Enteric Protozoa in the Developed World: a Public Health Perspective

Distribution in Developed Countries

One report (Fletcher et al., presented at the Communicable Disease Control Conference, Canberra, ACT, Australia, 2011) estimated that an enteric pathogen is isolated in an average of 40.9% (95% confidence interval [CI], 33.4 to 48.8%) of diarrheal cases in developed countries, among which enteric parasites represent less than 1% (95% CI, 1.1 to 3.5%) of cases. However, enteric parasites—mainly protozoa—are isolated from 1% to 65% of patients with diarrhea in various settings. The relative prevalences of enteric protozoa reported in several developed countries in outbreak and nonoutbreak settings among humans are reported in Table 1 (1, 11, 12, 24, 60, 85, 90, 96, 128, 144, 184, 192, 193, 204, 215, 225, 247, 248, 255, 264, 265, 301, 335, 355, 370, 395, 416, 417, 425, 442, 444, 452, 467). Giardia intestinalis (0.2% to 29.2% of cases), Cryptosporidium spp. (0.1% to 9.1% of cases), Entamoeba spp. (0.2% to 12.5% of cases), and Cyclospora cayetanensis (0.2% to 4.3% of cases) were the most common protozoa reported in developed settings. The common protozoa, however, were rivalled in prevalence by Blastocystis spp. (0.4% to 18.1%) and D. fragilis (0.4% to 6.3%). Both have the potential to cause illness but are more frequently associated with asymptomatic infection. Nevertheless, evidence from the literature suggests that Blastocystis spp. and D. fragilis have relatively high prevalences in developed settings (395; Fletcher et al., presented at the Communicable Disease Control Conference, Canberra, ACT, Australia, 2011). Infections are associated with recent travel to developing regions (394), with immigrants and refugees (164, 273), and with domestic transmission (387). The prevalence rates of D. fragilis vary widely, from 0.4% to 42%, and the incidence of this parasite was found to be second only to that of Blastocystis spp. and of similar or greater magnitude to those of the more commonly diagnosed parasites Giardia, Cryptosporidium spp., and Entamoeba spp. in many developed regions when diagnostic methods were implemented for these species. Several reports have also identified D. fragilis as the most common pathogenic protozoan found in stool when appropriate diagnostic methods are used (389). It is therefore recommended that both Blastocystis spp. and D. fragilis be considered in the differential diagnosis of gastrointestinal infections in developed settings (26).

Asymptomatic carriage of protozoan parasites is also common in developed countries, as several types have been isolated from healthy individuals without diarrhea (0.5% to 16.5%) (90, 301). For example, about 90% of individuals infected with Entamoeba are colonized by the nonpathogenic species Entamoeba dispar, and as such, they are asymptomatic; this is true even for immunosuppressed populations (14, 385). Table 2 presents an epidemiological summary of several protozoa that have been implicated in human illnesses in developed countries, and examples of stained enteric protozoa are shown in Fig. 1. Recommended treatment regimens for enteric protozoa are presented in Table 3, based on international standards (218, 390).

Cryptosporidium Species

Cryptosporidium was first recognized as an important cause of infection in AIDS patients (410). It is now well recognized and accounts for about 20% of diarrheal episodes in children in developing countries and up to 9% of episodes in developed settings and causes a considerable amount of diarrheal illness in young farm animals worldwide (340, 473). The environmentally resistant oocysts are fully sporulated when they are excreted in feces, and therefore they are immediately infectious (116, 441, 477). Infections are usually characterized by self-limiting diarrhea associated with severe abdominal pain in both immunosuppressed and immunocompetent persons, especially HIV-infected persons and children worldwide (16, 116, 390). The duration of clinical symptoms is highly dependent on the person's immunological competence (235). It is still an important cause of potentially life-threatening diarrhea in HIV-infected patients with limited access or poor compliance with highly active antiretroviral therapy (HAART) (228, 326). Cryptosporidium spp. are of importance to transplant patients, especially in regions where the organisms are endemic, where they can cause life-threatening prolonged diarrhea, dehydration, and malabsorption in transplant recipients (100). Species of Cryptosporidium and subtype families of Cryptosporidium hominis have been shown to induce different clinical manifestations and have different potentials to cause outbreaks (473). In developed countries, transmission occurs from person to person, especially in day care settings and between men who have sex with men (MSM), as well as through waterborne and zoonotic infections (473). Cryptosporidium spp. have been identified as a common cause of diarrhea in persons from developed countries visiting less developed areas (343, 473, 474, 476). Animal models have demonstrated the role of Cryptosporidium parvum in the formation of polyps and adenocarcinoma lesions in the guts of dexamethasone (Dex)-treated severe combined immunodeficiency (SCID) mice, suggesting the need to investigate whether a similar C. parvum-induced gastrointestinal cancer occurs in humans (50–52, 74, 459).

In developed countries, sporadic outbreaks due to fecal-oral transmission have been reported among children attending playgroups and day care centers and by ingestion of contaminated salads, contaminated water supply, or recreational water, contact with sick animals, swimming in public pools, and person-to-person transmission (55, 66, 325, 340). Epidemiological variations have been observed in the geographical, seasonal, and socioeconomic effects of the distribution of Cryptosporidium spp. in humans that may influence the sources and routes of transmission (473). Cryptosporidium spp. have a wide host range and cause infections in humans, livestock, domestic pets, and wildlife, among all four classes of vertebrates, and, most likely, in all mammalian species (55, 89, 290, 345, 441). Most animals are not infected with human-pathogenic species and thus play no role in zoonotic transmission of cryptosporidiosis. However, zoonotic transmission from direct contact with infected animals or their feces (55, 89, 345, 476) or indirectly through the consumption of contaminated water (63, 66, 327, 476) can occur.

The small size and subtle staining characteristics of Cryptosporidium spp. have contributed to the difficulties of identifying these parasites in routine stool preparations (116, 375). The diagnosis of cryptosporidiosis is generally undertaken by identification of oocysts in stool of the host/patient. Traditionally, the identification of Cryptosporidium oocysts was based on microscopic examination (300, 330). However, the identification of morphological characters of Cryptosporidium is unreliable and relatively time-consuming, even with light microscopy (209). Staining and preservation methods have been used to enhance the sensitivity of tests. Unfortunately, these do not identify species, and their analytical sensitivity can be poor, especially for samples containing small numbers of oocysts (138, 253, 329). The modified Ziehl-Neelsen technique (413) is used widely; however, one recent study found that it was less sensitive than PCR (75.7%; 95% CI = 68.3 to 81.8%) but was highly specific (100%; 95% CI = 96.5 to 100%) (56). Kinyoun's acid-fast staining technique (102), modified Sheather's flotation technique (76, 294, 296), and the iron-hematoxylin staining technique (119) have also been described.

Antigen detection by immunoassays has been used widely in the diagnosis of cryptosporidiosis, as these assays are thought to be more sensitive than conventional staining and more effective in cases where oocyst numbers are low (139, 141, 329). An evaluation of the Meridian Premier Cryptosporidium test and the Alexon ProSpecT Cryptosporidium microplate assay found that both systems performed according to the manufacturers' values for sensitivity and specificity (for the Meridian test, 91 and 99%, respectively; and for the Alexon test, 97 and 100%, respectively) (139). Highly rapid immunoassays have also been developed that can be used with fresh, frozen, or unfixed human fecal specimens. For example, the Biosite Diagnostics (San Diego, CA) Triage rapid qualitative enzyme immunoassay (EIA) for the detection of Giardia, E. histolytica/E. dispar, and Cryptosporidium antigens has been demonstrated to have a 98.3% sensitivity and 99.7% sensitivity in the detection of Cryptosporidium spp. from stool specimens (140). A multicenter French study evaluated the sensitivity and specificity of four immunochromatographic (ICT) assays and found that RIDAQuick (mean sensitivity, 73.3%), Remel Xpect (mean sensitivity, 74.1%), and ImmunoCard STAT (mean sensitivity, 73.3%) were fairly sensitive for detecting C. parvum and C. hominis but were very limited in detecting other Cryptosporidium spp. (3). Fluorescence microscopy and direct fluorescent-antibody (DFA) assay have been used with relatively high specificities (96 to 100%) and sensitivities (98.5 to 100%) for the detection of Cryptosporidium oocysts in clinical and environmental samples (139, 158, 209). However, the sensitivity and specificity of monoclonal antibody (MAb)-based DFA assays have been affected by different factors, such as the purity of the Cryptosporidium antigen originally used to raise the MAb (139, 158, 209, 214, 329).

Molecular methods have been developed for the detection and differentiation of Cryptosporidium spp. at the species/genotype and subtype levels (9, 168, 290, 372, 386). These methods, including a nested PCR (348), real-time PCR, multiplex real-time PCR, reverse transcription–quantitative real-time PCR (372, 455), and multiplex tandem real-time PCR (386), have been used for identification of species in diarrheal stools in relatively short time frames. More recently, an automated multiplex tandem PCR using a robotic platform to simultaneously detect Cryptosporidium spp. and coinfecting diarrheal pathogens from human fecal genomic samples was described. The assay was rapid (taking <2 h, with ∼5 to 10 min of technical work following the extraction of genomic DNA), and due to automation, data analysis required little molecular biological expertise, making it well suited to various diagnostic facilities and settings (e.g., hospitals or quarantine facilities) (210). Other tools, based on the Cryptosporidium oocyst wall protein (COWP) gene, have been used to amplify DNAs of C. parvum, C. hominis, Cryptosporidium meleagridis, and species and genotypes closely related to C. parvum (9, 242). However, it has been suggested that these methods have limited usefulness in genotyping Cryptosporidium spp. in animals because of their narrow specificity (342, 473). The disadvantage of some PCR tools is that they are designed to detect the dominant Cryptosporidium genotype in the specimen and require a substantial amount of PCR product to be visible on an agarose gel, and when specific genotyping and subtyping tools are used, they fail to detect concurrent infections with mixed Cryptosporidium species/genotypes or fail to detect other divergent species/genotypes (168). New methods such as reverse line blot (RLB) hybridization, isothermal methods such as the loop-mediated isothermal amplification method (319, 372), and nucleic acid sequence-based amplification (NASBA) methods that amplify RNA from either RNA or DNA templates also provide additional diagnostic platforms for the detection of Cryptosporidium spp. (154, 372).

Cryptosporidiosis is usually self-limiting in immunocompetent persons, requiring little or no treatment, but it is especially challenging to treat among high-risk immunosuppressed groups (39, 83). Nitazoxanide has proven effective in the treatment of immunocompetent patients (133); however, a higher dosage and longer duration of treatment have been indicated for immunosuppressed patients (39, 129). Rehydration fluids and nutritional management may be required for immunosuppressed persons with dehydration (57, 83).

Dientamoeba fragilis

There has been much debate about the pathogenicity of Dientamoeba fragilis (26, 390). Unlike the case for other protozoa, a cyst stage has not been demonstrated, and trophozoites degenerate within hours of being passed in stool (26, 391–393). Although the mode of transmission remains unknown, based on high rates of coinfection with Enterobius vermicularis, it was suggested previously that infection occurs via the pinworm vector (25, 153, 191). Recent studies have discounted this idea (229). However, high rates of coinfection with other enteric pathogens and protozoa suggest that transmission occurs directly via the fecal-oral route (26, 191). Infection may be acute or chronic and has been reported in both children and adults and both immunocompetent and immunosuppressed populations (26, 127, 361, 389, 392, 444). The most common clinical symptoms include abdominal pain, persistent diarrhea, loss of appetite, weight loss, and flatulence (22, 355, 389, 444). These symptoms are akin to those of irritable bowel syndrome (IBS), so D. fragilis should be considered in the differential diagnosis of IBS (399). Dientamoeba fragilis infection also presents with similar symptoms to those of Giardia infection and is estimated to occur with a similar or greater prevalence to that of Giardia infection in Belgium (444) and Australia (127, 355, 394, 395, 401). Dientamoeba fragilis trophozoites have been reported in nonhuman primates, but the limited host range suggests that human infection may not involve transmission from other animal species (396).

Traditional diagnosis relies on the microscopic detection of trophozoites in fresh or fixed stool specimens. The demonstration of the characteristic nuclear structure of D. fragilis needed for a definitive diagnosis cannot be achieved with unstained fecal material (392); as such, prompt fixation and permanent staining are necessary for definitive diagnosis (355, 388, 391). Microscopy of fixed smears with permanent staining (modified iron-hematoxylin or trichrome staining) (127, 393, 403) is considered the gold standard for diagnosis of D. fragilis infection, but this method is time-consuming and relatively insensitive compared to molecular methods (391). More recently, diagnostic tests based on various conventional and real-time PCRs to detect the small-subunit (SSU) rRNA gene of D. fragilis have been developed to facilitate rapid, sensitive, and specific diagnosis in fresh stools (386, 388, 401, 455), and the successful culture and cryopreservation of viable D. fragilis trophozoites from clinical specimens have been achieved (27, 355). It was demonstrated that when microscopy is combined with other methods, the success of detection of D. fragilis is increased significantly (26, 386, 388). However, while molecular methods are more sensitive than microscopy and staining, many of the necessary products are not commercially available and the methods are not employed routinely, even in developed countries.

Treatment is recommended in symptomatic patients and asymptomatic family members to prevent reinfection (153, 444). Various antimicrobial therapies have resulted in the successful clearance of D. fragilis and total resolution of gastrointestinal symptoms in infected patients (152, 387, 445). Paromomycin, secnidazole, iodoquinol, tetracycline, ornidazole, and metronidazole have been used successfully to treat D. fragilis infections (22, 152, 305, 445). However, there is emerging evidence of treatment failures of metronidazole among D. fragilis isolates, suggesting the increased need for combination therapy for these protozoa (22, 389). Combination therapy has been effective in the complete eradication of the parasite and in resolution of symptoms (387, 389).

Entamoeba Species

Six species of the genus Entamoeba have been described in humans, including Entamoeba histolytica, Entamoeba dispar, Entamoeba moshkovskii, Entamoeba poleki (also called Entamoeba chattoni), Entamoeba coli, and Entamoeba hartmanii. Among these, E. histolytica is the only pathogenic species (201, 236, 351). Improvements in the understanding of the biochemical, immunological, and genetic differences of the members of this genus have resulted in the confirmation of three species, E. histolytica, E. dispar, and E. moshkovskii, that are morphologically identical in both their cyst and trophozoite stages (41, 169). The vast majority (about 90%) of individuals infected with Entamoeba spp. are colonized by the nonpathogenic strain E. dispar (324, 385). In developed countries, infections of E. histolytica (a true pathogen) are largely confined to immigrants from or travelers to areas of endemicity, MSM, HIV-infected patients, and institutionalized populations (164, 174, 315). The WHO reports that approximately 500 million people worldwide are infected annually with E. histolytica, resulting in symptomatic illnesses and death in about 50 million and 100,000 persons, respectively (470). However, it is believed that since 90% (450 million) of infections are due to E. dispar, while 10% (or 50 million) are infections with E. histolytica, the worldwide incidence of invasive disease is more likely to be 5 million cases annually, with global mortality still at 100,000 per annum (201). Approximately 4 to 10% of carriers infected with E. histolytica develop clinical disease within a year, and amoebic dysentery is considered the third leading cause of death from parasitic disease worldwide (178, 280, 385). Entamoeba histolytica causes a range of disease manifestations, including (i) dysentery or dysenteric syndrome, characterized by small volumes of bloody, mucoid stools without fecal leukocytes; (ii) amoebic colitis, characterized by ulcerations of the colonic mucosa with typical flask-shaped abscesses; (iii) amoeboma, the formation of a fibrotic mass in the intestinal wall; and (iv) invasive disease, resulting in amoebic abscesses in the brain, lung, or liver (84, 315, 324, 449). Amoebic brain abscess occurs when Entamoeba histolytica trophozoites travel to extraintestinal tissues through the bloodstream, invading the central nervous system and producing amoebic brain abscesses that are frequently lethal (257). Other rare forms of amoebiasis have been reported, including cutaneous amoebiasis (amoebiasis cutis), arising as a complication of amoebic dysentery (232, 323). This usually results from contamination of damaged skin or continuous contact with exudates containing virulent trophozoites that stick to the traumatized skin, such as in the perianal or perigenital area, resulting in lysis of the skin and subcutaneous tissue and, subsequently, ulceration and necrosis (36, 323, 454).

While asymptomatic E. histolytica infections are equally distributed between the genders, invasive disease is more common in men (which may be due to a male-related susceptibility to invasive disease). Higher rates of carriage have been observed in HIV-infected MSM in the Asia Pacific region (194, 203, 280, 400). Patients with amoebic colitis present with a gradual onset of bloody or profuse watery diarrhea and abdominal pain and tenderness (324, 385). Symptoms may last for several weeks, and fulminant necrotizing colitis, the most severe form of intestinal disease, is often fatal (174, 324, 379). In children, <40% of patients present with fever and rectal bleeding without diarrhea. Some patients develop fulminant amoebic colitis, with profuse bloody diarrhea, fever, pronounced leucocytosis, and widespread abdominal pain, often with peritoneal signs (280, 399).

The virulence of pathogenic E. histolytica is based on its ability to secrete enzymes and proteases that contribute to the invasion of the epithelial cells penetrating the intestinal mucosa and to degradation of the extracellular matrix proteins and subsequently to interfere with the host's humoral immune response (14, 315). More recently, the emergence of clear roles for human and parasite genetics and environmental factors in the virulence of E. histolytica has increased our understanding of infections. It is thought that some persons are genetically resistant to infection, while malnourished children are more susceptible, and a polymorphism in the leptin receptor increases susceptibility to amoebiasis in both adults and children (14, 277, 332). The evidence suggests that not all strains are capable of causing liver abscess, and the observed higher incidence in men may be a result of gender-based differences in the complement system (379). For example, higher gamma interferon and functional natural killer T cell levels in females might underlie their resistance to liver abscess (280, 332). Based on the many presentations of amoebiasis, a high level of clinical suspicion is necessary for the early diagnosis of invasive and extraintestinal amoebiasis (221, 454). The specific diagnosis of E. histolytica infection is important in order to minimize undue treatment of individuals infected with nonpathogenic species of Entamoeba (131).

Diagnosis is difficult based on the fact that the pathogenic species E. histolytica is morphologically identical to the nonpathogenic species E. dispar and E. moshkovskii; hence, microscopy is generally considered insufficient for differentiation of these species (397). Various methods are employed in the diagnosis of Entamoeba. In many countries, microscopy is widely used for protozoan identification (131, 204, 249). Microscopic techniques utilized in the clinical laboratory include the screening of wet preparations, concentrated samples, and permanently stained smears for the identification of E. histolytica/E. dispar/E. moshkovskii in feces (131). The CDC recommends that in order to maximize the recovery of cysts, stool samples in formalin or other fixatives should be concentrated prior to microscopic examination (49). Wet mount preparations and trichrome-stained smears of stool specimens are routinely used for identification of E. histolytica/E. dispar (49). Various fixatives utilized for the preservation of the morphology of the parasites can be used in conjunction with various stains. The pathogenic species E. histolytica can sometimes be differentiated from the nonpathogenic, morphologically identical species by microscopy on the basis of ingested red blood cells within the cytoplasm of the trophozoites; however, this phenomenon does not occur commonly and is rarely seen in clinical samples (49, 131). Therefore, when a definitive diagnosis is not possible via microscopy, the presence of the E. histolytica/E. dispar/E. moshkovskii complex should be reported (49). Microscopy is less reliable at identifying Entamoeba species than culture or antigen-based and molecular tests (179, 410).

While cultivation is more sensitive than microscopy and isoenzyme analysis can effectively distinguish between E. dispar and E. histolytica, these methods are time-consuming, are not cost-effective, and are not routinely utilized by most diagnostic laboratories (132, 177). EIA kits are commercially available that detect E. histolytica only, while others detect both E. histolytica and E. dispar (49, 131, 460). A rapid ICT assay is available that detects antigens of E. histolytica and E. dispar in stool; however, this assay does not distinguish between E. histolytica and E. dispar (49). This assay also detects antigens of Giardia and Cryptosporidium. Borderline positive results and questionable negative results obtained by this technique should be confirmed further by additional testing. This assay is quick and easy to perform, and no special equipment is needed (49). EIAs and rapid ICT tests require the use of fresh or frozen stool specimens and cannot be used with the majority of preserved specimens because antigens are lost during the concentration procedure (49, 397). However, some newer single-vial collection systems that utilize nontraditional fixatives can be used in these assays. Many stool antigen assays have been shown to be as sensitive and specific as culture with isoenzyme analysis and outperform microscopy for the detection of E. histolytica in areas of endemicity (177, 179, 316). One study found that in comparison to PCR, some stool antigen tests lacked sensitivity but were highly specific in diagnosing E. histolytica/E. dispar in areas where these parasites are not endemic (460), while another study found that antigen detection tests can be both rapid and technically simple to perform (177).

A number of molecular assays have been described in the scientific literature for detection and/or differentiation of Entamoeba species and are now considered the gold standard for diagnosis (177, 397). Both conventional and real-time PCRs have proven to be more sensitive and specific than microscopic examination for the detection of E. histolytica and E. dispar in single stool samples (455, 460). In developed settings, PCR is more useful for detection of E. histolytica in stools than antigen detection tests due to the higher sensitivities observed for PCR and the reduced chance of cross-reactivity with other Entamoeba species (167, 397, 460). However, PCR has yet to become the mainstream for the detection of parasites in the clinical laboratory, even in developed countries, due to the need for specialized equipment, dedicated molecular areas, specified workflow, and associated costs. It must be emphasized that when species differentiation is not possible, the E. histolytica/E. dispar complex should be reported.

Serological methods such as indirect hemagglutination (IHA), latex agglutination, immunoelectrophoresis, immunofluorescence assay (IFA), and enzyme-linked immunosorbent assay (ELISA) are highly sensitive at detecting E. histolytica antibodies in human serum and are useful for detecting invasive disease (41, 131, 236). However, serology is of limited use in areas of endemicity because of the difficulty in distinguishing between past and present infections (167, 236); hence, it is of more value in nonendemic settings in establishing the diagnosis of E. histolytica infection if antibodies are present (460). A combination of microscopy, culture, and serology should be complemented with a PCR assay or with abdominal imaging (when PCR is unavailable) for detection of invasive disease (41, 131). Diagnosis of amoebic brain abscesses usually requires computerized axial tomography scans of the brain (257). Identification of E. histolytica antigens in tissue aspirates or PCR analysis of tissue aspirates is also effective (390).

All infections with E. histolytica should be treated because of its potential for causing invasive disease and the risks to public health (110, 324, 385). Asymptomatic carriers should be treated with a luminal agent to minimize the spread of disease. The treatment of choice differs for intestinal and invasive disease; hence, diagnosis is important before treatment begins. Metronidazole is a highly effective tissue amoebicide and is used in the treatment of invasive amoebic disease. However, a luminal agent should be used in combination, as treatment does not eliminate intestinal colonization in up to 50% of patients with invasive amoebiasis, resulting in relapse of invasive infection months later (174, 222, 236). Other nitroimidazole derivatives, such as tinidazole and ornidazole, are equally effective for treatment of invasive disease (218, 324). The luminal agents paromomycin, iodoquinol, and diloxanide furoate are strictly recommended for treatment of patients with intestinal and asymptomatic infections, as they are effective in eliminating cysts from the intestinal tract (174, 218).

Giardia intestinalis

Giardia intestinalis (syn. Giardia duodenalis and Giardia lamblia) is a common cause of parasitic diarrhea, with prevalences ranging from 2 to 7% in developed countries to 20 to 30% in most developing countries worldwide (134, 207). Because of the burden of illness from Cryptosporidium spp. and Giardia, their ability to impair development and socioeconomic improvements, and their associations with poverty, they were included in the WHO Neglected Diseases Initiative in 2004 (354). Giardia intestinalis consists of seven genetically distinct genotypes, designated A to G, but a novel lineage designated assemblage H has been identified in marine vertebrates (233). Assemblages A and B infect mammalian species and are the only two assemblages known to infect humans; hence, they are considered to be zoonotic (118, 134, 146, 196, 236, 262, 433). Recent developments in the study of protein coding capacities have found genomic differences between strains WB (assemblage A) and GS (assemblage B), which may explain some of the observed biological and clinical differences between the two isolates. These observations led to the suggestion that Giardia assemblages A and B may be two different species (134, 207, 285).

It has been suggested that Giardia trophozoites may remain in the small intestine for weeks to years (315). However, this is unsupported, since many long-term infections are suspected to represent either persisting abdominal symptoms elicited post-Giardia infection or reinfections (176, 196, 484). The fecal-oral route still remains the most important mode of infection (33), and various studies have found evidence of zoonotic transmission (89, 118, 237). The symptoms of giardiasis can be variable, but it presents mainly as acute or chronic diarrhea associated with abdominal pain, nausea, malabsorption, and weight loss. In malnourished children, infection can lead to growth retardation, and asymptomatic illness is also possible (37, 40). There have been a few reports suggesting that giardiasis may be a risk factor for zinc deficiency in school-aged children (80, 328). High rates of asymptomatic carriage of Giardia have been reported for humans and animals in developing settings (213).

In developed settings, Giardia intestinalis has also been isolated from various animals, including livestock (17, 31, 65), fish (475), and nonhuman primates (262). Giardia infestation is common in dogs and cats, with prevalences of approximately 8% to 16% and 4% to 11%, respectively (44, 302). While there is limited evidence for direct transmission from companion animals to people, rare infections and the isolation of zoonotic genotypes from cats and dogs suggest that they are a potential source of human infection that may be acquired through handling, sleeping together, licking, and kissing (61, 308, 346, 424, 480). Giardiasis is frequently associated with waterborne and day care center disease outbreaks and is related to travel-associated diarrhea (471). A study of travel- and migration-associated illnesses in Europe revealed that G. intestinalis was the second most common pathogen and the most common parasite as a cause of gastrointestinal illnesses (122). Giardia is frequently isolated from diarrheal stools from MSM, with or without HIV/AIDS, in whom transmission is most likely via the fecal-oral route (33, 324). In the United States, it is suspected that seasonal peaks are related to increased use of recreational water venues, such as lakes, rivers, swimming pools, and water parks, in summer months (188).

Diagnosis is usually based on the microscopic detection of Giardia cysts or trophozoites in a stool specimen. Stools may be examined either directly as fresh smears, preserved in formalin or polyvinyl alcohol and stained with iodine, trichrome, or hematoxylin, or after formalin-ethyl acetate concentration (222, 236, 262, 289, 305, 457, 465). Several antigen detection assays are available for G. intestinalis, including EIAs, ELISAs, and monoclonal antibody and direct fluorescent-antibody tests, which are widely used (130, 140, 141, 145, 234, 431). Many have proven to be cost- and labor-effective, and many of them are designed for the sensitive and specific testing of several protozoa simultaneously (140, 141, 465). These antigen detection tests are useful in screening settings and are more sensitive than routine microscopic examinations for detection of ova, cysts, and parasites (OCP) (137). For example, one rapid qualitative EIA has been demonstrated to have a 95.9% sensitivity and 97.4% sensitivity for the detection of Giardia in stool specimens (140). The use of real-time PCR as a tool for the detection of Giardia is increasing in developed settings. Conventional single, nested, and multiplex PCRs have also been developed but are utilized more often in specialized centers involved in molecular studies of Giardia (40, 236, 475). These molecular methods have proven to provide higher sensitivity than conventional methods; however, many of them are still not commercially available (236). The use of PCR in molecular epidemiology studies has advanced the knowledge of the population structure of Giardia, which has improved the understanding of zoonotic transmission and the molecular epidemiology of giardiasis (430).

Treatment is usually the same for both immunosuppressed and immunocompetent patients (83). Metronidazole or tinidazole has been used as the therapy of choice against giardiasis; however, treatment failures and clinical relapses have been known to occur (37) and could be due to the emergence of resistant isolates of Giardia (37). Furazolidone, albendazole, nitazoxanide, and paromomycin are suitable alternatives when available (83). One vaccine (GiardiaVax) has been licensed for use in the United States to prevent clinical disease in dogs and to significantly reduce the incidence, severity, and duration of cyst shedding in cats (267). However, more recent data have shown limited vaccine efficacy (15), and no human vaccines are currently available (434).

Cyclospora cayetanensis

Cyclospora cayetanensis has emerged as an important cause of endemic or epidemic diarrheal illness in children and adults worldwide (53). Cyclospora cayetanensis is the only species of this genus found in humans and is host specific (79). An important feature of the biology of C. cayetanensis is that oocysts excreted in feces require days to weeks outside the host to sporulate and to become infectious; hence, direct fecal-oral transmission from relatively fresh stool does not occur (189, 259). Clinical illness is characterized by persistent diarrhea, bloating, flatulence, abdominal cramps, constipation, and fatigue (305). Illness associated with travel to nonindustrialized countries has been reported in the United Kingdom (291) and Netherlands (456) and is a common cause of illness among returned international travelers (417). However, non-travel-related and waterborne cases of cyclosporiasis have been reported in several developed countries (12, 450). Food-borne outbreaks have been reported in North America (189, 291, 450) and Germany (92). Infections in immunosuppressed patients have been reported in Turkey (350), and although symptoms may be similar to those in immunocompetent individuals, they may be prolonged (410). Of the 1,110 laboratory-confirmed cases of sporadic cyclosporiasis captured by U.S. disease surveillance over the 1997-2008 period, approximately one-third of cases occurred in persons with a known history of international travel, the vast majority of whom traveled to countries in Southern and Central America (173). Domestically acquired cases were concentrated in time (late spring and summer) and were probably linked to an undetected outbreak (173).

Standard laboratory procedures for ova and parasites do not identify Cyclospora; therefore, the laboratory must be notified when Cyclospora is being considered (368, 461).

Diagnosis is made by the demonstration of Cyclospora oocysts by examination of their autofluorescence and staining characteristics. Oocysts appear as acid-fast round or ovoid structures and autofluoresce white-blue under an epifluorescence microscope, using a 330- to 380-nm dichromatic (DM) excitation filter, or blue or green with a 450- to 490-nm DM filter (53, 137, 305, 306, 330, 456). However, detection is often difficult because the pathogen is very small, measuring 8 to 10 μm in diameter; hence, oocyst measurement is important to differentiate the oocysts of this species from smaller Cryptosporidium oocysts, which measure 4 to 6 μm (137, 259, 330). The oocysts can be seen in concentrated or nonconcentrated feces by light microscopy using various stains, but the disadvantage is that C. cayetanensis oocysts stain only variably with Gram, Giemsa, and hematoxylin-eosin stains and results are variable with modified Ziehl-Neelsen staining (259, 368, 456). A safranin-based stain has been described that uniformly stains oocysts of Cyclospora a brilliant reddish orange when the smears are heated in a microwave oven prior to staining (305, 461). Other routine procedures in use include concentration by the formalin-ethyl acetate technique followed by either (i) UV epifluorescence and bright-field microscopy, (ii) examination of a modified acid-fast-stained stool slide, or (iii) examination using a modified safranin-based technique (151, 307, 368, 377, 461). Samples can be stored in 2.5% potassium dichromate for 14 days at temperatures ranging from 22 to 37°C for sporulation or molecular detection (306, 377). In comparison to UV detection, the sensitivity of the acid-fast technique is about 78% (93).

Various molecular techniques have been developed for the identification of Cyclospora. These include spectrophotometry-based detection with an oligonucleotide ligation assay. Various PCR tools have been used that target the internal transcribed spacer region, using primers for the 18S rRNA gene (93, 306). These include conventional PCR, reverse transcriptase PCR in combination with agarose gel electrophoresis, and nested PCR (281, 304, 306, 314, 450). Some of these techniques for the identification of C. cayetanensis are time-consuming, labor-intensive, and subject to problems of contamination; hence, further studies are needed for the development of highly sensitive rapid tests for this protozoan. On a cautionary note, it was reported that PCRs for Cyclospora cross-amplify DNAs from other coccidia, especially those belonging to the genus Eimeria. This cross-reactivity with Eimeria is problematic mainly in outbreak settings and with environmental specimens, since human infections by Eimeria are currently unknown (259, 314). A restriction fragment length polymorphism (RFLP) protocol is required to distinguish between these species in environmental samples (259).

Laboratory diagnosis is important before empirical treatment commences, since the organism requires different treatment from that for some of the other protozoa of similar presentation (368). Trimethoprim-sulfamethoxazole (TMP-SMZ; also known as cotrimoxazole) is the drug of choice for managing cyclosporiasis. Infection can be treated effectively with cotrimoxazole for 10 days, with results occurring in a few days (218, 222, 306). Oral or intravenous rehydration may be appropriate, based on the degree of dehydration (368).

Blastocystis Species

Blastocystis spp. are commonly found in stools from humans and numerous animal hosts (341, 418). There is considerable genetic heterogeneity within Blastocystis organisms, and currently, human, mammalian, avian, and reptilian isolates have been assigned to 1 of 13 subtypes (312, 341, 384, 402). While it is unclear whether any of these subtypes are specific to human disease, Blastocystis sp. subtype 3 is most commonly associated with illness in human prevalence studies (90, 341, 418, 479). The name Blastocystis hominis refers to approximately 10 different genetically diverse populations that are all morphologically identical and cannot be differentiated on the basis of microscopy alone (406, 435). In many reports, B. hominis refers to the parasite isolated from humans and Blastocystis spp. represent those isolated from other animal hosts (293). Based on the discrepancies in the use of these terms, several experts in the field have come to a consensus that based on published small-subunit rRNA gene analyses, all mammalian and avian isolates should be designated Blastocystis spp. and assigned to one of nine subtypes (408). Knowledge about the life cycle of Blastocystis spp. remains elusive, and various morphological forms have been described (82, 320). These are the vacuolar, granular, amoeboid, and cyst forms, and other, less frequently encountered forms are the avacuolar and multivacuolar cells and cells containing filament-like inclusions (191, 274, 371, 409, 419).

The role that Blastocystis spp. play in eliciting gastrointestinal pathology and symptoms remains uncertain and controversial (191, 421). Boorom et al. argue that there may be some pathogenic variants of Blastocystis spp. and that the limitations in their detection by existing diagnostic methods may have added to the controversy about their pathogenicity (32). Another thought is that the amoeboid form of Blastocystis spp. could be either an indicator of or contributor to the pathogenicity of these protozoa in patients exhibiting symptoms (422), but this suggestion remains unsupported (341, 420). However, more recent in vivo and in vitro studies suggest that some of the “strains” or potentially different species of this organism may be pathogenic (32, 399, 418, 479). The genetic and antigenic diversity among Blastocystis spp. complicates the epidemiological understanding of these parasites; however, emerging evidence—despite still being controversial—suggests that pathogenicity may be subtype dependent (419, 421). It is thought that the cyst is probably the infectious form and is transmitted by the fecal-oral route: when the parasite passes down the intestinal tract, small vesicles within its cytoplasm coalesce into a vacuole, eventually forming a cyst which is passed into the environment in the stool of the host (191, 274, 371).

Clinical features of illness which have been attributed to Blastocystis spp. include nausea, anorexia, abdominal pain, flatulence, and acute or chronic diarrhea (216, 381). In several instances, Blastocystis spp. have been the most common enteric organisms isolated from diarrheal patients but have been reported as noninfectious pathogens (422, 438, 444). Blastocystis spp. are the most common enteric protozoa isolated from diarrheal patients in most developed countries (247, 309, 395, 416). For example, they are commonly isolated from immigrants in Italy (164), associated with chronic gastrointestinal illness of unknown etiology (216), and often associated with IBS-like symptoms (32, 90, 91, 211).

However, numerous studies have found no link between Blastocystis spp. and active disease (90, 91, 404, 420). It has also been argued that identification of Blastocystis from patient samples is not clinically significant but should be used as a marker of potential exposure to other pathogenic protozoa (315). Another study suggested that immunosuppression plays an important role in the display of clinical symptoms (418). More recently, clinical and molecular investigations demonstrated an association with Blastocystis spp. in IBS patients that supports a pathogenic role of this parasite in Latin America (211). However, other studies have failed to establish an association between Blastocystis spp. and IBS (334, 414, 440). It has been suggested that since IBS is a functional disorder that can be caused by various microbiological, genetic, and environmental factors, these factors could also explain the discrepancy in the literature (321). Cysts may be waterborne, food borne, or passed from person to person, especially in child care centers or other institutional settings. It has also been suggested that since many zoonotic subtypes exist, there is increased potential for zoonotic transmission (191, 420, 438).

The diagnosis of infection with Blastocystis spp. is usually based on the detection of the vacuolar, granular, amoebic, or cystic form in stool samples, using wet mount smears, iodine staining, trichrome staining, or iron-hematoxylin staining (309, 341, 390, 402, 404, 416, 481). However, identification by direct wet mounts is difficult and has resulted in false-negative results due to the polymorphic nature of Blastocystis spp. (404, 419). It has been suggested that trichrome-stained fecal smears or xenic in vitro culture systems offer the best sensitivity (90, 418). However, slower-growing subtypes (e.g., subtype 7) may be missed with this procedure (418). The formol ether concentration technique (FECT), which is commonly used for laboratory identification of OCP, is not recommended for Blastocystis due to its inability to isolate Blastocystis spp. (309, 404, 419).

Several molecular techniques with increased sensitivity have been developed (341). Advancements in the sequence analysis of Blastocystis-specific PCR products and subtype-specific PCR primers have led to progress in identifying several subtypes (268, 312, 341, 402, 418, 451). PCR using the SSU rRNA gene is being used increasingly for detection of Blastocystis spp. Despite being more costly, PCRs have demonstrated much higher sensitivities than more commonly used methods such as permanent staining (48%) (211, 341), direct light microscopy (29%), and xenic in vitro stool culture analysis (52%) (320). Owing to the wide genetic diversity among Blastocystis spp., the choice of primers is crucial from a diagnostic perspective (341).

Although disagreement about the pathogenicity of Blastocystis spp. still exists, treatment is indicated when there is no alternative explanation for symptoms (109, 218). Metronidazole is the suggested drug of choice, although failures of this drug in eradicating the organism are common (275, 407, 409). Cotrimoxazole, nitazoxanide, and a combination of paromomycin and metronidazole have also been used (222, 485). A recent study found extensive differences in drug sensitivities among two clinically important zoonotic subtypes (subtypes 4 and 7) and identified four new potential therapeutic options against Blastocystis spp., namely, mefloquine, cotrimoxazole (trimethoprim-sulfamethoxazole) (1:2), ornidazole, and furazolidone; the study also confirmed the antiprotozoal activities of 10 compounds already reported to be effective against Blastocystis spp. (272). It is clear that antimicrobial eradication of Blastocystis spp. is possible; however, because of the difficulty of the problem, a reevaluation of current treatment regimens is required, with a view to defining clear new treatments. Consideration should also be given to the fact that improvements in symptoms after treatment with metronidazole or trimethoprim-sulfamethoxazole may be due to clinical effectiveness of these drugs against coinfecting pathogens (71). There is therefore a need for further investigations to better understand drug efficacy, resistance, and reinfection issues (407).

Cystoisospora belli (formerly Isospora belli)

Cystoisospora belli causes intestinal disease in several mammalian hosts (42). Infections are thought to be acquired through the ingestion of mature environmentally resistant sporulated oocysts in contaminated food or water, although good evidence for the source of infection in most infected patients is limited (109, 219). Infection is almost indistinguishable from cryptosporidiosis (208) and is usually self-limiting and characterized by watery diarrhea, abdominal cramps, anorexia, and weight loss (175, 311). Cystoisospora belli is often implicated in traveler's diarrhea in travelers to developing countries with high levels of endemicity (2, 156, 311, 313, 427). It is more common in AIDS patients (165, 230, 453), other immunocompromised patients (163, 227, 266, 336, 344), and indigenous populations in the United States (291, 315). In HIV-infected patients, infection may be characterized by chronic diarrhea, acalculous cholecystitis cholangiopathy, and extraintestinal infection (337, 453). Other Cystoisospora species are important causes of diarrhea in domestic animals (108), and Cystoisospora suis is an economically important parasite causing severe diarrheal illness in pigs (8, 212, 282).

Diagnosis is made by direct microscopic observation of the oocyst in feces, with acid-fast staining, since the oocysts are large (20 to 23 μm by 10 to 19 μm) and morphologically distinctive. Diagnosis has also been done from mucosal biopsy specimens (227, 453). Molecular techniques such as conventional and real-time PCR can be used to augment diagnosis where possible, since these methods can be more sensitive in detecting infection (283). However, while PCRs have shown excellent sensitivity and specificity for the detection of C. belli in fecal samples, these are neither widely nor commercially available (283, 337, 347, 390, 427, 453). Infection usually responds to treatment with oral cotrimoxazole, and where resistance or intolerance exists, ciprofloxacin is a good alternative (109).

Balantidium coli

Balantidium coli is a ciliate and is the largest protozoan that infects humans (114, 383). Pigs are thought to be the natural host of this parasite, despite showing no clinical disease (111, 359). Infections in humans are acquired via the fecal-oral route from the ingestion of cysts present in untreated or minimally treated water and on uncooked or undercooked food (114, 374). Human infection is common mainly in communities that live in close proximity to pigs (114, 359). The vast majority of infections are asymptomatic, but mild diarrhea and abdominal discomfort have been reported in symptomatic patients. A few patients develop fulminating acute balantidiasis, with intestinal perforation leading to a case fatality rate of about 30%, or fulminating dysentery associated with hemorrhage and shock, resembling amoebic dysentery (120, 359). Balantidiasis is uncommon in developed countries, but infections have been reported as far north as Sweden, Finland, and Northern Russia, with the highest prevalence rates in tropical and subtropical regions (383). In developed countries, clinicians and laboratory technologists should consider balantidiasis as an alternate diagnosis for patients presenting with watery diarrhea who have a recent travel history to developing countries, especially those in Southeast Asia, the Western Pacific islands, and rural South America (111, 120, 359, 360).

Because of its large size (cysts are 50 to 70 μm; trophozoites are 30 to 200 μm by 40 to 70 μm) and spiraling motility, Balantidium is easily recognized in wet mount slide preparations. Trophozoites are visible with a hand lens, and sometimes with the naked eye, in freshly collected diarrheic stools (114, 359) as well as in bronchoalveolar wash fluid; cysts are more common in formed stools. Collection of stool samples over several days is recommended because excretion of parasites can be erratic (359). Treatment is done with tetracycline or metronidazole for 5 or 10 days, respectively (109).

Intestinal Microsporidiosis (Enterocytozoon bieneusi and Encephalitozoon intestinalis)

Enterocytozoon bieneusi and Encephalitozoon intestinalis are species of microsporidia that cause enteric disease. These parasites, historically considered spore-forming protozoa, are now classified as fungi (69, 150, 157). However, based on their importance, especially in immunocompromised populations, they are included in this review. Intestinal microsporidiosis was first identified in AIDS patients, with E. bieneusi being the more common of the two causative species (81, 398). Many identical genotypes of E. bieneusi from humans and animals are known (81), raising concerns over waterborne, food-borne, and zoonotic transmission. The importance of this parasite in human infections increased as it became a cause of opportunistic infections in HIV/AIDS and other immunosuppressed patients during the 1980s (6, 29, 114, 248, 315). However, with increasing access to antiretroviral therapy, infection rates in HIV/AIDS patients have reduced significantly (390, 448), with prevalences ranging from 0 to 42% (13). Infections have also been found in non-HIV/AIDS and immunocompetent patients (1, 248) and in persons with corneal infections (263), and there are also many infections thought to be associated with protracted traveler's diarrhea, during or after travel (156). Infections with Encephalitozoon spp. are believed to occur through ingestion or inhalation of spores (86), with the primary infection developing in the epithelium of the small intestinal or respiratory tract (86). Severe ulceration of the small bowel is associated with mucosal atrophy and acute and chronic inflammation (13). In the case of E. bieneusi, infection occurs mainly through ingestion of spores, which subsequently develop within the epithelial cells lining the duodenum and jejunum of the small intestine (86). Gastrointestinal symptoms include persistent diarrhea, abdominal pain, and weight loss, especially in immunosuppressed individuals (86). In otherwise healthy individuals such as travelers, E. bieneusi infection has resulted in self-limiting diarrhea of approximately 1 month (86). Fecal-oral transmission is implicated most often as the means of infection (96).

The diagnosis of intestinal microsporidiosis is difficult and is usually based on microscopic detection of the spores in stool samples, requiring special fluorescent or trichrome stains to detect the small spores (114, 200, 222). However, the detection of the spores and species determination can be difficult due to their small size (222), requiring reliable immunological diagnostics to supplement PCR or histochemistry when sporadic spore shedding is suspected (88). One study found that microscopy and staining demonstrated low sensitivity for the detection of microsporidian species compared with the calcofluor white technique and staining with DAPI (4′,6-diamidino-2-phenylindole), which demonstrated a higher sensitivity and specificity (97.12% and 98.55%, respectively) (439). However, the disadvantage of calcofluor white staining is that it requires a fluorescence microscope and is a nonspecific stain leading to the fluorescence of other organisms and artifacts which can be mistaken for microsporidia (78, 423). PCR-based methods have now been used successfully for detection of microsporidian infections, exhibiting excellent sensitivity and specificity (186, 248, 349).

Where treatment is indicated, albendazole, administered at 400 mg every 12 h for 1 month, is effective against Encephalitozoon species (13, 114, 222). Enterocytozoon bieneusi is less responsive to albendazole, and while symptoms may improve, microsporidian spore shedding is likely to continue (136). Fumagillin is the only treatment that has demonstrated consistent efficacy against E. bieneusi infection; however, neutropenia and thrombocytopenia have been reported as major side effects in both HIV-infected patients and transplant recipients, requiring monitoring of tacrolimus levels (58, 276). Supportive fluid therapy in immunocompromised patients and early initiation of HAART in HIV-infected patients are crucial to help restore the immune status of the host (87, 114, 117). Significant reductions have been seen in the incidence of intestinal microsporidiosis in HIV-infected patients since the introduction of HAART (398, 448).

Food-Borne Transmission

The majority of the enteric protozoa discussed herein can be transmitted by food. Although the risk of obtaining a food-borne protozoan infection is lower in developed settings, the significance and impact of these infections cannot be ignored (125, 183, 222, 288). In developed countries, large-scale food production, distribution, retailing, and importation of raw food ingredients increase the risk for the spread of food-borne infectious disease and often result in costly recalls (250). Intestinal protozoa can contaminate food through a variety of routes (42). Food can become contaminated with parasites during the production stage, from contaminated irrigation water, soil, untreated manure, or biosolids used as fertilizers (54). Food may also become contaminated during the harvesting, handling, and preparation processes, from cross-contamination with soiled implements, animal manure, or contaminated water used for preparation or by the hands of the food handlers themselves (79, 157, 174). The risk of food-borne transmission is increased when food is consumed raw, undercooked, or in a semicooked form (198, 322). Food becomes contaminated directly from feces, soil, irrigation water, sewage, and human handling during various phases of the food production chain (115, 288, 383).

Food-borne outbreaks cost individuals and families thousands of dollars in medical costs and lost wages. The food industry suffers financially even more due to recalls of food products, litigation, and, sometimes, closure (38, 184, 197). In recognizing the public health and trade implications of pathogens in fresh fruit and vegetables, the FAO and WHO have provided scientific advice to the Codex Committee on Food Hygiene (CCFH) on the fresh produce commodities of greatest concern from a global perspective (113). The submission looked at microbial hazards in produce that is marketed fresh, and often ready-to-eat, throughout the production-to-consumption continuum, with the development of specific management guidance (113).

Cryptosporidium, C. cayetanensis, and Giardia are the main enteric protozoa associated with food-borne infections in developed countries (291). A U.S. report for the years 2000 to 2008 indicated that a significant proportion of laboratory-confirmed cases of food-borne parasitic diseases were due to G. intestinalis (356). In addition, of a total of 53 cases of seafood-associated outbreaks of parasitic infection reported in the United States from 1973 to 2006, Giardia was responsible for 43%; more than half (55%) of these were associated with the consumption of fish (199). It was reported that from 1990 to 2000, there were 11 food-borne outbreaks of cyclosporiasis in North America that affected at least 3,600 people (259). Almost all cases of C. cayetanensis infection in the United States are food borne (173, 356). Several cases of food-borne cyclosporiasis related to the consumption of salads have also been reported in European countries (92, 198, 350). According to one U.S. report, source attribution is often affected by insufficient numbers of cases for definitive case-control studies and a lack of tools for molecular epidemiology (173). Approximately 10% of Cryptosporidium infections are ascribed to food-borne transmission (171), and outbreaks have been associated with eating raw produce or salads (66, 187, 322, 476). Food-borne outbreaks associated with imported foods have been reported in several industrialized countries (92, 189, 271, 291, 322, 450). It is therefore important to have adequate food safety standards in place to govern locally produced and imported foods, which is essential to the safety of the food supply chain (220). In addition, industrialized countries should give priority to the institution of sensitive disease surveillance systems and the development of molecular methods for linking cases of protozoan infections (162, 173, 258).

Waterborne Transmission

The role of water resources in the transmission of enteric protozoa cannot be overemphasized, especially where there is the potential for water supply contamination. Despite ongoing investment in better sanitation infrastructure, water quality, and environmental protection legislations and the subsequent reduction in pathogen loads in public water supplies, waterborne disease outbreaks still pose significant risks to human health in developed countries (34). Small water supplies in rural communities in developed countries may be more vulnerable to contamination than the larger public water supplies, resulting in outbreaks of infectious disease (195, 317). In the United States, it is suspected that seasonal peaks of cryptosporidiosis are related to increased use of recreational water venues such as lakes, rivers, swimming pools, and water parks in summer months (188, 463). Cryptosporidium hominis subtype IbA10G2 was the causative agent identified in the largest waterborne outbreak of cryptosporidiosis reported in Australia to date and was associated with public swimming pools in densely populated coastal cities (463). Surveillance-based data indicate a significant increase in onset of cryptosporidiosis and giardiasis during summer through early fall in the United States (476, 477). These are likely associated with increased outdoor activities and increased exposures, such as camping and use of communal swimming venues (e.g., lakes, rivers, swimming pools, and water parks) by young children, during the summer recreational water season (476, 477). Cryptosporidium spp. remain the leading cause of diarrheal illness outbreaks in treated recreational water venues in the United States (476). One study determined that acute diarrheal illness associated with small community water supplies could cost an estimated $4.671 billion (95% CI, $1.721 to $9.592 billion), with the capital costs of intervention amounting to $13.703 billion (95% CI, $6.670 to $20.735 billion) and those of postinfectious IBS resulting from these acute gastroenteritis outbreaks amounting to $11.896 billion (95% CI, $3.118 to $22.657 billion) (195).

The oocysts of several protozoa are highly resistant to chlorination, a conventional water treatment method (43). It is suspected that when water becomes contaminated, oocysts can become trapped by chlorine and UV-resistant biofilms in water pipelines and constantly shed into the water supply (68, 123, 466). While availability of potable water and water treatment standards are generally better in developed countries, remote and isolated areas often have deficient water systems, resulting in waterborne outbreaks, for example, in South Bass Island, OH (303), and in France (135). Giardia intestinalis and Cryptosporidium spp. have been implicated in several large waterborne outbreaks in the United States (72, 252, 303, 352), Norway (297), Australia (295, 463), and elsewhere (360).

Many countries worldwide have legislation or follow standard guidelines to monitor microbial indicator organisms to determine the microbiological quality of water (68, 317). Traditionally, fecal indicator bacteria (FIB)—total coliforms, fecal coliforms, and enterococci—are used as microbial indicators, and there are very few reports outlining the use of protozoa as indicators of fecal pollution (190, 269, 310, 353). Unfortunately, many waterborne pathogens are still difficult to detect, and despite advances in molecular diagnostics, such methods are not widely available or used even in developed countries (123). Increased water shortages in some developed countries have caused authorities to resort to the use of recycled water from highly contaminated sources such as sewage (67, 239). The increased use of biosolids from sewage as soil conditioners as a means of sustainable disposal has raised issues about the potential for the transmission of infectious pathogens, including the protozoa G. intestinalis, Cryptosporidium spp., Balantidium, Entamoeba spp., Blastocystis spp., and Dientamoeba fragilis (23, 104, 217). These issues have been the subject of extensive debate in many countries, such as Australia, New Zealand, the United States, and countries in the European Union, highlighting an extensive gap in knowledge on the spread of enteric protozoa through recycled sewage and biosolids (67, 243).

General Measures

In domestic settings, simple prevention and control measures are effective against most enteric parasites. These include personal hygiene; hand washing after using or handling the toilet, changing diapers, or caring for a person with diarrhea and before preparing or handling food (366); proper disposal of excreta; and washing of soiled materials such as clothing or bedding. The use of adequately treated water, for example, by boiling, for drinking, food preparation, and washing of fruits and vegetables is essential (57). In the food industry, some protozoa get into food through fecal contamination of raw materials and inadequate treatment of the food before consumption or through posttreatment contamination. This is true for the oocysts or sporocysts of Cryptosporidium spp. and C. cayetanensis (291, 374). Control measures must therefore be aimed at all aspects of the food chain, including minimizing dissemination of cysts and oocysts in the farming environment and via human waste management (373). This may include abstinence from using untreated human and animal manure in farming.

Food hygiene and management practices in food service and catering industries should include restricting the purchase of raw materials to suppliers with good agricultural practices (95) and having adequate facilities for washing and cleaning of raw fruits and vegetables before storage and before preparation (220). A system for identifying and controlling microbial hazards, such as the Hazard Analysis Critical Control Point (HACCP), should be employed by food and water production companies, in industries or sectors that use fresh produce, and in operations in which contaminated water or ingredients could end up in the product (79, 172, 202).

Persons working in high-risk environments such as schools, day care centers, food-handling facilities, and aged-care institutions should be educated properly on their risk of infection (297), and where infection occurs, persons should be excluded for up to 48 h after the last diarrhea episode (10). Infected persons such as travelers, campers, and hikers should refrain from passing of feces into rivers and streams and should not use recreational waters such as swimming pools and public baths for up to 2 weeks after symptoms cease (57). Cryptosporidium spp. and Giardia intestinalis in swimming pools are difficult to control (325). Open farms and petting zoos should have guidelines in place for the handling of animals and should ensure that adequate hand-washing facilities are in place for visitors and staff. Wilderness backpackers and travelers to developing countries must exercise precautions with food and drinking water safety (331). Basic hygiene, including hand washing and cleaning of cookware, is essential in the prevention of diarrhea (10). Long-term groups, including military, missionary, aid worker, volunteer, and tourist groups, should seek travel advice. They should also take with them adequate supplies to treat water for domestic use (e.g., filtration and boiling), as chlorination by itself cannot remove the oocysts of protozoa such as Cryptosporidium spp. and C. cayetanensis from water (12, 19). Routine boiling should be used to augment chlorination in less developed settings (331). Since domestic house flies can play a role in the efficient transmission of human protozoan parasites, care must be taken with food exposed to flies (160). In the case of cyclosporiasis, it is important that it be considered by microbiologists and physicians as a possible cause of illnesses in cases of prolonged diarrhea that are not associated with travel, as well as in food-borne outbreaks associated with imported foods (306).

Water and Wastewater Quality Control

Detection of Protozoa in Water Samples

Detection of protozoa in water samples is important for the monitoring of contaminants and for the prevention and control of parasitic infections from water. Their small size and dispersion in water and the difficulty in efficiently concentrating oocysts from environmental samples while excluding extraneous materials make it difficult to detect protozoa in water by traditional methods, with detection requiring well-trained and experienced personnel (45, 47). The biggest problem with identifying protozoa in water supplies is that cultivation from cysts or oocysts is difficult and it is almost impossible to concentrate oocysts from environmental samples while limiting the presence of extraneous materials. As a result, enteric bacteria are traditionally used as surrogate markers for fecal contamination of water sources (47). However, the development of new assays and methods for the purification and concentration of cysts or oocysts by the use of immunomagnetic separation (IMS) has addressed this problem (375). Currently, the U.S. EPA and other such bodies commonly use detection methods for isolation of protozoa, from either large (up to 1,000 liters) or small (10 to 50 liters) samples, involving four sequential steps: (i) the filtration of water, resulting in the cysts or oocysts and extraneous materials being retained on the filter; (ii) the elution and separation process, involving purification and concentration of cysts or oocysts by IMS and discarding of the extraneous material; (iii) staining with specific fluorescent antibodies (FA); and (iv) enumeration using fluorescence and differential interference contrast microscopy (45, 48, 106, 375). Other techniques that can also be used for cyst or oocyst purification purposes include density gradient, saturated-salt flotation, and continuous flow centrifugation, continuous flow filtration, and flow cytometry with cell sorting (45, 251, 299). Additional molecular methods are currently available for the identification of protozoa in water samples by detection of pathogen-specific genes or SSU rRNA (292, 375). For example, nested PCR-RFLP assays are used to target different loci (1 and 2) of the hypervariable region of the 18S rRNA gene for identification of different Cryptosporidium spp. in water (292). Other PCRs have been developed to amplify the COWP gene (9). Species of microsporidia have been identified in various water sources by use of Weber's chromotrope-based stain, with positive samples analyzed by PCR (200). Several authors suggest that molecular techniques are the most promising methods for the sensitive, accurate, and simultaneous detection of waterborne protozoa, with much benefit to the water industry and public health (47, 101), compared with conventional staining and microscopy (205, 386, 387).

Another approach is the use of alternative bioindicators together with conventional fecal markers to identify the source of fecal pollution and associated pathogens in water (353). Water can become contaminated by point sources (raw sewage, storm water, wastewater effluent, and industrial sources) and non-point-source discharges (agriculture, forestry, wildlife, and urban runoff). The identification of the source of fecal contamination and pathogens could aid in the management and remediation of water resources when other control strategies are not feasible (353). However, some scientists suggest that fecal indicators do not adequately indicate the presence of all fecal pathogens in natural waters because their presence is affected by different environmental conditions (147, 241, 310). Since the cysts and oocysts of enteric protozoa are hardy and chlorine resistant and can survive for longer periods in water, these could be evaluated for use as alternative fecal indicators (310). The WHO suggests that the spores of the anaerobic bacteria Clostridium perfringens and Bacillus spp. can be used to evaluate the effectiveness of protozoan cyst or oocyst removal by sewage/water treatment, since they are highly resistant in the environment and their vegetative cells do not reproduce in aquatic sediments (18).

Evaluating the presence of fecal indicator organisms in water or the bioaccumulation of encysted protozoa in shellfish has value for identifying source attribution for surveillance and in outbreak settings (115). Bivalve shellfish have been used as bioindicators of fecal protozoan contamination in water bodies. For example, Cryptosporidium and Giardia in clams (Corbicula fluminea) have been detected by PCR and DFA assays (270) or IFA (148). When it is not possible or practical to test for individual protozoan parasites, it may be useful to use a combination of fecal indicator source tracking methods to enhance identification of contaminants of public health interest in surface waters (121, 270, 353).