compelling evidence that Blastocystis infections give rise to cutaneous disorders, primarily urticaria [1••]. Recently, a specific association was found between acute urticaria and the amoeboid forms of Blastocystis sp subtype 3 . The authors suggested several possible modes through which Blastocystis may give rise to such cutaneous symptoms, namely by disrupting immune homeostasis as the host mounts an inflammatory response against the amoeboid forms of Blastocystis. Possible antigens might include carbohydrates found on its surface coat, leading to inflammatory cell recruitment, which release histamine-activating factors that prime mast cells and basophils. IgE might also be a possible mediator in urticaria through mast cell activation and degranulation, and subsequent release of inflammatory mediators. However, the mechanism by which a Blastocystis infection gives rise to such symptoms remains to be elucidated. The identification of Blastocystis-specific IgE in patients with urticaria would be useful in defining the mechanism by which the parasite causes these lesions.
Several recent surveys suggest that certain populations may be more susceptible to Blastocystis infections.
Patients with HIV/AIDS
Patients infected with HIV have a higher incidence of parasitic infections, and such infections pose a higher risk of disseminated disease compared with healthy populations. Studies often report Blastocystis to be the most commonly isolated protozoan parasite from HIV/AIDS patients . Diarrhea is the most common presenting symptom in such cases. The authors of a recent study investigating the prevalence of Blastocystis subtypes in HIV and cancer patients suggested that subtype 3 was pathogenic because it was the most frequently isolated subtype in both groups . However, the subtype distributions were typical of most surveys in healthy populations. The lack of a control group and the failure to recognize that subtype 4 was five to six times overrepresented in their study, compared with the average of 16 other major studies [24•], indicates that conclusions reported in this study resulted from misinterpretation of data.
Patients with Cancer
Blastocystis is one of the most frequent parasites isolated from cancer patients . The most common symptoms in these patients are abdominal pain, diarrhea, and flatulence . One study reported an overgrowth of Blastocystis after carcinoma-induced intestinal obstruction . A recent case study described Blastocystis in abdominal fluid of a patient with adenocarcinoma and associated bowel perforation .
Children are highly susceptible to Blastocystis infections . A high incidence of Blastocystis infections was observed in immunocompetent  and immunocompromised  minors. Children receiving corticosteroid therapy for nephrotic syndrome are reported to be susceptible to Blastocystis infections .
Exposure to Animals and Animal Products
Most studies advocating a lack of association between Blastocystis infections and intestinal disease focus on the distribution of the parasite between asymptomatic and symptomatic groups. They indicate either no significant difference in the prevalence of the parasite between the two groups or a higher incidence in the asymptomatic group. Such studies wrongfully assume that Blastocystis infections are biologically and pathogenically homogeneous. Clinical outcomes of parasitic infections are multifactorial and are influenced by diverse host and parasite factors; therefore, such studies may not truly reflect the pathogenic potential of the parasite [1••].
Results of placebo-controlled treatment trials involving symptomatic patients infected solely with Blastocystis would better reflect Blastocystis pathogenic potential. Only two such reports have been published; both concluded that chemotherapy successfully eradicated the parasite with concomitant resolution of symptoms [36, 37]. One of these studies used metronidazole and reported clinical cure in 88% of the patients in the treatment group with parasitologic clearance in 80% of cases . Similar outcomes were observed in studies using nitazoxanide as a chemotherapeutic agent . However, symptoms persisted in patients in whom Blastocystis infection could not be eradicated, suggesting that these patients were harboring subtypes that are resistant or less responsive to the antiprotozoal agents used. Resolution of symptoms with concomitant parasite clearance in symptomatic patients with Blastocystis infection provides compelling evidence for the parasite’s pathogenic potential. However, the outcomes of such studies are influenced by several factors. The drugs used in these studies are all broad-spectrum antibiotics, and clinical cure thus could be attributed to the clearance of some unidentified enteric pathogen or to lack of comprehensive exclusion of other possible enteric pathogens, especially viruses or bacterial toxins.
Animal Infection Studies
A major obstacle in establishing Blastocystis pathogenic potential is the absence of an appropriate animal infection model. Several experimental infection studies involving rats, mice, guinea pigs, and chickens were reported [1••]. Animal surveys have reported that laboratory mice generally do not harbor Blastocystis, suggesting that they are not suitable animal models for Blastocystis infections. Additionally, experimental infections in mice are generally mild and self-limiting. Rats and domestic fowl, particularly chickens, are often infected with the parasite. Therefore, animal infection studies in recent years have focused particularly on rats and chickens as potential animal models for Blastocystis infections. More recently, the infectivity of various zoonotic Blastocystis genotypes from humans was tested in rats and chickens . Variability was observed in the infectivity of rodent subtype 4 and avian subtype 6 isolates. Curiously, isolates of subtype 3—the most common subtype found in humans—could not infect chickens and rats, whereas avian subtype 7 isolates could only infect chickens, suggesting that these subtypes exhibit some level of host specificity. In another study, Blastocystis isolates from symptomatic and asymptomatic individuals were used to experimentally infect rats [39•]. Isolates from symptomatic patients induced moderate to severe pathologic changes in infected rats, whereas parasites from asymptomatic individuals induced only mild pathology. Authors concluded that the subtype 1 isolate, which induced mortality in 25% of rats, was pathogenic, and that pathogenic potential for subtypes 3 and 4 was variable. A recent Blastocystis animal infection study, using rats as hosts, reported up-regulation of proinflammatory cytokine interferon-γ, interleukin (IL)-12, and tumor necrosis factor-α 2 to 3 weeks after infection . Despite significant up-regulation of proinflammatory cytokines, local tissue pathology was moderate and the authors suggested that the subtype 4 isolate used in their study was a weak pathogen.
The results of recent animal infection studies are encouraging. However, the observation that rodents do not naturally harbor subtypes other than subtype 4, whereas birds harbor mostly subtypes 6 and 7, may limit the usefulness of these animals in infection studies [24•]. The lack of pathology might therefore reflect resistance to colonization in rats or chickens rather than avirulence of the parasite. In fact, humans harbor the greatest diversity of subtypes, and would perhaps make the ideal host for host–pathogen interaction studies.
Cellular and Molecular Basis for Virulence
Blastocystis exerts immunologic effects on cultured colonic epithelial cells. These observations may provide insights into the roles Blastocystis may play in relation to host-pathogen interactions and its influence on other microorganisms present in the intestines. Long et al.  observed that Blastocystis significantly increase IL-8 and granulocyte-macrophage colony-stimulating factor levels after 24 h. At 6 h, IL-8 levels did not increase and were reduced in the presence of the bacteria Escherichia coli and the bacterial endotoxin lipopolysaccharide. The authors suggested that Blastocystis down-regulates the host immune responses in the early phase of the infection to improve its survival, which would indirectly facilitate the progress of infection by other opportunistic pathogens.
It has been suggested that Blastocystis cysteine proteases are virulence factors. Puthia et al.  observed that cell lysates and secretory products of Blastocystis subtypes 4 and 7 contain IgA-degrading protease, suggesting that Blastocystis proteases may play a role in parasite survival in vivo. Recently, cysteine proteases from Blastocystis subtype 4 were observed to induce IL-8 production from human colonic epithelial cells in a nuclear factor-κB-dependent manner, showing for the first time a specific proinflammatory role for the Blastocystis cysteine proteases . We recently cloned and characterized legumain, an unusual cysteine protease found on the surface of Blastocystis spp subtype 7  and are presently investigating its role as a virulence factor. Considering emerging evidence for subtype-dependent virulence in Blastocystis, it is crucial that future studies focus on the roles of different subtypes in host tissue inflammation.
Cysteine proteases are known to modulate the activity protease-activated receptors (PAR) found on epithelial cell surfaces, resulting in proinflammatory responses. Increased fecal protease activity and activation of PAR-2 receptors were reported in several intestinal disorders, including IBS , and the possibility that Blastocystis proteases modulate PARs should therefore be investigated. Pathogenic strains of E. histolytica show higher cysteine protease activity compared with nonpathogenic strains . We recently observed that subtype 7 cells exhibit markedly higher cysteine protease activity when compared with subtypes 4 cells [15•], suggesting the possibility that subtype 7 parasites are more virulent than subtype 4 parasites. Interestingly, subtype 7 parasites are also morphologically larger and grow more slowly than subtype 4 parasites. Two recent studies reported that cells of Blastocystis parasites from symptomatic patients were larger than those from asymptomatic ones [48, 49], and one of the studies  also showed that parasites from symptomatic patients displayed slower growth rates. Although the pathogenic mechanisms are unclear, it would be interesting to speculate that cell size and generation time influence parasite virulence. Collectively, these results suggest a subtype-dependent variation in Blastocystis virulence and provide a tentative explanation that subtypes may influence clinical outcomes of Blastocystis infections (Table 1). A model for the pathogenesis of Blastocystis spp is shown in Fig. 1.
Treatment options and regimens for blastocystosis
• 750 mg tid for 10 d
• 15 mg/kg/d for 7 d
• 500 mg tid for 10 d
• 20–30 mg/kg/d for 10 d
• 1.5-g single dose/d for 10 d
• 320 mg TMP and 1,600 mg SMX daily in two equal doses for 7 d
• 6 mg/kg TMP and 30 mg/kg SMX daily in two equal doses for 7 d
• 500 mg bid for 3 d
• 100 mg bid for 3 d (1–3 y)
• 200 mg bid for 3 d (4–11 y)
Although Blastocystis is one of the most common intestinal protists found in humans, its pathogenic role is controversial; however, accumulating evidence from recent studies supports the notion that it is an emerging pathogen. The primary reasons for the controversies surrounding this parasite are the lack of a standardized laboratory detection method, existence of genetic and biologic heterogeneity, absence of an animal infection model, reports of treatment failure, and studies with small sample sizes and occasional misinterpretation of data. Recent studies clearly highlight the pathogenic potential of this parasite, but more supporting data are needed before Blastocystis can be classified as a human pathogen. Although clinical outcome of the infection is multifactorial and involves host and parasite factors, a major research priority is to clearly identify pathogenic and nonpathogenic Blastocystis subtypes. Currently, reports suggest that subtypes 1, 4, and 7 are pathogenic whereas subtypes 2 and 3 represent nonpathogenic parasites.
Research from Dr. Tan’s laboratory is supported by generous grants from the Academic Research Fund, National Medical Research Council (NMRC), and Biomedical Research Council. Dr. Wu is a postdoctoral fellow funded by the NMRC. Haris Mirza and Joshua Teo are postgraduate students funded by National University of Singapore Research Scholarships.
No potential conflict of interest relevant to this article was reported.