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3.1 Infections Acquired Through the Gastrointestinal Tract
The Flagellates belong to the Magistophora and possess more than one flagellum. Beating these flagella enable them to move. A cytosome may be present which helps in the identification of the species.
Flagellates possess one advantage over their ameboid relatives in that they can swim. Therefore, enabling them to invade and adapt to a wider range of environments unsuitable for other amoebae. They are able to change from a flagellated free-swimming environment to a non-flagellated tissue dwelling stage and vice versa.
Flagellates are known to inhabit the reproductive tract, alimentary canal, tissue sites and also the blood stream, lymph vessels and cerebrospinal canal.
There are pathogenic and commensal species of flagellates. The flagellates which are encountered in the intestinal tract are Giardia lamblia, Dientamoeba fragilis, Chilomastix mesnili, Trichomonas hominis, Retortamonas intestinalis and Enteromonas hominis (the latter two being less common). The trophozoites are easily recognized in saline preparations by their motility. However, accurate identification is done on a permanently stained fecal smear. Cysts are more commonly seen than the trophozoite.
Giardia lamblia is a flagellate of world-wide distribution. It is more common in warm climates than temporal climates. It is the most common flagellate of the intestinal tract, causing Giardiasis. Humans are the only important reservoir of the infection. The infection is most common in parts of the world where sanitation is at its lowest. Giardiasis is an infection of the upper small bowel, which may cause diarrhea. Only Giardia spreads disease.
Morphology of the Trophozoites
The trophozoites of G. lamblia are flattened pear shaped and are an average size of 15mm long, 9mm wide and 3mm thick. When stained, the trophozoite is seen to have two nuclei, two slender median rods (axostyles), and eight flagella arising from the anterior end. They have been described as looking like tennis rackets without the handle (they are often seen has having a comical face-like appearance when looking at the front view).
The movement of the trophozoites is described as tumbling leaf motility, using their four pairs of flagella for locomotion. They attach themselves to the surface of the jejunal or duodenal mucosa by their disc-like suckers which are found on their ventral surface. They multiply in the gut by binary fission. Once the trophozoites drop off the mucosal surface they are normally carried in the intestinal contents down the gut where they usually encyst.
Illustration 3-1. Life cycle of Giardia lamblia. Cysts are resistant forms and are responsible for transmission of Giardiasis. Both cysts and trophozoites can be found in the feces (diagnostic stages) . The cysts are hardy and can survive several months in cold water. Infection occurs by the ingestion of cysts in contaminated water, food, or by the fecal-oral route (hands or fomites) . In the small intestine, excystation releases trophozoites (each cyst produces two trophozoites) . Trophozoites multiply by longitudinal binary fission, remaining in the lumen of the proximal small bowel where they can be free or attached to the mucosa by a ventral sucking disk . Encystation occurs as the parasites transit toward the colon. The cyst is the stage found most commonly in nondiarrheal feces . Because the cysts are infectious when passed in the stool or shortly afterward, person-to-person transmission is possible. While animals are infected with Giardia, their importance as a reservoir is unclear. (SOURCE: PHIL 3394 - CDC/Alexander J. da Silva, PhD/Melanie Moser)
The cysts of G. lamblia are 8-12mm in length and are ellipsoid in shape. They contain four nuclei which tend not to be obvious. Longitudinal fibrils consisting of the remains of axonemes and parabasal bodies may also be seen. Cysts may appear to shrink from the cell wall. The cysts are infective as soon as they are passed.
Image 3-3. A, B, C Three trophozoites of Giardia intestinalis, stained with trichrome (A) and stained with iron hematoxylin (B and C). Each cell has two nuclei with a large, central karyosome. Cell size: 9 to 21 µm in length. (SOURCE: CDC)
Image 3-4. This scanning electron micrograph (SEM) revealed some of the external ultrastructural details displayed by a flagellated Giardia lamblia protozoan parasite. G. lamblia is the organism responsible for causing the diarrheal disease "giardiasis". Once an animal or person has been infected with this protozoan, the parasite lives in the intestine, and is passed in the stool. Because the parasite is protected by an outer shell, it can survive outside the body, and in the environment for long periods of time. (SOURCE: PHIL 8698 - CDC / Janice Carr)
Giardia lamblia colonizes the small intestine where the trophozoites adhere to the mucosal surface by means of their sucking disc. Cysts are produced as the parasites descend the intestinal tract although trophozoites can be passed in the feces in severe infections. G. lamblia is transmitted through ingestion of cysts in contaminated water or food. Cysts can survive outside the body for several weeks under favorable conditions. The main symptoms are abdominal pain, flatulence, and episodic diarrhea with steatorrhea and periodical soreness in severe cases. No blood or mucus is normally seen. However 50% of G. lamblia infections are symptomless, although severe infections may develop in immunocompromised hosts. What determines susceptibility is poorly understood. After swallowing cysts for the first time, symptoms commonly develop 2-6 weeks later.
Cysts can be found by examination of the deposit of a formol-ether concentrate of a stool preparation. The oval cysts with thick walls serve as characteristic features for these organisms. (Keys 3-1. & 3-2.) The flagella disintegrate and form a central ‘streak’ which becomes visible when stained with iodine or MIF (merthiolate-iodine-formaldehyde). Cysts may be excreted intermittently; therefore it is important to examine more than one stool. Stools are usually passed 3-8 times / day and are usually pale, offensive, rather bulky and accompanied by much flatus.
Trophozoites are found by examination of saline wet preparations of fresh, diarrheic stool, duodenal or jejunal aspirate or in a permanently stained fecal preparation.
Trophozoites can also be found in the jejunal aspirate. These can be recovered by the String Test or Enterotest capsule and the material examined microscopically for motile trophozoites.
Trophozoites and cysts can be found to be scarce in chronic infections. Serological methods of diagnosis are proving to be useful as means of diagnosis. An ELISA to detect IgM in serum provides evidence of a current infection. A polyclonal antigen-capture ELISA can be used to demonstrate submicroscopic infections in feces and an IgA-based ELISA will detect specific antibodies in saliva. Table 3-1. details useful morphological features that are similar between species of flagellate and are used in laboratory diagnosis. Detection of the trophozoites and cysts can also be achieved by using rapid antigen detection testing kits that specifically detect antigen released by Giardia lamblia. These tests are now commercially available and offer a high degree of sensitivity even on specimens that are preserved and can be detected days before any trophozoites and cysts are shed.
Dientamoeba fragilis is an amoeba-flagellate with a cosmopolitan distribution. The life cycle is not completely known.
Morphology of Trophozoites and Lifecycle
D. fragilis are relatively small, varying from 3-22mm in diameter and there can be considerable variation in size among organisms in the same fecal sample. The organisms have only a trophozoite stage and in a permanently stained preparation, one, two or rarely three nuclei can be seen, two being the most common. The nuclear chromatin is usually fragmented into three to five granules but these have not been visualized by Giemsa Stain, and there is normally no peripheral chromatin on the nuclear membrane. The cytoplasm is usually vacuolated and may contain ingested debris as well as some large uniform granules. The cytoplasm can also appear uniform and clean with a few inclusions. D. fragilis live in the lumen of the cecum and upper colon.
Illustration 3-2. This is an illustration of the assumed life cycle of Dientamoeba fragilis, the cause of a protozoan parasitic infection. (SOURCE: PHIL 3389 - CDC/Alexander J. da Silva, PhD/Melanie Moser)
Image 3-5. Dientamoeba fragilis. Trichrome stain. (SOURCE: PHIL 548 – CDC)
This is a controversial area. The organism has been reported in association with mucous diarrhea, abdominal pain and tenderness. Nausea, vomiting and low-grade fever have also been reported in a number of cases. The precise role of this organism as a cause of disease remains to be determined.
Diagnosis is dependent on examination of the fresh direct wet preparation or permanently stained smears prepared from unformed or formed stools with mucus. It is particularly important that permanently stained smears of stool preparations should be examined, because survival times of the organism in terms of morphology, is very limited and specimens must be examined immediately or preserved in a suitable fixative as soon as possible after defecation. The recommended stains are Fields’ and Giemsa stain (trophozoites are destroyed in a formol-ether concentrate). Table 3-1. details useful morphological features that are similar between species of flagellate and are used in laboratory diagnosis.
This flagellate is cosmopolitan in its distribution. It is thought to be non-pathogenic although it has been associated with diarrheic stools. It is the most commonly found flagellate next to G. lamblia and D. fragilis. Found in a wide host range including non-human primates, cats, dogs and various rodents.
Diagram 3-3. This is an illustration of the life cycle of Trichomonas vaginalis, the causal agent of Trichomoniasis. (SOURCE: PHIL 3423 - CDC/Alexander J. da Silva, PhD/Melanie Moser)
Trichomonas hominis do not have a cystic stage. The trophozoites measure from 5-15mm in length by 7-10mm in width. The shape is pyriform and has an axostyle which runs from the nucleus down the centre of the body and extends from the end of the body. They also possess an undulating membrane which extends the entire length of the body and projects from the body like a free flagellum (this feature distinguishes it from other trichomonads). The characteristic number of flagella is five; there is some deviation from this number. They also have a single nucleus at the anterior end. Trichomonads swim with a characteristic wobbly movement, which makes them unmistakable during diagnosis.
Image 3-6. Two trophozoites of Trichomonas vaginalis obtained from in vitro culture. Smear was stained with Giemsa. (SOURCE: CDC)
In a fresh stool, the flagellates move very rapidly in a jerky, non-directional manner. The axostyle and undulating membrane are diagnostic. The flagellates are difficult to stain; however, the axostyle can be seen on a stained preparation and is diagnostic. Table 3-1 details useful morphological features that are similar between species of flagellate and are used in laboratory diagnosis.
Illustration 3-4. The life cycle of C. mesnili (Cuomo)
The trophozoites of C. mesnili are pear shaped and measure 6-20mm in length. They have one large nucleus with a small karyosome and three flagella that extend from the nucleus at the anterior end of the parasite. A distinct oral groove or cytosome can be seen near the nucleus with its sides being supported by two filaments. They are known to move in a directional manner.
Image 3-7. Chilomastix mesnili cyst.(SOURCE: PHIL 426 - CDC /Dr. Mae Melvin)
Morphology of cysts
The cysts are 6-9mm; they have a large single nucleus with a large karyosome. They also have a prominent side knob giving it a characteristic lemon shape. The cytosome is evident with a curved shepherds crook fibril. It also has a characteristically coiled filament which when stained is darker in color.
Image 3-8. Chilomastix mesnili cysts are excreted with feces and constitute the transmission form of the micro-organism. The uninucleated lemon shaped cysts are seen with a little proturberance at one end and a prominent cytostome. (Iodine stained). (6µm) (CDC)
The characteristic lemon shaped cysts can be seen in a formol-ether concentrate. Motile organisms can be seen in a wet preparation of a fresh stool however the characteristic morphology is evident in a permanently stained preparation. Table 3-1 details useful morphological features that are similar between species of flagellate and are used in laboratory diagnosis.
Enteromonas hominis is a small flagellate and is rarely encountered in man. It is found in both warm and temperate climates and is considered to be non-pathogenic.
Illustration 3-5. The life cycle of E. hominis (Cuomo)
The trophozoites are oval and 4-10mm in length. They have four flagella, three anterior flagella and one adheres to the body ending in a tail, producing a jerky rotational movement. They have one nucleus with a large karyosome that is evident in a stained preparation.
Morphology of the Cyst
The cysts are oval and range between 6-8mm in length. They have up to four nuclei with a bipolar tendency.
The cysts are seen in a formol-ether concentrate. The cysts have no distinguishing characteristics and thus can be confused with E. nana or even yeasts. The characteristic trophozoites can be seen in a permanently stained fecal smear. Table 3-1. details useful morphological feature’s that are similar between species of flagellate and are used in laboratory diagnosis.
Retortamonas intestinalis like Enteromonas hominis is a small flagellate and is rarely encountered. It is found in both warm and temperate climates and is considered to be non-pathogenic.
Illustration 3-6. Life cycle of Retortamonas intestinalis (Cuomo)
The trophozoite is small, measuring between 4mm and 9mm. Its movement is jerky and rotational and has two anterior flagella and a prominent cytosome that can be seen in an unstained preparation. It has a relatively large nucleus at the anterior end with a small compact karyosome.
Morphology of the Cyst
The cysts are small and pear shaped. They range in size between 4-7mm with one large nucleus frequently near the centre. The fibril arrangement from the nucleus is suggestive of a birds beak. This is characteristic of R. intestinalis cysts.
The small pear shaped cysts are uncharacteristic in an unstained formol-ether preparation. However, the addition of iodine reveals the characteristic bird beak fibrillar arrangement in the pear shaped cyst. In a fresh stool preparation, the two anterior flagella and cytosome can be seen in the trophozoite. In a permanently stained preparation, the large nucleus with small central karyosome is diagnostic. The below Tables detail useful morphological features which are similar between species of flagellate and are used in laboratory diagnosis.
3.2 Identifying Flagellates
It is important to know and understand the morphological features which differentiate each species of flagellate from one other. The below Tables details the important features that are used when identifying flagellates found in human stool samples. Trophozoites and cysts can be seen in saline mounts of fresh feces. On occasions, species identification may require stained preparations.
* Not a normal feature for identifying species in routine stool samples
Table 3-1. Differential Morphology of Protozoa Found in Stool Specimens of Humans: Flagellates-Trophozoites (SOURCE: CDC)
Table 3-2. Differential Morphology of Protozoa Found in Stool Specimens of Humans: Flagellates-Cysts (SOURCE: CDC)
Flagellate trophozoites are best identified in fresh saline mounts, allowing you to observe the way that they move. Use Key 3-1. to help to identify stained flagellate trophozoites. Iodine solutions are used primarily to stain flagellate cysts, this makes it possible to see the structure of the nuclei. Use Key 3-2. to help to identify amebic and flagellate cysts:
Flagellates (Adapted and redrawn, WHO, 1991)