Living trophozoites (motile feeding stage) of E. histolytica vary in size from about 12 to 60 µm in diameter. Organisms recovered from diarrheic or dysenteric stools generally are larger than those in formed stool from an asymptomatic individual. The motility has been described as rapid and unidirectional. Although this characteristic motility is often described, amebiasis rarely is diagnosed on the basis of motility seen in a direct mount. The cytoplasm is differentiated into a clear outer ectoplasm and a more granular inner endoplasm.

E. histolytica has directional and progressive motility, whereas the other amebae tend to move more slowly and at random. However, motility is rarely seen even in a fresh wet mount from a patient with diarrhea or dysentery. The cytoplasm is generally more finely granular, and the presence of red blood cells (RBCs) in the cytoplasm is considered diagnostic for E. histolytica (Figure 48-2).

Permanent stained smears demonstrate accurate morphology compared with other techniques. When the organism is examined on a permanent stained smear (trichrome or iron-hematoxylin stain), the morphologic characteristics of E. histolytica/E. dispar are readily seen. The nucleus is characterized by evenly arranged chromatin on the nuclear membrane and a small, compact, centrally located karyosome (condensed chromatin). As mentioned, the cytoplasm usually is described as finely granular, with few ingested bacteria and scant debris in vacuoles. As stated previously, in organisms isolated from a patient with dysentery, RBCs may be visible in the cytoplasm, a feature diagnostic for E. histolytica (Figure 48-3). Most often, infection with E. histolytica is diagnosed on the basis of the organism’s morphology, without the presence of RBCs.

As part of the life cycle, the trophozoites may condense into a round mass (precyst), and a thin wall is secreted around the immature cyst. Two types of inclusions may be found in this immature cyst: a glycogen mass and highly refractile chromatoidal bars (refractile chromatin structure) with smooth, rounded edges. As the cyst matures (metacyst) (see Figure 48-3; Figure 48-4), nuclear division occurs, with the production of four nuclei. Often chromatoidals may be absent in the mature cyst. Cyst morphology does not differentiate E. histolytica from E. dispar. Cyst formation occurs only in the intestinal tract; once the stool has left the body, cyst formation does not occur. The one-, two-, and four-nucleated cysts are infective and represent the mode of transmission from one host to another.

Epidemiology

Amebiasis is caused by infection with the true pathogen, Entamoeba histolytica. Recent evidence from molecular studies confirms the differentiation of pathogenic E. histolytica and nonpathogenic E. dispar (Figure 48-5) as two distinct species. E. histolytica is considered the etiologic agent of amebic colitis and extraintestinal abscesses (amebic liver abscess), whereas nonpathogenic E. dispar produces no intestinal symptoms and is not invasive in humans.

Infection is acquired through the fecal-oral route from infective cysts contained in the feces. These cysts can be ingested in contaminated food or drink or contracted from fomites or various sexual practices that could include accidental ingestion of fecal organisms. Flies and cockroaches have been implicated as mechanical vectors of contaminated fecal material.

The infection occurs worldwide, particularly in areas with poor sanitation. It is estimated that E. histolytica infection kills more than 100,000 people each year.

The life cycle of E. coli is identical to that of E. dispar. After digestion of infective cysts, the organisms excyst in the intestinal tract and produce trophozoites. Cyst formation occurs as the gut contents move through the intestinal tract; the excreted cysts are the infective form that is transmitted to humans and some animals.

E. coli trophozoites are somewhat larger than those of E. histolytica and E. dispar and range from 15 to 50 µm in diameter (see Table 48-1; Figures 48-6 and 48-7; see Figure 48-3). Motility is sluggish with broad, short pseudopods. In wet preparations, differentiating nonpathogenic E. coli from pathogenic E. histolytica is almost impossible. On the permanent stained smear viewed at a higher magnification, the cytoplasm is granular with vacuoles containing bacteria, yeasts, and other food materials. The nucleus has a large blotlike karyosome that may be eccentric rather than centrally located. The chromatin on the nuclear membrane tends to be clumped and irregular. Although rare, if RBCs are present in the intestinal tract, E. coli may ingest them rather than bacteria.

Early cysts often contain chromatoidal bars, which tend to be splinter shaped and irregular. Eventually, the nuclei divide until the mature cyst, containing eight nuclei, is formed (see Table 48-2; see Figures 48-3 and 48-6). In rare cases, the number of nuclei reaches 16. The cysts measure 10 to 35 µm in diameter, and as they mature, the chromatoidal bars disappear. When the cyst of E. coli matures, it becomes more refractive to fixation; therefore, the cyst may be seen on the wet preparation but not on the permanent stained smear. Occasionally, on trichrome smears, the cysts appear distorted and somewhat pink (Figure 48-8).

The life cycle of E. hartmanni is similar to that of E. dispar, with differences in size (Figures 48-9 and 48-10). In wet preparations, E. hartmanni trophozoites range in size from 4 to 12 µm in diameter, and cysts range in size from 5 to 10 µm in diameter. On the permanent stained smear, the cysts, primarily, tend to shrink as a result of dehydration; therefore, the sizes of all the organisms, including pathogenic E. histolytica, may be somewhat smaller (1 to 1.5 µm) than the wet preparation measurements.

Trophozoites do not ingest RBCs, and the motility is usually less rapid (see Table 48-1; see Figures 48-3, 48-9, and 48-10). The morphologic characteristics of E. hartmanni are very similar to those of E. histolytica, with two exceptions. Frequently, E. hartmanni cysts may contain only one or two nuclei, even though the mature cyst contains four nuclei. Mature cysts of E. hartmanni also retain their chromatoidal bars, a characteristic not usually seen in E. histolytica/E. dispar. E. hartmanni’s chromatoidal bars are similar to those of E. histolytica and E. dispar but smaller and more numerous (see Table 48-2; see Figures 48-9 and 48-10). At the species level, differentiation between E. hartmanni and E. histolytica/E. dispar depends on size; therefore, laboratories are required to use calibrated microscopes that are checked periodically for accuracy.

Endolimax nana, one of the smaller nonpathogenic amebae, has a worldwide distribution and is seen as frequently as E. coli.

E. nana has the same life cycle stages as E. dispar and the other nonpathogenic amebae. The trophozoite usually measures 6 to 12 µm in diameter (normal range, 8 to 10 µm) (see Table 48-1; Figures 48-11 to 48-13). Although rarely seen, motility is sluggish and nonprogressive with blunt, hyaline pseudopods. In the permanent stained smear, normally no peripheral chromatin is seen on the nuclear membrane, and the karyosome is large, with either a central or an eccentric location in the nucleus (see Figures 48-12 and 48-13). E. nana shows more nuclear variation than any of the other amebae, and occasionally E. nana can mimic D. fragilis or E. hartmanni. The cytoplasm may have small vacuoles containing ingested debris or bacteria, but it also may appear relatively clean.

Cysts usually measure 5 to 10 µm in diameter (normal range, 6 to 8 µm) (see Table 48-2). Cysts as large as 14 µm have been seen. The cyst is usually oval to round, with the mature cyst containing four nuclei. The nuclei typically have no peripheral chromatin and are somewhat evenly distributed in the cyst. Occasionally, very small, slightly curved chromatoidal bars are present. The two-nucleated stage is not commonly seen, and frequently both trophozoites and cysts are present in clinical specimens.

Iodamoeba bütschlii, one of the nonpathogenic amebae, has a worldwide distribution. Generally, the acquisition rate for this organism is not as high as that for E. coli and E. nana.

The life cycle stages of I. bütschlii are exactly the same as those of E. nana. The trophozoite varies from 8 to 20 µm in diameter and has fairly active motility in a fresh stool preparation (see Table 48-1). The cytoplasm is granular, containing numerous vacuoles with ingested debris and bacteria. The cytoplasm is more vacuolated than in E. nana trophozoites. The nucleus has a large karyosome, which can be either centrally located or eccentric (Figures 48-14 and 48-15). On the permanent stained smear, the nucleus may appear to have a halo, and chromatin granules fan out around the karyosome. If the granules are on one side, the nucleus may appear to have a “basket nucleus” arrangement of chromatin, more commonly seen in the cyst stage. The trophozoites of I. bütschlii and E. nana may appear similar and are difficult to differentiate at the species level, even on the permanent stained smear. Both organisms are considered nonpathogenic. E. nana is recovered in clinical specimens much more frequently than is I. bütschlii.

I. bütschlii cysts are round to oval (see Table 48-2). The glycogen vacuole is so large that occasionally the cyst collapses on itself. Because nuclear multiplication does not occur in the cyst form, the mature cyst contains a single nucleus. The cysts measure approximately 5 to 20 µm in diameter and are rarely confused with those of other amebae (see Figures 48-14 and 48-15).

Blastocystis hominis (see Figure 48-1; see Table 48-6) comprises a number of different strains that are indistinguishable morphologically; some of which are pathogenic, and some are nonpathogenic. Although usually listed with the amebae, the organism’s classification is still under review; different strains eventually may be classified as different species. Although the true role of this organism in terms of disease has been controversial, it is now generally considered a causative agent of intestinal disease. The current recommendation is to report the presence of B. hominis and quantitate from the permanent stained smear (i.e., rare, few, moderate, many, packed); this information may be valuable in helping to assess the pathogenicity of the organism in the individual patient.

B. hominis consists of four major forms. The cyst form is the most recently described form of the life cycle stages. Thick-walled cysts are thought to be responsible for external transmission through the fecal-oral route; thin-walled cysts are thought to cause autoinfection. Cysts can vary in shape but are mostly ovoid or spherical. The central vacuole form (also referred to as the central body form) is the most common form found in clinical stool samples. The large central vacuole can occupy most of the cellular volume. The amoeboid form is rarely seen. The granular form can be seen in cultures of B. hominis.

G. lamblia is the most common cause of intestinal infection worldwide. Other than B. hominis, G. lamblia (also called G. duodenalis and G. intestinalis) is probably the most common protozoan organism identified in individuals in the United States. It causes symptoms ranging from mild diarrhea, flatulence, and vague abdominal pains to acute, severe diarrhea to steatorrhea and a typical malabsorption syndrome. Various documented waterborne and food-borne outbreaks have occurred during the past several years. A number of animals may serve as reservoir hosts for G. lamblia. Differentiation of flagellates is based on overall shape, numbers, and arrangements of flagella.

Both the trophozoite and cyst stages are included in the life cycle of G. lamblia. Trophozoites divide by means of longitudinal binary fission, producing two daughter trophozoites. The organism is found most commonly in the crypts in the duodenum. Trophozoites are the intestinal dwelling stage and attach to the epithelium of the host villi by means of the ventral disk. The attachment is substantial and results in disk “impression prints” when the organism detaches from the surface of the epithelium. Trophozoites may remain attached to or may detach from the mucosal surface. Because the epithelial surface sloughs off the tip of the villus every 72 hours, the trophozoites apparently detach at that time. G. lamblia trophozoites are teardrop shaped and have been described as “someone looking at you” (see Figures 48-16 to 48-18).

Cyst formation takes place as the organisms move down through the jejunum after exposure to biliary secretions. The trophozoites retract the flagella into the axonemes, the cytoplasm becomes condensed, and the cyst wall is secreted (see Figures 48-16 to 48-18). As the cyst matures, the internal structures are doubled, so that when excystation occurs, the cytoplasm divides, producing two trophozoites. Excystation occurs in the duodenum or appropriate culture medium.

C. mesnili has both trophozoite and cyst stages and is somewhat more easily identified than are some of the smaller flagellates, such as E. hominis and R. intestinalis (see Tables 48-3 and 48-4; see Figure 48-19). The C. mesnili trophozoite is pear shaped, measuring 6 to 24 µm long and 4 to 8 µm wide. It has a single nucleus and a distinct oral groove, or cytostome (mouth), close to the nucleus. Flagella are difficult to see without obvious motility in a wet preparation. The morphology can be seen on the permanent stained smear; the cytostome may be visible in some trophozoites. The cysts are pear or lemon shaped and range from 6 to 10 µm long and 4 to 6 µm wide (see Figure 48-20). They have a single nucleus and a typical curved cytostomal fibril, called the shepherd’s crook. The cyst’s definitive morphology can be seen on a permanent stain.

D. fragilis was described in 1918. It has a worldwide distribution, and surveys report incidence rates of 1.4% to 19%. Much higher incidence figures have been reported for patients in mental institutions, missionaries, and Native Americans in Arizona. D. fragilis tends to be common in some pediatric populations, and the incidence is higher for patients under 20 years of age in some studies. Some speculate that D. fragilis may be infrequently recovered and identified; a low incidence or absence from survey studies may be due to poor laboratory techniques and a general lack of knowledge about the organism.

The D. fragilis trophozoite is characterized as having one nucleus (20% to 40%) or two nuclei (60% to 80%). The nuclear chromatin usually is fragmented into three to five granules, and normally no peripheral chromatin is seen on the nuclear membrane. In some organisms the nuclear chromatin tends to mimic that of E. nana, E. hartmanni, or even C. mesnili, particularly if the organisms are overstained with trichrome or iron-hematoxylin stain. The cytoplasm is usually vacuolated and may contain ingested debris and some large, uniform granules. The cytoplasm can also appear uniform and clean with few inclusions. Size and shape vary considerably among organisms, even on a single smear.

Pentatrichomonas hominis

P. hominis is probably the most commonly identified flagellate, other than G. lamblia and D. fragilis. P. hominis has been recovered from all parts of the world, in both warm and temperate climates, and is considered nonpathogenic and noninvasive. It is not known to have a cyst stage (see Figure 48-16). P. hominis trophozoites live in the cecum and feed on bacteria. The trophozoite measures 5 to 15 µm long and 7 to 10 µm wide. It has a pyriform shape and has both an axostyle and an undulating membrane, which aid identification of the organism. The undulating membrane extends the entire length of the body, in contrast to that seen in the pathogen T. vaginalis (on which the membrane extends halfway down the body).

The life cycle of B. coli includes both the trophozoite and cyst stages (Figures 48-24 and 48-25). The cyst form is the infective stage. After ingestion of the cysts and excystation, trophozoites secrete hyaluronidase, which aids the invasion of the colonic tissue.

The trophozoite is quite large, oval, and covered with short cilia. It measures approximately 50 to 150 µm long and 40 to 70 µm wide. The organism can be seen in a wet preparation on lower power. The anterior end is somewhat pointed and has a cytostome (primitive mouth opening); in contrast, the posterior end is broadly rounded. The cytoplasm contains many vacuoles with ingested bacteria and debris. The trophozoite has two nuclei: one very large, bean-shaped macronucleus and a smaller, round micronucleus. The organisms live in the large intestine. The trophozoites have a rapid, rotatory, boring motion because of the movement of the cilia. The cyst is formed as the trophozoite moves down the intestine. Nuclear division does not occur in the cyst; therefore, only two nuclei are present, the macronucleus and the micronucleus. The cysts measure 50 to 70 µm in diameter (see Table 48-5).

Cryptosporidium spp. are intracellular parasites that primarily infect epithelial cells of the stomach, intestine, and biliary ducts. The organism previously called Cryptosporidium parvum, thought to be the primary Cryptosporidium species infecting humans, now is classified as two species, C. parvum (mammals, including humans) and C. hominis (primarily humans) (see Table 48-6, Figures 48-26 to 48-28). Differentiation of these two species based on oocyst morphology is not possible. Currently, more than 20 established Cryptosporidium species have been identified in vertebrates, and more than 10 Cryptosporidium spp. have been reported in humans.

Cryptosporidium infections begin with ingestion of viable oocysts. Upon contact with gastric and duodenal fluid, each oocyst releases four sporozoites, which invade the epithelial cells and develop into trophozoites surrounded by a parasitophorous vacuole (layers of endoplasmic reticulum around an intracellular parasite). In the epithelial cells, trophozoites undergo two or three generations of asexual amplification, called merogony, leading to the formation of different types of meronts containing four to eight merozoites. The merozoites differentiate into sexually distinct stages in a process called gametogony. New oocysts are then formed in the epithelial cells in a process called sporogony. About 20% of the oocysts are thin walled and may excyst in the digestive tract of the host, leading to the infection of new cells (autoinfection). The remaining 80% of the oocysts are excreted into the environment; are resistant to low temperature, high salinity, and most disinfectants; and can initiate infection in a new host. Cryptosporidium oocysts in humans measure 4 to 6 µm in diameter.

During the past few years, a number of outbreaks of diarrhea associated with Cyclospora cayetanensis have occurred; the distribution is worldwide (United States, Caribbean, Central and South America, Southeast Asia, Eastern Europe, Australia, Nepal). These organisms are acid-fast variable and have been found in the feces of immunocompetent travelers to developing countries, immunocompetent individuals with no travel history, and patients with AIDS. Cumulative evidence suggests that outbreaks in the United States and Canada during the spring months of 1996 and 1997 were related to the importation and ingestion of Guatemalan raspberries. An outbreak in Florida in 1995 quite likely was also attributable to contaminated food. The cases reported in all three outbreaks probably represented only a small fraction of incidences.

The life cycle of C. cayetanensis involves only humans as hosts. Oocysts are passed in the feces unsporulated (Figure 48-29). At room temperature (23° to 25°C), small numbers of oocysts may sporulate within 10 to 12 days.

In clean wet mounts, Cyclospora organisms are seen as nonrefractile spheres, which are difficult to recognize as parasites. Unless a high number of oocysts are present, they may easily be mistaken for artifacts. They are acid-fast variable with the modified acid-fast stain; those that are unstained appear as glassy, wrinkled spheres (wrinkled cellophane). The oocysts are twice the size of those of Cryptosporidium spp. and measure 8 to 10 µm in diameter. Because it takes 10 days to 2 weeks for the oocysts to sporulate, no internal structures are visible (sporozoites), as can be seen in Cryptosporidium organisms.

Although isosporiasis is found worldwide, certain tropical areas in the Western Hemisphere have specific locations where endemic infections occur. These organisms infect both adults and children, and intestinal involvement and symptoms are generally transient unless the patient is immunocompromised. I. belli has also been implicated in traveler’s diarrhea. However, unlike with Cryptosporidium spp. and C. cayetanensis, large outbreaks of isosporiasis have not been reported (see Table 48-6, Figure 48-30).

I. belli oocysts are passed in the stool. They are long and oval, measuring 20 to 33 µm long by 10 to 19 µm wide. Usually the oocyst contains one immature sporont, but two may be present. Continued development occurs outside the body, with the development of two mature sporocysts, each containing four sporozoites, which can be recovered from the fecal specimen. The sporulated oocyst is the infective stage that excysts in the small intestine, releasing the sporozoites, which penetrate the mucosal cells and initiate the life cycle.

Two well-described Sarcocystis species include S. bovihominis (cattle) and S. suihominis (pigs). (Some publications refer to S. bovihominis as S. hominis.) When uncooked meat from these infected animals is ingested by humans, gamogony (fission resulting in the production of sporozoan gametes) can occur in the intestinal cells, with eventual production of the sporocysts in stool.

Sarcocystis spp. have an obligatory two-host life cycle. Intermediate hosts (herbivores and omnivores) become infected through ingestion of sporocysts excreted in the feces of the definitive hosts (carnivores and omnivores). The definitive hosts become infected through ingestion of mature cysts found in the muscles of the intermediate hosts. In some intermediate hosts, such as cattle and sheep, all adult animals may be infected. Extraintestinal human sarcocystosis is rare, with a much lower incidence than is seen with the intestinal infection. Humans who have ingested meat containing the mature sarcocysts serve as the definitive hosts. Fever, severe diarrhea, abdominal pain, and weight loss have been reported in immunocompromised hosts, although the number of patients with these symptoms has been quite small.

The sporocysts found in the stool are broadly oval and slightly tapered at the ends. They measure 9 to 16 µm long and contain four mature sporozoites and the residual body (see Table 48-6). Normally, the oocyst contains two sporocysts (similar to I. belli); however, in Sarcocystis infections, the sporocysts are released from the oocyst and normally are seen singly. These sporocysts tend to be larger than Cryptosporidium oocysts that contain four sporozoites, so they look totally different. The oocysts are fully sporulated when passed in the stool.

General Characteristics

Human microsporidial infections have been documented worldwide. The spore, the only life cycle stage able to survive outside the host cell, is the infective stage (see Table 48-7 and Figures 48-31 to 48-35). Infection occurs with ingestion or inhalation of the infective spores, from which the infective sporoplasm (spore protoplasm) enters the host cell through the polar tubule. The microsporidia multiply extensively in the host cell cytoplasm; the life cycle includes repeated divisions by binary fission (merogony) or multiple fission (schizogony) and spore production (sporogony). Both merogony and sporogony can occur in the same cell at the same time. During sporogony, a thick spore wall is formed, providing environmental protection for the spore.

Microsporidial spores measure 0.7 to about 4 µm in diameter. Mature spores contain a tubular extrusion apparatus (polar tube or tubule) for injecting infective spore contents (sporoplasm) into the host cell.

Epidemiology

Transmission possibilities include human-to-human and animal-to-human routes. Many questions relating to reservoir hosts and possible congenital infections are still unanswered. Primary infection occurs through inhalation or ingestion of spores from environmental sources or by zoonotic transmission. The presence of Encephalitozoon intestinalis has been confirmed in tertiary sewage effluent, surface water, and groundwater; Enterocytozoon bieneusi has been confirmed in surface water; and Vittaforma corneae has been confirmed in tertiary effluent. This study represents the first confirmation, to the species level, of human-pathogenic microsporidia in water, indicating that these parasites are probably waterborne pathogens. Ingestion of the environmentally highly resistant spores is probably the normal mode of transmission.

E. bieneusi, an intestinal pathogen, serves as an example of infection potential. The spores are released into the intestinal lumen and are passed in the stool. These spores are environmentally resistant and can be ingested by other hosts. Zoonotic transmission of microsporidia infecting humans has not been verified but appears likely, because many microsporidial species can infect both humans and animals.

Pathogenesis and Spectrum of Disease

Microsporidia were recognized as causing disease in animals as early as the 1920s but were not recognized as agents of human disease until the AIDS pandemic began in the mid-1980s. Before then, several earlier human cases had been reported but were thought to be very unusual.

Laboratory Diagnosis

The most commonly used stains are chromotrope-based stains (modified trichrome) and chemofluorescent optical brightening agents, including calcofluor white and other chemofluorescent stains. Regardless of the staining technique selected, the use of positive control material is highly recommended. Detection of the small microsporidial spores requires adequate illumination and magnification (i.e., magnification using the oil immersion objective [×100] for a total magnification of ×1000).

Therapy

Albendazole therapy can result in clinical cure of HIV-associated infection with Encephalitozoon spp., along with elimination of spore shedding. Albendazole is not effective for Enterocytozoon infections, although clinical improvement occurs in some patients. Oral purified fumagillin appears to eradicate Enterocytozoon bieneusi in many patients, but serious adverse events and parasitic relapse have been seen. Antiretroviral combination therapy results in complete clinical response with elimination of intestinal microsporidia.

Prevention

The presence of infective spores in human clinical specimens suggests that taking precautions when handling body fluids and following personal hygiene measures, such as hand washing, may be important in preventing primary infections in the health care setting. However, the establishment of comprehensive guidelines for disease prevention requires more definitive information about sources of infection and modes of transmission.