Understanding Intestinal Spore-Forming Protozoa: Cryptosporidia, Microsporidia, Isospora, and Cyclospora

  1. Richard W. Goodgame, MD
  1. From Baylor College of Medicine and Ben Taub General Hospital, Houston, Texas. Acknowledgments: The author thanks Drs. Linda Rabeneck, Wayne Shandera, and Cynthia Chappell for critiques of the manuscript. Credit for the photoµgraphs goes to Dr. Robert Genta (Figure 2 and Figure 3, left), Dr. Charles Stager (Figure 3, right), Professor Sebastian Lucas (Figure 4, left), and Dr. P.L. Chiodini Figure 4, right, and Figure 5. Requests for Reprints: Richard W. Goodgame, MD, Baylor College of Medicine, One Baylor Plaza, Room 525-D, Houston, TX 77030.
     
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    Abstract

    Objectives: To summarize recent information about the “new” gastrointestinal protozoal pathogens (cryptosporidia, microsporidia, isospora, and cyclospora) and to help practicing clinicians integrate this information into their clinical databases by emphasizing the similarities among these organisms.

    Data Sources: Relevant English-language articles published between 1988 and 1995 were identified through a MEDLINE search done using the names of the intestinal spore-forming protozoa. Articles cited in the bibliographies of these and other articles were searched manually.

    Study Selection: Studies that contained information on the history, taxonomy, life cycle, epidemiology, pathogenesis, clinical manifestations, diagnosis, and treatment of the pathogens were reviewed.

    Data Extraction: Cryptosporidium parvum, Isospora belli, Cyclospora cayetanensis, Enterocytozoon bieneusi, and Septata intestinalis are intestinal spore-forming protozoa that cause intracellular infections, predominantly in the epithelial cells of the intestine. They are transmitted either by stool from person to person or through contaminated water or food by an infectious particle called a spore or oocyst. Asymptomatic infections occur; the most common symptom of infection is diarrhea. Infections have been associated with intestinal inflammation, disordered architecture (such as villus blunting), and abnormal function (for example, malabsorption). Mild to moderate, self-limited diarrhea is common in healthy persons, but patients with immune dysfunction can have severe intestinal injury and prolonged diarrhea. Diagnosis is made by a microscopic examination of the stool and the use of appropriate staining techniques. Effective antibiotic treatment for prolonged infection in immunocompromised patients is available for most of these infections.

    Conclusions: The intestinal spore-forming protozoa are four frequently identified gastrointestinal pathogens that have important similarities in epidemiology, disease pathogenesis, clinical manifestations, diagnosis, and treatment.

    For most primary care internists, gastrointestinal parasitology was, until recently, limited to a few endemic protozoa (giardia and amoebae) and the occasional roundworm infestation (hookworm, ascaris, trichuris, and strongyloides). Since the onset of the acquired immunodeficiency syndrome (AIDS) epidemic, the number of parasitic pathogens recognized and the frequency with which they are encountered in clinical practice have increased. The four intestinal protozoa that have been increasingly identified in patients with AIDS are cryptosporidia, microsporidia, isospora, and cyclospora. These organisms have also been implicated as important pathogens in otherwise healthy persons. Each has unique features that have recently been reviewed [1-6], but they also have many common characteristics.

    My primary objective is to point out the common characteristics of these organisms and the diseases that they produce. The shared features of these organisms, summarized in Table 1, provide a framework of understanding that practicing clinicians can use to easily integrate new information about these pathogens into their clinical databases. Once these four organisms are seen as members of a clearly defined class—the intestinal spore-forming protozoa—clinical recognition of them can be facilitated. Some of the differences among the intestinal spore-forming protozoa affect clinical decision making; these are also discussed and are summarized in Table 2.

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    Table 1. Similarities among the Intestinal Spore-Forming Protozoa
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    Table 2. Important Differences among the Intestinal Spore-Forming Protozoa*
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    History

    Several authors have referred to these organisms as “new” pathogens [2, 7-10]. This generalization is reasonably correct. Although human intestinal isosporal infection has been known for many decades [11-15], the first reports of human cases of intestinal cryptosporidia were reported only 20 years ago [16, 17]; those of cyclospora were published only 17 years ago [18, 19]; and those of the two intestinal microsporidia were published only 11 and 3 years ago [8, 20-22]. Throughout the 1980s, numerous articles showed that cryptosporidia are not rare opportunistic pathogens but rather are the cause of common, worldwide infections in healthy children and adults. Cyclospora were discovered [19] and rediscovered [23] as the cause of a “new” infection in otherwise healthy adults. They were, for a time, thought to be a blue-green algae (or cyanobacteria) [24, 25], but subsequent studies have shown that they are protozoa [2] with an epidemiology similar to that of cryptosporidia. The AIDS epidemic has played an important role in the recognition and clinical management of these “new” organisms, because all of the spore-forming protozoa are important AIDS-associated pathogens [3, 26-29]. The two organisms that cause intestinal microsporidiosis were first identified in patients with AIDS [8, 20-22]. Human intestinal microsporidial infections have been reported almost exclusively in patients with AIDS, but increasing awareness and recognition may change this [30].

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    Taxonomy, Biology, and Life Cycle

    Taxonomy

    All four organisms are protozoa and therefore share some characteristics with other medically important protozoa (see Table 3[31]). Although the intestinal spore-forming protozoa are classified into two separate phyla, the features that unify them are much more important to clinicians than the morphologic features that justify taxonomic separation. As Table 3 shows, the human intestinal spore-forming protozoa comprise five organisms called by four nontaxonomic names: cryptosporidia, microsporidia, isospora, and cyclospora. The use of the nontaxonomic terms “cryptosporidia,” “isospora,” and “cyclospora” in reference to human gut protozoal pathogens is unambiguous, because Cryptosporidium parvum, Isospora belli, and Cyclospora cayetanensis are the only members of their respective genera known to infect humans. The nontaxonomic term “human microsporidia” can actually refer to any of the five microsporidia known to cause disease in humans (Table 3). Three of these microsporidia are rare and nonintestinal: Encephalitozoon species, Nosema species, and Pleistophora species [4, 32]. The other two are common intestinal pathogens in patients with AIDS [33]: Enterocytozoon bieneusi and Septata intestinalis. Therefore, when human intestinal protozoal infections are discussed, the term “microsporidia” refers to these two organisms.

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    Table 3. Human Protozoal Pathogens*

    Biology and Life Cycle

    Many, perhaps most, animal species have infections with spore-forming protozoa [2, 14, 32]. However, the species of intestinal microsporidia, isospora, and cyclospora found in humans are different from those found in animals. Therefore, humans acquire infections from other humans. In contrast, the same species of cryptosporidia that causes disease in humans is an important cause of disease in cattle, goats, and other farm animals [34]. Unlike the other intestinal spore-forming protozoa, cryptosporidia has little host specificity, and acquisition from animals has been documented [35, 36]. However, most cryptosporidial infections do not result from animal contact [37, 38], and thus, most patients are infected with cryptosporidia and the other intestinal spore-forming protozoa through direct contact with another human or through food or water contaminated by another human.

    Ingestion of the spore begins a life cycle that is similar for all four of the intestinal spore-forming protozoa. The ingested spores release sporozoites, which invade enterocytes, primarily in the small intestine. The enterocyte infection progresses through two stages. The merogonic (or schizogonic) stage involves the maturation and development of meronts to reproduce and multiply in the infected cell or to infect other enterocytes of the host. This asexual stage allows the infection to spread to many enterocytes even if the host is not repeatedly exposed to the organism. The sporogonic (or gametogonic) stage involves the maturation and development of sporozoites enclosed in cysts or spores. As the infected enterocytes die, cyst or spore shedding occurs. The cysts or spores are sloughed into the gut lumen to be excreted in the stool.

    To emphasize the similarities among the organisms in life cycle and location of infection, Figure 1 shows a small-intestinal villus that is infected with all four of the intestinal spore-forming protozoa. This figure illustrates three main similarities. First, the spore-forming protozoa cause infections of the enterocyte, the epithelial-lining cell of the intestine. Invasion below the epithelial surface is not common. Second, the infections are intracellular; the organisms replicate and mature within the enterocytes. Third, the final product of maturation is the infectious particle, the oocyst or spore, which is sloughed into the lumen of the intestine. These particles pass out of the body with the stool. These similarities in biology and life cycle explain why these organisms have such similar pathophysiology and clinical manifestations. Figure 1 also shows some of the differences among these four organisms, including their relative sizes and various intracellular locations. These differences allow a specific morphologic diagnosis to be made when the stool specimen, duodenal aspirate, or small-bowel biopsy specimen is examined (Table 2). Figure 2, Figure 3, Figure 4, Figure 5 show real examples that correspond to Figure 1.

    Figure 1. All four organisms mature and multiply intracellularly in enterocytes, and all produce an infectious particle (spore or oocyst) that is excreted in the stool. However, the organisms vary in size and intracellular location.
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      Figure 1. All four organisms mature and multiply intracellularly in enterocytes, and all produce an infectious particle (spore or oocyst) that is excreted in the stool. However, the organisms vary in size and intracellular location. A small-intestinal villus infected with all four intestinal spore-forming protozoa.
      Figure 2. Left. As seen in jejunal biopsy specimen (hematoxylin and eosin stain; original magnification, × 400). Right. As seen in the feces (modified acid-fast stain; original magnification, × 1000). Actual size, 4 to 6 microns.
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        Figure 2. Left. As seen in jejunal biopsy specimen (hematoxylin and eosin stain; original magnification, × 400). Right. As seen in the feces (modified acid-fast stain; original magnification, × 1000). Actual size, 4 to 6 microns. Cryptosporidium parvum.
        Figure 3. Left. As seen in jejunal biopsy specimen as irregular oval bodies in the apical cytoplasm of most of the enterocytes (hematoxylin and eosin stain; original magnification, × 400). Right. As seen in the feces as red-pink oval spores mixed among blue bacteria (modified trichrome stain; original magnification, × 1000). Actual size, 1 micron.
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          Figure 3. Left. As seen in jejunal biopsy specimen as irregular oval bodies in the apical cytoplasm of most of the enterocytes (hematoxylin and eosin stain; original magnification, × 400). Right. As seen in the feces as red-pink oval spores mixed among blue bacteria (modified trichrome stain; original magnification, × 1000). Actual size, 1 micron. Enterocytozoon bieneusi.
          Figure 4. Left. As seen in jejunal biopsy specimen as large, densely staining, oval bodies in the apical cytoplasm of three enterocytes surrounded by an artifactual clear space (hematoxylin and eosin stain; original magnification, × 400). Right. As seen in feces (modified acid-fast stain; original magnification, × 1000). Actual size, 25 microns.
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            Figure 4. Left. As seen in jejunal biopsy specimen as large, densely staining, oval bodies in the apical cytoplasm of three enterocytes surrounded by an artifactual clear space (hematoxylin and eosin stain; original magnification, × 400). Right. As seen in feces (modified acid-fast stain; original magnification, × 1000). Actual size, 25 microns. Isospora belli.
            Figure 5. Left. Transmission electron photoµgraph of jejunal biopsy specimen showing six merozoites of within an enterocyte. Right. oocyst in feces (modified acid-fast stain; original magnification, × 1000). Actual size, 8 microns.
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              Figure 5. Left. Transmission electron photoµgraph of jejunal biopsy specimen showing six merozoites of within an enterocyte. Right. oocyst in feces (modified acid-fast stain; original magnification, × 1000). Actual size, 8 microns. Cyclospora cayetanensis.C. cayetanensisCyclospora
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              Epidemiology

              The epidemiology of the intestinal spore-forming protozoa is not fully understood. Work in this area has been hampered by a lack of complete surveillance, widespread serologic surveys, and extensive stool examinations done by trained observers using sensitive techniques. All of these organisms may be common enteric pathogens worldwide; their frequency may be related to the adequacy of sanitation in the environment. In general, the infections are more common in developing countries, but expanding poverty, urban migration, air travel, and immigrant populations in conjunction with other societal and environmental changes make the distinction between “tropical” and “Western” enteric diseases less clear-cut than it has been in the past [39]. Worldwide, the predominant method of transmission is the fecal-oral route; transmission can be direct or can occur through contaminated water and food [4, 5, 40, 41]. Specific epidemiologic features of each organism are discussed in the following paragraphs.

              Cryptosporidia cause substantial illness throughout the world [1, 38, 39]. The actual frequency of stool specimens found to be positive for cryptosporidia varies according to the characteristics, including symptom status and immunologic function, of the population studied. The generally quoted prevalences of cryptosporidia in stool specimens are 1% to 3% in Europe and North America and 5% to 10% in Asia and Africa [1, 42]. However, even in developed countries, where prevalence rates by stool examination have been found to be low, antibodies to cryptosporidia have been found in 32% to 58% of adults [43]. Cryptosporidia are highly transmissible in the family setting; transmission rates are similar to those of other highly infectious enteric pathogens, such as Shigella species [44]. For example, in one study, 19% of household contacts of index cases of cryptosporidiosis developed acute infection [44, 45]. Cryptosporidia have been implicated as a cause of diarrhea in travelers [46, 47] and of epidemic diarrhea in hospitals [48], day care facilities [49], urban centers [41, 50], and other institutions worldwide [1, 44].

              Isospora belli, which is less common than cryptosporidia, is endemic in many parts of Africa, Asia, and South America [13]. Exact prevalence rates and details of epidemiology are not known. The higher prevalence of isospora in developing countries is shown by the frequency with which isospora complicate AIDS in those countries. For example, among Haitian patients with AIDS, the frequency of isosporiasis is 15%, but among U.S. patients with AIDS, it is only 0.2% [28, 51]. Different frequencies of infection in patients with AIDS often reflect the different prevalences of pathogens in different environments [52]. Even in the United States, however, sporadic outbreaks have occurred in mental institutions and day care centers [5, 11, 12].

              Knowledge of epidemiology is much more limited for human microsporidia [33]. The widespread occurrence of intestinal microsporidial infection in patients with human immunodeficiency virus (HIV) disease and the potential for human-to-human fecal-oral transmission of microsporidia [32] suggest that microsporidia may be common, worldwide intestinal pathogens of humans [4]. The most compelling evidence showing that microsporidia have an epidemiology similar to that of the other intestinal spore-forming protozoa is the prevalence of E. bieneusi infection in patients with AIDS around the world, which is high despite the absence of any known nonhuman source of this infection [29].

              Cyclospora have now been identified worldwide in the feces of both immunocompetent and immunocompromised patients with diarrhea [2, 6, 25, 53]. At the Hospital for Tropical Diseases in London, cyclosporal infections are now diagnosed more often than isosporal infections [54]. Details of the life cycle, the host range, and the mode of transmission are unknown. Preliminary data suggest similarities with other intestinal spore-forming protozoa. For example, a study in Peru [2] found that both cryptosporidial and cyclosporal infections occurred at the same time of year and affected primarily children 1 to 2 years of age; this suggests that these organisms have a common mode of transmission. A recent report from Massachusetts [55] showed that cyclospora may cause community-acquired diarrhea in otherwise healthy persons in the United States. Fecal-oral transmission through infected water caused an epidemic of cyclosporal infections in Chicago [40, 53].

              All four intestinal spore-forming protozoa are frequent AIDS-related pathogens. Cryptosporidia are found in the stools of 10% to 20% of patients with AIDS-associated diarrhea [1, 27, 56-63]. In many series (but not all [64]), microsporidia are even more commonly associated with chronic diarrhea in patients with AIDS than are cryptosporidia; the prevalence ranges from 6% to 50% [4, 26, 33, 63]. The frequency with which cryptosporidia and microsporidia are identified in the stools of patients with AIDS is related to the CD4 lymphocyte count (identification is more frequent when the count is less than 100 cells/mL) and the presence of gastrointestinal symptoms [1, 62, 65]. In the recent series by Kotler and Orenstein of 141 patients with AIDS and diarrhea [62], 35% of patients had microsporidia (51 had E. bieneusi and 5 had S. intestinalis; this is similar to the distribution found by Field and coworkers [9]), and 23.4% had cryptosporidia.

              Isospora are infrequently associated with AIDS-related diarrhea in the United States and Europe (about 2% [62, 64]) but are common in patients with AIDS and diarrhea in Brazil (9.9% [66]), Zaire (12% [67]), Zambia (16% [68]), and Haiti (12% [3]). These rates are similar to the 11% prevalence rate found for cyclospora in Haitian patients with AIDS and diarrhea [3]. Cyclospora has only occasionally been seen in U.S. patients with AIDS [24, 69]). Pape and colleagues [3] give four reasons why this prevalence is so low: An untrained observer might confuse cyclospora with cryptosporidia; an acid-fast stain is not always done on the stool specimen; trimethoprim-sulfamethoxazole prophylaxis is frequently used; and cyclospora have an underlying low prevalence in developed countries. The last three reasons may also explain the low prevalence of isospora in U.S. patients with AIDS.

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              Pathogenesis

              Infection by all four intestinal spore-forming protozoa has been associated with substantial alterations in intestinal structure and function, but the pathogenesis of the predominant symptom, diarrhea, is not completely understood. New paradigms for the pathophysiology of infectious diarrhea may explain both the morphologic and the physiologic abnormalities found in intestinal spore-forming protozoal infections [70]. The hypothesized sequence of events is as follows: After the initial invasion of the enterocytes by the spore-forming protozoa, the epithelial cells release cytokines (such as interleukin-8) that activate resident phagocytes and recruit new phagocytes into the lamina propria from the blood [71]. These activated leukocytes release soluble factors that increase intestinal secretion of chloride and water and inhibit absorption [72, 73]. Some of these mediators, such as histamine, serotonin, and adenosine, affect secretion and absorption by acting directly on epithelial cells [71]. Others, such as prostaglandins, leukotrienes, and platelet-activating factor, act on the enteric nerves to induce neurotransmitter-mediated intestinal secretion. Enterocyte damage or death may be a direct consequence of parasite invasion, multiplication, and extrusion. Alternatively, cell damage could be a consequence of inflammation, mediated by T cells or proteases and oxidants secreted by mast cells [71]. Even without direct damage, activated T cells can affect epithelial cell growth and produce villus atrophy and crypt hyperplasia [74]. Regardless of the specific mechanism, marked distortion of villus architecture is accompanied by nutrient malabsorption and osmotic diarrhea.

              Specific experimental evidence that this general paradigm may relate to the pathogenesis of diarrhea in intestinal spore-forming protozoal infection is only available for cryptosporidiosis. Evidence exists for both immune-mediated abnormalities and direct cytotoxic damage to enterocytes. In the pig experimental model of cryptosporidial infection, decreased intestinal sodium absorption has been related to both decreased villus surface area and inhibition by prostaglandin E2 produced by inflammatory cells [75-77]. Stool specimens from calves [78] and humans [79] with cryptosporidial infection contain secretagogue activity that may be produced by the organisms or the host inflammatory response. Conclusive evidence showing that the organism itself produces an enterotoxin is lacking [80]. In in vitro cell culture systems, cryptosporidial infection alters cell permeability and produces cell death in the absence of host inflammatory response [81, 82]. Additional evidence indicates that the number of organisms infecting the mucosa is an important determinant of disease severity. The intensity of intestinal infection with cryptosporidia varies from patient to patient [83], and the severity of intestinal structural abnormalities and gut function defects are related to the intensity of infection [84, 85]. Heavy infections in patients with AIDS are associated with an intense inflammatory response. These patients, however, lack an effective immune response that can limit or reduce the number of parasites. Thus, the pathogenesis of diarrhea caused by cryptosporidia (and probably by the other intestinal spore-forming protozoa) is due to a complex interaction between host and parasite factors [80].

              Specific experimental evidence elucidating the pathogenesis of diarrhea in intestinal spore-forming protozoal infection is limited, but pathologic studies have documented a broad range of structural abnormalities in all four of these infections. Even in the presence of obvious infection with cryptosporidia [84], microsporidia [4], isospora [15], and cyclospora [86], small-bowel morphology can be normal. However, severe pathologic changes have also been described, including villus shortening or flattening, crypt hyperplasia, and increased numbers of leukocytes in the lamina propria and epithelium [14, 15, 54, 84, 86, 87]. Invasion and ulceration (except for that with S. intestinalis[88] do not occur. It is likely that the severe lesions are associated with heavy infection, at least for microsporidia [87] and cryptosporidia [84]. Heavy intestinal infection with microsporidia in humans is associated with severe structural alterations of the enterocyte, including changes in the shape of the cell from columnar to cuboidal to pleomorphic; atrophy of the microvilli; swelling of the mitochondria, Golgi apparatus, and endoplasmic reticulum; and accumulation of lysosomes and lipid vacuoles [89]. Areas of heavy microsporidial infection commonly show the sloughing of strips of enterocytes, denuding the tips of villi [32, 89]. Similar morphologic abnormalities have been reported in cryptosporidial [90], cyclosporal [86], and isosporal [14, 15] infections.

              The morphologic abnormalities described above have been associated with severe functional abnormalities. More than 20 years ago, clinical case studies showed that chronic isosporiasis can be associated with marked steatorrhea [14, 15]. Impaired D-xylose absorption has been documented in a few cases of cyclosporal infection in normal hosts [86, 91]. Small-bowel dysfunction, including malabsorption of vitamin B12, D-xylose, and fat, has been found in patients with AIDS and cryptosporidia, microsporidia, and isospora [92-94]. Malabsorption of vitamin B12 and D-xylose is related to the intensity of infection in AIDS-related cryptosporidiosis [85], and the morphologic abnormalities of the small intestine parallel the abnormalities of intestinal function [85].

              In summary, although the pathogenesis of diarrhea and intestinal dysfunction is unknown, current evidence suggests that when parasite numbers increase, progressive morphologic and functional abnormalities of the small intestine occur. The most severe and prolonged infections occur in immunodeficient hosts.

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              Immunity

              Epidemiologic evidence for protective immunity exists for cryptosporidial and cyclosporal infection. Among cattle workers, repeated infections with cryptosporidia occur, but the second and third infections are much milder than the first [95]. Similarly, in areas where cryptosporidiosis is common, permanent residents frequently acquire asymptomatic or clinically mild infections, but travelers become very sick [45]. A similar observation has been made about cyclosporal infection [2, 18]. Duration of diarrhea due to cyclosporal infection averaged about 20 days in Peruvian children [2] but more than 40 days in visitors to Nepal [40]. The existence of protective immunity is supported by the observation that most symptomatic cyclosporal and cryptosporidial infections in residents of an endemic area occur in infants and young children. Although severe and even lethal infections occur in children with apparently normal immunologic function [96], both T-cell dysfunction (as in HIV infection) and antibody defects (as in IgA deficiencies [97]) can predispose patients to protracted cryptosporidial infection. That infections with intestinal spore-forming protozoa occur with increased frequency and severity in immunodeficient hosts shows that immune mechanisms effectively keep parasite numbers low in most normal persons.

              Specific immune responses have been measured in cryptosporidial infections. Acute cryptosporidial infection [48, 98-101] and long-term exposure [100-102] elicit humoral antibody in both immunocompetent persons and patients with AIDS [98, 103]. Human studies indicate that intestinal antibodies can reduce parasite numbers [97, 104-107], but these antibodies do not seem able to protect patients with AIDS from heavy parasite burdens [108]. Cell-mediated immunity is necessary to prevent heavy cryptosporidial infections in humans [109, 110]. The more severe the immunodeficiency, the more likely the patient is to develop chronic infection [111]. Improved T-cell counts and immune function have been associated with reduced numbers of parasites in patients with AIDS [112-115].

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              Clinical Manifestations

              Asymptomatic Infections

              Asymptomatic infection is part of the clinical spectrum of disease produced by intestinal spore-forming protozoa. Asymptomatic infections with cryptosporidia occur in both normal and immunodeficient hosts [47, 116-124]. In one case–control study of HIV-positive patients with severe immunodeficiency [125], microsporidial infection (based on electron microscopic examination of the small-bowel biopsy specimen) was found in about 15% of patients, regardless of the presence or absence of intestinal symptoms. Some preliminary data [29] suggest that asymptomatic enteric carriage of microsporidia in patients with AIDS may precede wasting and diarrheal illness. Only 11% to 28% of immunologically normal Peruvian children with acute cyclosporal infection had diarrhea [2]. The reported frequency with which asymptomatic infection occurs is controversial, especially for microsporidia [4, 126, 127] and cyclospora [128]. The link between infection with these protozoa and clinically apparent disease is strong [40, 129, 130]. The frequency with which asymptomatic infection is identified depends on the sensitivity of the assay used to detect infection and on the immunologic status of the patients studied.

              Normal Hosts

              Acute diarrhea in normal hosts has been shown to be a prominent clinical manifestation of infection with intestinal spore-forming protozoa, except for microsporidia. Only one well-documented case of acute diarrhea caused by microsporidia in a normal host (traveler's diarrhea) has been reported [30]. The other intestinal spore-forming protozoa have been implicated as causes of acute diarrhea in infants and children living in underdeveloped countries [1, 2, 13, 131], medical personnel [12, 25, 48], travelers [25, 40, 46, 47, 54, 132-134], and persons in institutions [1, 11, 12, 25, 44]. In normal hosts, symptomatic illness caused by cryptosporidia [41, 48, 130, 131, 135], isospora [5, 11-13], and cyclospora [2, 40] is usually characterized by 3 to 25 days of diarrhea (diarrhea lasts longer with cyclospora), abdominal pain, and malaise and occasionally by nausea, vomiting, and fever. The absence of leukocytes or erythrocytes in the stool helps to differentiate this diarrhea from that caused by entero-invasive bacteria and invasive protozoa (amoebae). Prolonged diarrhea is frequent. For example, in 45% of index cases with acute cryptosporidiosis in Brazil, diarrhea lasted for more than 2 weeks [44]. One case series, published in 1970, described severe diarrhea and malabsorption lasting for months to years in six adults with isosporiasis [15]. Similar observations were made in soldiers with isosporiasis in the Philippines during World War I [13]. Prolonged (often 4 weeks) but self-limited diarrheal illness caused by cyclospora has been described in travelers to Mexico [23, 69, 136], Nepal [91, 137], South America [24], Southeast Asia [24], the Caribbean [24, 69], Pakistan [54], and India [24, 54]; in U.S. medical personnel [25]; and in infants and children from Peru [2].

              Immunodeficient Hosts

              Persons with immunodeficiency are predisposed to more frequent and prolonged infections with spore-forming intestinal protozoa. Most reported cases are in patients with AIDS, but severe infections have also been described in patients with other immunodeficiency states. Cryptosporidiosis has been reported in persons with IgA deficiency [97], and isosporiasis has been reported in persons with cancer and persons having chemotherapy for cancer [138, 139].

              The broad clinical spectrum of disease caused by these infections in immunodeficient patients ranges from asymptomatic infection to severe, life-threatening diarrhea, dehydration, and malabsorption. The clinical features of 128 patients with AIDS-related cryptosporidiosis showed four patterns of disease: transient (28.7%), chronic (59.7%), fulminant (7.8%), and asymptomatic (3.9%) [124]. Transient, self-limited diarrhea was more common in patients with less severe immunosuppression [111]. In one study [154], fulminant, large-volume diarrhea (2 L/d) occurred in patients with CD4 counts less than 50 cells/mL, and it was frequently associated with other enteric opportunistic infections. Mean survival for all patients was 25 weeks, but patients with chronic disease survived for 20 weeks, and those with fulminant disease survived for 5 weeks [124]. Patients frequently have weight loss and cramping abdominal pain in proportion to the severity of the diarrhea. A similar spectrum of disease severity has been described in AIDS-related intestinal infection with microsporidia [4, 65, 140], isospora [28], and cyclospora [3].

              Extra-Intestinal Disease

              The primary location of all intestinal spore-forming protozoal infections is the small intestine; the distal small bowel probably predominates [1, 85, 87, 94]. However, several of the organisms tend to involve contiguous epithelial surfaces, especially in heavy infections. Colonic infection is common with cryptosporidiosis and has been reported with microsporidiosis (E. bieneusi) [127]. Infection in the biliary tract in patients with AIDS occurs with cryptosporidia [141-143], microsporidia [22, 144-146], and isospora [147]. Biliary infection produces two syndromes. The first is a sclerosing cholangitis-type lesion that causes progressive, irregular obstruction and dilation of the intra- and extra-hepatic bile ducts [141, 142, 144, 145]. Patients have right upper-quadrant pain and an increasing alkaline phosphatase level. The second syndrome is acalculous cholecystitis caused by infection of the gallbladder wall, which occurs in patients with AIDS. Cryptosporidia [143], microsporidia [22], and isospora [147] have caused this lesion. Rarely, cryptosporidia involve the pancreatic duct [143, 148].

              Invasion beneath the epithelial surface and dissemination to other parts of the body is a regular feature only of S. intestinalis, which infects lamina propria macrophages, fibroblasts, and endothelial cells and can disseminate to other organs, including the liver [146], respiratory tract [149], and kidney [22, 88]. Enterocytozoon bieneusi may also be a respiratory pathogen in patients with AIDS [150].

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              Diagnosis

              The four intestinal spore-forming protozoal infections cannot be clinically differentiated solely on the basis of medical history or physical examination. Specific diagnosis depends on laboratory evaluation; the stool examination is the most important diagnostic test in patients suspected of having these infections. In some laboratories, the general request of “stool for O & P” means that the specimen will be carefully screened for the spores and oocysts of these four organisms. In most laboratories, however, tests for each organism must be specifically requested. Table 2 shows the stains recommended for the adequate visualization of spores or oocysts in the stool and the specific morphologic features that allow identification.

              The modified acid-fast stain can be routinely used to visualize the oocysts of cryptosporidia [151], cyclospora [3], and isospora [5]Figure 2, Figure 3, Figure 4, Figure 5 in stool or duodenal aspirate. Differentiating among these three organisms requires expertise and attention to detail [18, 152]. The exact sensitivity of the acid-fast stain is not known. In one report, examination of single stool specimens only identified 30% of intestinal cryptosporidial infections [124]; examining multiple specimens increases diagnostic yield. Enhanced sensitivity can be obtained by concentrating oocysts with various techniques [51] or, for cryptosporidia, by using the monoclonal antibody-based immunofluorescent stain [153-155].

              The first step in diagnosing intestinal microsporidiosis is to examine the stool specimen using the modified trichrome stain [33, 129]. The spores are small (the same size as many intestinal bacteria) and are hard to distinguish from fecal debris. A nonspecific fluorescence method [156] may enhance speed and sensitivity.

              The appearance of each of the intestinal spore-forming protozoa on small-bowel biopsy specimens is summarized in Table 2. Both cryptosporidia and isospora are easily seen in intestinal biopsy specimens with routine light microscopy Figure 2 and Figure 3, but many patients whose stools test positive for these organisms may have duodenal biopsy specimens that test negative. Cyclospora have not been seen with light microscopy in duodenal biopsy specimens [54, 86], but in one study, electron microscopy showed the organisms [54]. Small-bowel biopsy may be more sensitive than stool examination for the diagnosis of intestinal microsporidiosis [125, 157]. Orenstein and coworkers [87] showed that the hematoxylin and eosin stain is adequate in most cases (Figure 5). Various other tissue stains, including tissue Gram stain [33], toluidine blue [32], Warthin Starry [9], and Giemsa stain of a touch preparation [157], have been advocated for visualizing microsporidia in small-bowel biopsy specimens.

              As more sensitive diagnostic tests are used, more low-intensity infections will be identified. Some of these infections may have little clinical significance. This has been shown for cryptosporidia [83, 85] and possibly for microsporidia [125].

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              Treatment

              Table 2 summarizes antimicrobial chemotherapy options for each of the intestinal spore-forming protozoa. Because most acute infections with these protozoa are self-limited, most treatment trials have been done with chronic infections in persons with AIDS. Both uncontrolled case series [158-163] and a randomized, controlled trial [164] indicate that paromomycin may be effective in treating chronic AIDS-related cryptosporidiosis. Treatment is associated with decreased intensity of infection and improved intestinal function and morphology [85]. Relapse after treatment is common, and maintenance therapy is frequently required. The response rate varies from 30% to 70%. The cost of treating active infection with paromomycin is about $15.00 per day; maintenance therapy costs about one half that amount.

              A 10-day course of trimethoprim-sulfamethoxazole therapy treats isosporiasis in immunologically normal patients [14] and patients with AIDS [165]. Prophylaxis with either trimethoprim-sulfamethoxazole or sulfadoxine-pyrimethamine prevents recurrent disease in patients with AIDS [165]. Other treatment options include pyrimethamine and metronidazole [5]; these are especially useful for patients allergic to sulfonamides. Trimethoprim-sulfamethoxazole [3, 137, 152] also effectively treats cyclosporal infection.

              Descriptive case series suggest that S. intestinalis infection can be cured with albendazole [140, 166]. No therapy has been proven effective for E. bieneusi infection, although albendazole may improve clinical status without eliminating organisms from the stool or small bowel [167]. Albendazole is only available on a compassionate-use basis from SmithKline Beecham.

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              Summary

              The human intestinal spore-forming protozoa known as cryptosporidia, isospora, cyclospora, and microsporidia have many characteristics in common. They all cause intracellular infections of intestinal epithelial cells and thereby interfere with intestinal absorption and secretion. All are important causes of AIDS-related diarrhea. As a group, they are now recognized as important causes of traveler's diarrhea, institutional and community outbreaks of diarrhea, and acute sporadic childhood gastroenteritis. The different clinical situations in which each of these organisms should be sought are summarized in Table 2. The immune status of the host is an important determinant of the length and severity of the diarrhea: Immunocompetent hosts usually acquire acute, self-limited diarrhea; immunodeficient hosts are at risk for chronic, debilitating diarrhea. In patients with prolonged diarrhea, specific diagnosis through microscopic examination of the stool can prompt specific treatment (Table 2).

              Some of these organisms are attracting more attention as important public health hazards [25, 42, 50, 53, 168-170]. In January 1995, the Council of State and Territorial Epidemiologists [168] recommended that each state or city report cases of cryptosporidiosis to the Centers for Disease Control and Prevention's National Notifiable Disease Surveillance System. No such recommendation exists for the other intestinal spore-forming protozoa. Recommendations to prevent the spread of cryptosporidia in child care settings have been published [169]. No specific recommendations have been made about the complex issues of repeated stool testing for infected persons or about the testing of asymptomatic contacts. Because immunocompromised patients are at greater risk for severe disease caused by intestinal spore-forming protozoal infection, specific measures to reduce exposure to cryptosporidia have been recommended for these patients [168, 170]. These include minimizing oral exposure to water from lakes, streams, and public swimming pools and treating drinking water by boiling it for 1 minute or by using a filter capable of removing particles of 1 micron or less. The safety of bottled water varies from supplier to supplier [168, 170]. It is generally agreed that both public health officials and practicing clinicians have only begun to see the beginning of the disease caused by these organisms. The principles discussed in this article should enhance the general understanding of these organisms and improve the recognition, diagnosis, and treatment of the infections they cause.

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