INF2/parasit/handout (10/11)

Intestinal protozoa

AMOEBAE & CILIATES

Intestinal protozoa of humans can be grouped as given belowAmoebae Ciliates:Entamoeba histolytica Balantidium coliE.dispar CoccdiaE.hartmaniE.coli Cryptosporidium spp.E.gingivalis(oral cavity) Cyclospora cayetanensisEndolimax nana Isospora belliIodamoeba butschliiDientamoeba fragile Sarcocystis hominisFagellatesGiardia intestinalis(lamblia)Trichomonas tenax (Oral cavity)Trichomonas hominis

Intestinal amoebae

Amoebae are unicellular organisms, common in the environment; many are parasites of vertebrate and invertebrates. Relatively few species inhabit the human intestine and of them majority are non-pathogenic commensals. Of the several species parasitizing the alimentary tract of humans, only E.histolytica is recognized as pathogenic ( the pathogenic potential of Dientamoeba fragilis and that of Iodamoeba butschlii has not been conclusively proved).There are two stage in life cycle of these amoebae, the motile, feeding trophozoites which divide by binary fission and the dormant resistant form the cyst. However, no cystic stage has yet been identified in E.gingivalis and Dientamoeba fragilis. Only the cyst stage is important in transmission. These amoebae can be differentiated on the morphological features of either the trophozoite or the cyst. The differentiating features of trophozoite include the size; motility; character of the pseudopodia; inclusion bodies found in the cytoplasm; nuclear number and in the nucleus, the arrangement of chromatin and the karyosome.In the cystic stage, the size is very important identification feature. The other features include the shape, presence of glycogen mass, nuclear number, arrangement of chromatin and karyosome and chromatid bodies.Genus: EntamoebaThese organisms inhabit the alimentary tract of humans, monkeys and many other species of vertebrate and invertebrate. This genus includes all amoebae that have more or less spherical nucleus with a distinct nuclear membrane lined with chromatin granules and a relatively small karyosome situated at or near the center of nucleus. Trophozoite has a single nucleus. Genus: Endolimax and Iodamoeba - have a relatively large karyosomeEntamoeba histolyticaE.histolytica is pathogenic amoeba, which inhabit the large intestine of human. Infection caused by this organism is called amoebiasis. Infection can lead to amoebic dysentery, resulting from trophozoites invading the intestinal wall, and amoebic liver abscess and other extraintestinal lesions resulting from spread of trophozoites from the intestine via the blood stream. E.histolytica has worldwide distribution with a high prevalence associated with poor environmental sanitation.

Objectives:1. Name the amoebae and ciliates that infect humans2. Of these name the organisms that are pathogenic to humans3. Outline the lifecycle of Entamoeba histolytica and Balantidium coli4. Indicate the stages that cause pathogenic effects and those that are of diagnostic importance in

the above5. Identify the points in the life cycle where preventive measures are applicable6. Describe the mechanisms of pathogenesis7. Describe the pathological and clinical consequences of the diseases caused by these organisms 8. Describe the mode(s) of transmission, prevention and control of amoebiasis9. Describe laboratory methods of diagnosis of these organisms

Life cycleInfection is through ingestion of mature cysts. Cyst is resistant to gastric acid and on ingestion it passes into the small intestine. Excystment (digestion of cyst wall) occurs in the small intestine and each cyst gives rise to 8 immature troiphozoites. Maturation of trophozoites takes place in the caecum. Trophozoites feed, grow and divide and are responsible for causing pathological effects by invading intestinal mucosa. As the trophozoite pass down the colon, it assumes a spherical shape and secretes a resistant wall around itself forming a cyst. Subsequently nucleus divides twice to produce quadrinucleated (4 nuclei) mature cyst. Cyst evacuated through faeces. Cyst is the infective stage to human. When voided these are carried with the faeces resulting in contamination of soil, water and can infect other humans either directly or indirectly. Trophozoites are non infective. Primary known reservoir for E.histolytica is the human although morphologically similar amoebae may be found in primates, dogs and cats.Pathogenesis:Infection with E. histolytica does not necessarily lead to disease. About 90% of people who become infected with E. histolytica are asymptomatically colonized. The pathogenic effects are due to tissue invasion, which depend on both host and parasite factors. Unfortunately, many are incompletely understood. The major host factors include:

1. Presence of bacteria in gut which provide an environment with low oxygen tension2. The degree of immunological resistance.

Parasite factors:Virulent factor CharacteristicsAdhesion molecules (-N- acetyl-D-galactosamine-inhibitable lectin( Gal/Gal Nac adherence lectin) Target cell adherence, not directly cytotoxic. Enhance target cell lysis by reducing galactose concentration. Channel forming peptides (Amoebapores) - Stored in cytoplasmic granules, and released following

target cell contact; forms ion channels in the plasma membranes thus lysing the target cell.

Cysteine proteinase - Aid in penetration of host tissue by digesting extracellular matrix, cleaving collagen, elastin,fibrinoge in extracellular matrix by stimulating host cell proteolytic cascadeInactivates the complement factors and are thus resistant to complement mediated lysis. Limit the effectiveness of humoral response by degrading both IgA and IgG Intestinal disease: Invasion of intestinal mucosa by E. histolytica is an active process mediated by the parasite. Trophozoites adhere to colonic mucins and host cells through adhesion molecules. Once the mucus barrier has been broken down E. histolytica reaches the luminal surface of enterocytes and initially produces a contact –dependent focal and superficial epithelial erosions of the large intestine. Cell lysis is produced by the channel forming peptides of E histolytic (amoebapores). The parasite then penetrate the muscularis mucosa and gain entry to the submucosal tissues where it multiplies and spread laterally.Spread is probably helped by cysteine protinases which can cleave extracellular structural matrix. The typical lesion is a flask-shaped ulcer, with its large base in the submucosa and small opening on the mucosal surface. The mucosal lining may appear normal between ulcers. Amoebic ulcers most often develop in the caecum, appendix or adjacent portion of ascending colon. Rarely, an amoeboma or amoebic granuloma may result from repeated invasion of the colon by E. histolytica. Amoebomas are more frequently found at the caecum and rectosigmoidal junction. Trophozoites from the submucosa can spread to other sites through the blood stream or the lymphatic to other organs such as liver, lung, skin and brain with abscess formation in these organs. Direct extension to skin may occur with rectal lesions. Extension through the intestinal wall can lead to perforation of colon.Extraintestinal amoebiasis: Hepatic amoebiasis is the most common extraintestinal form of invasive amoebiasis. Amoebic abscesses may be found in all age groups but are more frequent in adults than in children, and are more frequent in males than in females. The trophozoites invade the liver cells, producing focal necrosis and oedema. The necrotic foci enlarge and coalesce, forming one or two large abscesses. Pulmonary infection could either be due to direct extension of liver abscess or due to blood stream spread. In severe cases, especially in patients treated with corticosteroids, amoebic trophozoites can be found in every organ of the body.Clinical features:Intestinal amoebiasis: Clinical spectrum of intestinal E.histolytica infection ranges from asymptomatic carrier state or symptomatic nondysenteric infection, acute amoebic dysentery, to fulminant colitis with perforation. Onset is insidious, except in fulminant cases with colicky abdominal pain, frequent bowel movements, and tenesmus may present. With the onset of dysentery bowel movements characterized by blood tinged mucus. Intestinal ulceration causes a blood and mucus diarrhoea with tenesmus. In severe cases, symptoms may begin very suddenly and include profuse diarrhoea, fever, dehydration. In extraintestinal amoebiasis, which usually present as non-purulent abscesses, the clinical features depend on the organ affected.

Amoebic liver abscess: onset is abrupt with pain in the upper abdomen and high fever and on physical examination the cardinal sign is painful hepatomegaly. Diagnosis: Confirmatory diagnosis is by demonstrating the cyst and trophozoites in faeces, sigmoidoscopy , tissue biopsy, hepatic aspirates or by immunological methods. The diagnosis of invasive intestinal amoebiasis is based on the identification of E. histolytica trophozoites in recently evacuated faeces or in rectal smears. Fresh sample should be examined for detection of trophozoites. Two types of wet mount preparation should be made from each specimen: mount in saline (to observe amoebic motility in warm specimen): mount in saline and iodine (to distinguish E. histolytica cysts from other amoebic species).Refer the table below for characteristic morphological features of the different amoebae. The minimum of three faecal samples using concentration and confirm the diagnosis by permanent stain is recommended. The presence of trophozoites containing ingested red blood cells is strongly suggestive of E.histolytica. Non-invasive imaging techniques (eg., ultrasound, CT, MRI) can be used to detect hepatic abscesses. It is also possible to aspirate hepatic abscesses. However, this is rarely done and only indicated in selected cases (eg. serology and imaging is not available, therapeutic purposes). The aspirate is usually a thick reddish brown liquid that rarely contains trophozoites. Trophozoites are most likely to be found at the abscess wall and not in the necrotic debris at the abscess center. In extraintestinal amoebiasis , organism may or may not be found in the faeces. Thus, serology is especially useful for the diagnosis of extraintestinal amoebiasis. Of the serological test available ELISAs are the most sensitive and specific. Kits for differentiation of E.histolytica and E.dispar directly from faecal samples, based on ELISA are available. Sensitivity and specificity of tests of diagnosis for amoebiasis

TestColitis Liver abscess

Sensitivity Specificity Sensitivity

Microscopy (stool) <60% 10-50% <10%

Microscopy (abscess fluid) <25%

Stool antigen detection (ELISA) >95% >95% Usually negative

Serum antigen detection (ELISA) 65% (early) >90% ∼75% (late), ∼100% (first 3 days)

Abscess antigen detection (ELISA) ∼100% (before treatment)

PCR (stool) >70% >90% Not doneTransmissionInfection is by the ingestion of mature cysts. Animal reservoirs are not important. Infection occurs via the faeco-oral root, food and water contaminated through exposure to infected human faeces. Food handlers excreting cysts are an important source of contamination. Waterborne outbreaks can occur through contamination of water supply with sewage. Houseflies also play an important role as mechanical vectors contaminating food. Sexual transmission also occurs.Cysts cannot withstand desiccation, heat (96° C for one min) and freezing (.10°C for 24h). Cysts can survive for up to 45 minutes in faecal material lodged under fingernails. Cysts can survive in water and milk for weeks. Standard chlorination of water is not effective in complete elimination of cysts. Food disinfectants such as iodine((220ppm), full strength vinegar(5-10%acetic acid) and sodium chloride , boiling water for 10 minutes are effective in killing all cysts.Prevention:Personal protection in endemic areas includes drinking only bottled, boiled water. Drinks with ice cubes should be avoided. Drinking water can also made safe by filtering (1 micron or less filter) .Good personal hygiene is very important to prevent faecal- oral transmission.Ciliates

What are ciliates?Ciliates are protozoa that can be recognized by their hairlike ‘cilia’. They use them for locomotion and for feeding. Cilia are structurally identical to flagella but typically shorter and present in much larger numbers. Ciliates have two nuclei: a small micronucleus, and a large macronucleus. Reproduction is by binary fission. Balantidium coliLargest protozoan parasite of man and is the only pathogenic ciliate that parasitize humans. It inhabits distal ileum and colon. Organism invades the intestinal mucosa, multiply and spread in the submucosa. The commonest habitat is the colon of pigs, rodent and monkeys.Morphology:

Have two stages, the trophozoite and the cystTrophozoite: This is the pathogenic stage to human. It is quite large measuring 60 –70 x 40 -60 μm. Trophozoite is oval shape and covered with cilia. At the anterior end there is a cytostome (a mouth) leading to cytopharynx. There are two nuclei; a large kidney shaped macronucleus and a small rounded micronucleus. Reproduction is by binary fission.Cyst: This is the infective stage to human. It is large and spherical measuring 50 –60 μm in diameter. Two nuclei are present, the macronucleus and the micronucleus.Life cycle:The cyst which is the infective stage is excreted in faecesWhen the cyst is ingested with contaminated food or water, the cyst enters a new host and forms the trophozoite, which multiplies inside the host.Pathogenesis: The trophozoite is able to invade the distal ileal and colonic mucosa to produce intense mucosal inflammation and ulceration. Clinical features : Infection is closely resembles amoebic colitis. It causes blood and mucus diarrhoea.

Diagnosis:Microscopic examination of fresh or fixed faecal smear for trophozoites and cysts

Morphologic features and pathogenicity of intestinal amoebaeCharacteristics E. histolytica, E. dispar E. hartmanni E. coli E. nana I. bütschlii

Trophozoites (size, nucleus, and movement)

20 - 40 μm; 1 nucleus; actively motile cytoplasmic protrusions, finger shaped pseudopodium

6-10 μm; 1 nucleus; non-successive

15-50 μm; 1 nucleus; sluggish, non-directional, short and blunt pseudopodium

6-15 μm; 1 nucleus, blunt and hyaline pseudoodium, s non-directional slow movements

6-20 μm; 1 nucleus, slow movement, nonsuccessive, hyaline pseudopodium

Cysts (size, nucleus)

10-20 μm; mature cyst has 4 nuclei, immature cyst has 1 or 2 nuclei

5-8 μm; mature cyst has 4 nuclei; immature cyst has 1 or 2 nuclei; 2 nucleated cysts very common

10-35 μm; mature cyst has 8 nuclei

6-8 μm; 4 nuclei 10-12 μm; 1 nucleus

Appearance of trophozoites

Stained trophozoites fine, uniform granules of peripheral chromatin, and small central karyosome in nucleus; ingested RBC (E. dispar are similar to E. histolytica trophozoites, NoIngested RBC

Nuclear structure similar to E. histolytica; ingested bacteria; cytoplasm finely granular

Nucleus with irregular cluster of peripheral chromatin; large, , eccentric karyosome

Nucleus with large karyosome; no peripheral chromatin

Large granular karyosom, no peripheral chromatin

Appearance of cysts

nuclear structure, similar to trophozoite, chromatoid bars with rod shaped with rounded ends,glycogen mass see in immature cysts

nuclear structure, similar to trophozoite,, chromatoid bars with rounded or squared ends

nuclear structure, similar to trophozoite, chromatoid bars infrequent needle shaped when present

nuclear structure, similar to trophozoite, chromatoid bars absent

nuclear structure, similar to trophozoite ,large compact glycogen, no peripheral chromatin

Pathogenicity Pathogen E.histolytica,E. dispar are nonpathogen)

Nonpathogen Nonpathogen Nonpathogen Nonpathogen

Task: Tabulate the characteristic morphological features of the different amoebae, trophozoites and cyst forms from the specimens shown at the demonstration classPractice questions:

1. List the intestinal amoebae found in humans2. Describe the morphology of different stages of these parasites3. Explain the basis of clinical presentations caused by amoebiasis4. Describe the laboratory diagnosis of amoebiasis5. Discuss the advantages and disadvantages and limitations of these laboratory procedures

Recommended Text books and web sitesManson’s Tropical Didease By Gotfon C Cook and Alimuddin ZumlaAtlas of Human parasitology by Lawrence R Ash. Thomas C Orihelhttp://www.cdc.gov.

Dr.D.Iddawela,