Chapter 2. The Ciliates, Coccidia, and Microsporidia

In vertebrates, by far the most favorable sites for intestinal parasites are the duodenum, ileum, cecum and large intestine. To survive to reproduce in the gastrointestinal tract the parasites have to adapt to continuous physiological changes relative to the feeding habits of the host, the battery of protein, fat and carbohydrate-splitting enzymes, pH changes and the almost oxygen-free environment. Despite these features many parasites, both protozoa and helminths are capable of colonizing the gastrointestinal tract successfully.

Protozoa are single-celled animals which resemble a single cell of a higher organism. However, the protozoan cell is capable of carrying out vital functions such as reproduction, feeding, and locomotion. Intestinal protozoa include species which can live in the lumen of the intestine and others which additionally live and reproduce in the cells of the intestinal walls.

The protozoa make up a wide spectrum of organisms which have different life cycles and variable characteristics.

The ciliates belong to the family Ciliophora. They possess simple cilia or compound ciliary organelles, two types of nuclei and a large contractile vacuole. The only member of the ciliate family to cause human disease is Balantidium coli.

Introduction

Balantidium coli is widely distributed in warmer climates, which is where human infections most commonly occur. The organisms inhabit the large intestine, cecum and terminal ileum where they feed on bacteria. The most common hosts being humans, pigs and rodents. Human infection is usually from pigs and is rare.

Illustration 2-1. Life cycle of Balantidium coli. (SOURCE: PHIL 3380 - CDC/Alexander J. da Silva, PhD/Melanie Moser)

The cyst is spherical or ellipsoid and measures from 30-200�m by 20-120�m. It contains 1 macro and 1 micronucleus. The cilia are present in young cysts and may be seen slowly rotating, but after prolonged encystment, the cilia disappear. Cysts form when diarrhea subsides and the rectal contents become formed. The cyst, ingested by a fresh host, excysts to liberate the trophozoite.

Image 2-1. Balantidium coli cyst. (SOURCE: PHIL 584 - CDC/Dr. L.L.A. Moore, Jr.)

Trophozoites of B. coli measure approximately 30-150�m in length x 25-120�m in width but have been known to attain lengths of up to 200�m. They are oval in shape and covered in short cilia. A funnel shaped cytosome can be seen near the anterior end. Multiplication is by binary fission in the trophozoite stage. In an unstained preparation, the organisms are easily recognized because of their size and rapid revolving rotation. In a stained preparation, the characteristic macro and micronuclei may be observed.

Image 2-2. Balantidium coli cyst with Trichuris egg inside. Unstained wet mount. (SOURCE: PHIL 1460 - CDC/Dr. Mae Melvin)

Severe B. coli infections may resemble amebiasis. Symptoms include diarrhea, nausea, vomiting, and anorexia. The diarrhea may persist for long periods of time resulting in acute fluid loss. Balantidium coli also has the potential to penetrate the mucosa resulting in ulceration just as those of Entamoeba histolytica, but perforation is more common. Metastatic lesions do not occur. Extra-intestinal disease has also been reported, but is rare.

Wet preparations of fresh and concentrated stool samples reveal the characteristic cysts and motile trophozoites. They are easier to identify in direct-smear saline preparations than permanently stained fecal smears.

The Coccidia are a group of organisms which parasitize the epithelial cells of the intestinal tract. This group includes Cryptosporidium parvum, Cyclospora cayetanensis and Isospora belli. Most of the coccidian infections in man are zoonoses (having the potential to infect animals or arise from animals). In immunocompetent individuals, they usually produce mild, self-limiting infections.

Cryptosporidium species, are coccidian protozoa, which are cosmopolitan in distribution, occurring in both developed and underdeveloped countries and causing infection in both humans and their livestock. Cryptosporidium parvum is the species responsible for human infection.

Development of Cryptosporidia occurs in a parasitophorous vacuole located on the microvillous surface of the epithelial cells.

Illustration 2-2. Life cycle of Cryptosporidium sp. Sporulated oocysts, containing 4 sporozoites, are excreted by the infected host through feces and possibly other routes such as respiratory secretions. Following ingestion (and possibly inhalation) by a suitable host, excystation

occurs. The sporozoites are released and parasitize epithelial cells (

) of the gastrointestinal tract or other tissues such as the respiratory tract. In these cells, the parasites undergo asexual multiplication (schizogony or merogony) (

) and then sexual multiplication (gametogony) producing microgamonts (male)

and macrogamonts (female)

. Upon fertilization of the macrogamonts by the microgametes (

), oocysts (

) develop that sporulate in the infected host. Two different types of oocysts are produced, the thick-walled, which is commonly excreted from the host

, and the thin-walled oocyst

, which is primarily involved in autoinfection. Oocysts (measuring 4-5�m in diameter and containing 4 sporozoites) are infective upon excretion, thus permitting direct and immediate fecal-oral transmission. (SOURCE: PHIL 3386 - CDC/Alexander J. da Silva, PhD/Melanie Moser)

C. parvum is now widely recognized as a cause of acute gastro-enteritis, particularly in children. The infection produces a persistent, watery, offensive diarrhea often accompanied by abdominal pain, nausea, vomiting and anorexia. In immunocompetent persons, symptoms are usually short lived (one to two weeks). The small intestine is the site most commonly affected, symptomatic Cryptosporidium infections have also been found in other organs including other digestive tract organs, the lungs, and possibly conjunctiva.

Image 2-3. Cryptosporidium parvum oocysts. This photomicrograph revealed the morphologic details of Cryptosporidium parvum oocysts, i.e., encapsulated zygotes, which had been stained using the modified acid-fast method. These oocysts exhibit a bright red coloration when using this staining technique, and in this case, you�ll note the sporozoites that were made visible inside the two oocysts on the right. Sporozoites are the nucleated, motile stage of development through which many protozoans pass such as C. parvum, on their way to becoming adults, and represent a very infectious form of these organisms. The sporozoites will be released from these C. parvum oocysts. (SOURCE: PHIL 7829 - CDC/ DPDx - Melanie Moser)

Cryptosporidiosis in immunocompromised individuals, especially in HIV patients, can be life threatening, as many as 10% may pass oocysts of C. parvum. Infections are characterized by the production of frequent, large volume watery stools and sometimes there is invasion of the pancreas, biliary or respiratory tract.

Oocyst excretion and symptoms may fluctuate during the course of infection. Asymptomatic infections are commonly found in developing countries with poor hygiene, where there is close contact with livestock.

Definitive diagnosis of cryptosporidiosis is by finding the characteristic spherical oocysts in fecal samples. They do not concentrate well using standard concentration techniques and are identified using various staining techniques.

Using the modified Ziehl-Neelsen staining method (fuschin followed by methylene blue), the oocysts are acid fast. However, staining results within a smear and between specimens are diverse, varying from unstained to partial red staining and complete staining. Fully sporulated forms can be seen in which the red staining sporozoites are within an unstained oocyst wall. When staining the fecal smear with phenol-auramine/carbol-fuchsin, the oocysts appear as bright yellow discs with an "erythrocyte" pattern of staining against a dark red background.
Detection of the oocysts can also be achieved by using specific polyclonal or monoclonal antibodies conjugated to fluorescein. These tests are now commercially available and offer a high degree of sensitivity. However, caution must be exercised when they are used to detect oocysts in the fecal smears distributed by NEQAS parasitology. Such specimens are preserved in formalin, which interferes with the fluorescent staining of the parasites, and are thus difficult to detect.

Oocysts in stool specimens (fresh or in storage media) remain infective for extended periods. Thus stool specimens should be preserved in 10% buffered formalin or sodium acetate-acetic acid-formalin (SAF) to render oocysts non-viable. (Contact time with formalin necessary to kill oocysts is not clear; we suggest at least 18 to 24 hours).

Image 2-4. Staining of Cryptosporidium parvum oocysts in a stool smear with monoclonal antibodies conjugated to fluorescein. The Cryptosporidium oocysts appear with a peripheral green fluorescence. This technique could be of interest in epidemiological inquiries. (x 670) (SOURCE: Unknown)

Detection of the oocysts can also be achieved by using rapid antigen detection testing kits that specifically detect antigen released by Cryptosporidium parvu. These tests are now commercially available and offer a high degree of sensitivity even on specimens that are preserved.

Isospora belli is a coccidian protozoan of cosmopolitan distribution, occurring especially in warm regions of the world infecting both humans and animals.

The life cycle of I. belli involves an asexual (schizogonic stage) and a sexual (sporogonic stage).

Illustration 2-3. Isospora belli life cycle. At time of excretion, the immature oocyst contains usually one sporoblast (more rarely two)

. In further maturation after excretion, the sporoblast divides in two (the oocyst now contains two sporoblasts); the sporoblasts secrete a cyst wall, thus becoming sporocysts; and the sporocysts divide twice to produce four sporozoites each

. Infection occurs by ingestion of sporocysts-containing oocysts: the sporocysts excyst in the small intestine and release their sporozoites, which invade the epithelial cells and initiate schizogony

. Upon rupture of the schizonts, the merozoites are released, invade new epithelial cells, and continue the cycle of asexual multiplication

. Trophozoites develop into schizonts which contain multiple merozoites. After a minimum of one week, the sexual stage begins with the development of male and female gametocytes

. Fertilization results in the development of oocysts that are excreted in the stool

. Isospora belli infects both humans and animals. (SOURCE: PHIL 3398 - CDC/Alexander J. da Silva, PhD/Melanie Moser)

Infection with Isospora belli occurs in both immunocompetent and immunocompromised patients and begins when the mature oocyst is ingested in water or food.

Morphology of oocysts. The mature oocyst contains 2 sporocysts, each containing 4 sporozoites measure on average 35 x 9�m.

Figures 6. and 7. demonstrate fecal smears of oocysts. The sporulated oocyst is the infective stage of the parasite and they excyst in the small intestine releasing sporozoites which penetrate the epithelial cells, thus initiating the asexual stage of the life cycle. The sporozoite develops in the epithelial cell to form a schizont, which ruptures the epithelial cell containing it, liberating merozoites into the lumen. These merozoites will then infect new epithelial cells and the process of asexual reproduction in the intestine proceeds. Some of the merozoites form macrogametes and microgametes (sexual stages) which fuse to form a zygote maturing finally to form an oocyst.


A	B	C

Image 2-5. A, B, C: Oocysts of Isospora belli. The oocysts are large (25 to 30 �m) and have a typical ellipsoidal shape. When excreted, they are immature and contain one sporoblast (A, B). The oocyst matures after excretion: the single sporoblast divides in two sporoblasts (C), which develop cyst walls, becoming sporocysts, which eventually contain four sporozoites each. (Images contributed by Georgia Division of Public Health. (CDC))

In the immunocompetent, infection is generally asymptomatic or a self-limiting gastro-enteritis. However, in chronic infections, severe non-bloody diarrhea with cramp-like abdominal pain can last for weeks and result in fat malabsorption and weight loss. Eosinophilia may be present (atypical of other protozoal infections).

In immunocompromised individuals, infants and children, infection ranges from self-limiting enteritis to severe diarrheal illness resembling that of cryptosporidiosis.

Oocysts are thin walled, transparent and ovoid in shape. They can be demonstrated in feces after a formal ether concentration where they appear as translucent, oval structures.

Alternatively, oocysts can be seen in a fecal smear stained by a modified Ziehl-Neelsen method, where they stain a granular red color against a green background, or by phenol-auramine.

Cyclospora cayetanensis, a coccidian protozoan, has been described in association with diarrheal illness in various countries, in particular Nepal, Pakistan and India. Infection results in a disease with non-specific symptoms. Quite often the disease is the cause of unexplained summer diarrheal illness and similar illness following travel to tropical areas.

The life cycle of this organism is unknown, however environmental data suggest that Cyclospora, like Cryptosporidium species, is a water-borne parasite. The oocysts of C. cayetanensis are spherical, measuring 8-10�m in diameter and the mature oocyst contains 2 sporocysts. Oocysts of Cyclospora cayetanensis, are twice as large in comparison with C. parvum and are not sporulated (do not contain sporocysts - upon excretion).

Some of the elements in this figure were created based on an illustration by Ortega et al. Cyclospora cayetanensis. In: Advances in Parasitology: opportunistic protozoa in humans. San Diego: Academic Press; 1998. p. 399-418.

Illustration 2-4. When freshly passed in stools, the oocyst is not infective

(thus, direct fecal-oral transmission cannot occur; this differentiates Cyclospora from another important coccidian parasite, Cryptosporidium). In the environment

, sporulation occurs after days or weeks at temperatures between 22�C to 32�C, resulting in division of the sporont into two sporocysts, each containing two elongate sporozoites

. Fresh produce and water can serve as vehicles for transmission

and the sporulated oocysts are ingested (in contaminated food or water)

. The oocysts excyst in the gastrointestinal tract, freeing the sporozoites which invade the epithelial cells of the small intestine

. Inside the cells they undergo asexual multiplication and sexual development to mature into oocysts, which will be shed in stools

. The potential mechanisms of contamination of food and water are still under investigation. (SOURCE: CDC)

Image 2-6. This photomicrograph of a fresh stool sample, which had been prepared using a 10% formalin solution, and stained with modified acid-fast stain, revealed the presence of four Cyclospora cayetanensis oocysts in the field of view. Compared to wet mount preparations, the oocysts are less perfectly round and have a wrinkled appearance due to this method of fixation. Most importantly, the staining is variable among the four oocysts. (SOURCE: PHIL 7827 - CDC/ DPDx - Melanie Moser)

Image 2-7. This photomicrograph of a fresh stool sample, which had been prepared using a 10% formalin solution, and stained with safranin, revealed the presence of three uniformly stained Cyclospora cayetanensis oocysts in the field of view. (SOURCE: PHIL 7828 - CDC/ DPDx - Melanie Moser)

Patients from whose stools the organism has been isolated have reported nausea, vomiting, weight loss and explosive watery diarrhea. Flatulence and bloatedness, nausea and vomiting, myalgia, low-grade fever, and fatigue are associated symptoms. The site of infection is the small bowel. The disease is usually self-limiting to three to four days but untreated infections can last from several days to a month or longer, and may follow a relapsing course. Some infections are asymptomatic.

Image 2-8. Four Cyclospora oocysts from fresh stool fixed in 10% formalin and stained with modified acid-fast stain. Compared to wet mount preparations, the oocysts are less perfectly round and have a wrinkled appearance. Most importantly, the staining is variable among the four oocysts. (SOURCE: CDC)

The oocysts of C. cayetanensis are spherical as can be seen in formol-ether concentrated stool samples by light microscopy. They are refractile spheres which exhibit blue autofluorescence under ultraviolet light. It is important to note that UV microscopes set up for FITC and auramine microscopy only (450-500nm) will fail to detect the autofluorescence of the oocyst. Iodine-quartz microscopes do not produce UV wavelength below 400nm, while both mercury vapor and xenon vapor microscopes must be fitted with a 340-380nm excitation filter to demonstrate autofluorescence.

The oocysts are variably acid-fast when stained by the modified Ziehl-Neelsen method. Some cysts are acid-fast whereas others appear as round holes against a green background. They do not stain well with phenol-auramine.

The term microsporidia is also used as a general nomenclature for the obligate intracellular protozoan parasites belonging to the phylum Microsporidia. To date, more than 100 genera and 1,000 species have been described as parasites infecting a wide range of vertebrate and invertebrate hosts. There are at least seven microsporidian species that are well characterized as human pathogens. (Table 2-1.)

Microsporidia are characterized by the production of resistant spores and the polar tubule (or polar filament) which is coiled inside the spore as demonstrated by its ultrastructure.

They have recently come to medical attention as opportunistic pathogens in humans with Acquired Immune Deficiency Syndrome (AIDS) and have been implicated in conditions ranging from enteritis to keratoconjunctivitis.

Microsporidia are primitive organisms. They possess no mitochondria and have prokaryotic like ribosomes. Classification is based on the ultrastructural features, which include the number of coils in the polar tubes, the configuration of nuclei and the spore size 1-4�m, depending on the species.

Illustration 2-5. Life cycle of Microsporidia sp. The infective form of microsporidia is the resistant spore and it can survive for a long time in the environment

. The spore extrudes its polar tubule and infects the host cell

. The spore injects the infective sporoplasm into the eukaryotic host cell through the polar tubule

. Inside the cell, the sporoplasm undergoes extensive multiplication either by merogony (binary fission) or schizogony (multiple fission)

. This development can occur either in direct contact with the host cell cytoplasm (e.g., E. bieneusi) or inside a vacuole termed parasitophorous vacuole (e.g., E. intestinalis). Either free in the cytoplasm or inside a parasitophorous vacuole, microsporidia develop by sporogony to mature spores

. During sporogony, a thick wall is formed around the spore, which provides resistance to adverse environmental conditions. When the spores increase in number and completely fill the host cell cytoplasm, the cell membrane is disrupted and releases the spores to the surroundings

. These free mature spores can infect new cells thus continuing the cycle. (SOURCE: CDC)

Microsporidia	Size	Associated Disease
*Enterocytozoon bieneusi*	1�m x 1.5�m	Gastrointestinal and biliary tract infections
*Encephalitozoon intestinalis*	1.5�m x 2.5�m	Gastrointestinal tract and systemic infections
*Encephalitozoon hellem*	1.5�m x 1�m	Keratopathy (corneal edema), respiratory tract infection
*Encephalitozoon cuniculi*	1.5�m x 1�m	Central nervous system disease
*Nosema connori*	2�m x 4�m	Systemic infections
*Nosema corneum*	2�m x 4�m	Keratopathy
Pleistophora species	2.8�m x 3.4�m	Myositis

Table 2-1. Microsporidia found in humans and their associated disease. (Cuomo)

In addition to the species in Table 2-1., above, there are other, not well-characterized microsporidian human pathogens. These are designated as Microsporidum, a collective taxon that includes Microsporidum africanum and Microsporidium ceylonensis.

The most common microsporeans found in patients with AIDS are Enterocytozoon bieneusi, Encephalitozoon intestinalis and Encephalitozoon hellem. Patients with these infections tend to be severely immuno-deficient with a CD4 count less than 100 x 10⁶/L. Additionally, cases of microsporidiosis have been reported in immunocompromised persons not infected with HIV and in immunocompetent individuals. The clinical manifestations of microsporidiosis are very diverse, varying according to the causal species, with diarrhea being the most common.

Infections with E. bieneusi are restricted to the enterocytes of the small intestine, resulting in villous atrophy and malabsorption. Clinical symptoms include chronic watery, non-bloody diarrhea, malaise and weight loss.

Image 2-9. Electron micrograph of an Enterocytozoon bieneusi spore. Arrows indicate the double rows of polar tubule coils in cross section which characterize a mature E. bieneusi spore. (SOURCE: CDC)

Infection with Encephalitozoon intestinalis occurs in the enterocytes of the small intestine but is more widely disseminated than E. bieneusi and has been found in the colon, liver and kidney.

Image 2-10. Electron micrograph of an eukaryotic cell with Encephalitozoon intestinalis spores and developing forms inside a septated parasitophorous vacuole. The vacuole is a characteristic feature of this microsporidian species. (SOURCE: CDC)

These organisms have also been found in disseminated microsporidiosis. Clinical symptoms may include sinusitis, nephritis, hepatitis, keratoconjunctivitis and peritonitis.

Initially, the diagnosis of intestinal microsporidiosis depended on tissue biopsies which were stained with Gram�s stain and examined by light microscopy. However, in order that ill patients were not subjected to unnecessary invasive procedures, non-invasive diagnostic procedures were developed. The modified Trichrome stain and the Fungiqual fluorescent stain are the stains of choice. Immunofluorescence assays (IFA) using monoclonal and/or polyclonal antibodies are being developed for the identification of microsporidia in clinical samples.

Many intestinal disorders are due to intestinal parasites which cannot be diagnosed symptomatically. Laboratory investigation is therefore required and the staff responsible should have adequate expertise in examining fecal specimens for parasitic organisms.

The request form should always state the patients clinical symptoms and signs and whether the patient had recent overseas travel. If the patient has had no recent history of overseas travel, examination for Cryptosporidium, Giardia and Microsporidia, if immunocompromised should be considered. If overseas travel has been undertaken, it is important to note is the patient ill or whether a routine post-tropical screen is requested. The geographical location is also important as it may indicate these parasites which could be present.

If a fecal sample is not properly collected and taken care of before examination, it will be of little or no value for accurate diagnosis. This is especially true if protozoa are present. Amoebic trophozoites begin to degenerate one to two hours after passage, as do flagellate trophozoites. Cysts will deteriorate if the fecal specimen is left standing for many hours or overnight, especially at high temperatures.

Helminth eggs and larvae are less affected by the age of the specimen than are protozoa. Nevertheless, changes may occur that could affect their identification; hookworm larvae may become embryonated and larvae may hatch from the eggs risking confusion with Strongyloides larvae. Larvae themselves may disintegrate thus making their identification difficult.

To ensure that good specimens are provided for examination, it is important to note the following points.

It is important to observe the macroscopic appearance of the stool as this can give a clue to the type of organisms present. Therefore the consistency; formed, unformed or liquid; the color and the presence or absence of the exudate are reported. The presence of adult worms can also be seen in a freshly passed stool e.g. adult stage of Ascaris lumbricoides and Enterobius vermicularis. Proglottids of Taenia species can also be seen.

2.7 Routine procedure for the microscopic examination of fecal samples for parasites

The principal of the successful identification of fecal parasites is based upon,

The addition of iodine to formol ether concentrates highlights the internal structure of cysts and helps distinguish between vegetable matter and cysts. Permanently stained fecal smears are useful in demonstrating the nuclear pattern of cysts.

Epithelial cells and macrophages can be confused with amoebic trophozoites, especially macrophages that show slight amoeboid movement and may contain red blood cells. Pus cells can be confused with amoebic cysts. The nuclei appear as 3 or 4 rings and usually stain heavily. The cytoplasm is ragged and the cell membrane is often not seen. Amoebic cysts have a distinct cell wall.

Hair and fibers may be confused with larvae, but they do not have the same internal structure as larvae.

Plant cells can be confused with cysts or eggs. Though plant cells usually have a thick wall and cysts have a thin wall.

Ideally, the presence of all parasites should be reported, whether they are pathogens or non-pathogens. This particularly applies to the presence of cysts. However, if it is laboratory practice to report all cysts, the report should state whether they are pathogenic or non-pathogenic.

The stage of the parasite should always be reported. For the protozoa, whether cysts or trophozoites are present; the stage of larvae as in Strongyloides; and whether adult stages or eggs of helminths are present.