Lecture 2: Protozoa None pathogenic Amoebae - PowerPoint Presentation

1. Lecture 2: ProtozoaNone pathogenic AmoebaeFlagellates/ Part IMedical ParasitologyProf. Dr. Ahmed Ali Mohammed

2. Nonpathogenic Amoebae A number of species of the genus Entamoeba are of worldwide distribution but do not appear to cause disease. The knowledge of these species is of value in differentiating the harmless commensals from potentially pathogenic E. histolytica.1. Entamoeba dispar Formerly, it was believed that there is a pathogenic invasive strain and a nonpathogenic strain of E. histolytica, and Entamoeba dispar representing the nonpathogenic strain, but using the isoenzyme-electrophoretic techniques, and based on the antigenic differences, genomic DNA and ribosomal RNA, it is recognized now as a separate species. The two species are morphologically identical but the trophozoites of E. histolytica contain ingested red blood cells. The cysts of E. histolytica and E. dispar cannot be differentiated microscopically and should therefore be reponed as E. histolytica / E. dispar.

3. 2. Entamoeba hartmanni It is cosmopolitan in distribution, morphologically similar to E. histolytica but both its trophozoites and cysts are smaller and the cyst never contains ingested red blood cells. Therefore, it was earlier regarded as a small race of E. histolytica. It is a nonpathogenic amoeba acquired by the ingestion of food or water contaminated with cysts. Its life cycle is similar to that of E. histolytica. The diagnosis can be established by the measurement of the size of the trophozoites and cysts and the absence of red blood cells in the endoplasm of the trophozoite.

4. 3. Entamoeba coli Generally considered nonpathogenic in humans (commensal). The trophozoite does not ingest or invade the host tissues. Its presence is evidence that the host has ingested fecal material. It lives in the lumen of the caecum and the lower level of the large intestine. The parasite has two stages trophozoite and cyst. The trophozoite has a spherical shape. The food vacuoles contain bacteria, yeast and other enteric microbes and fragments of intestinal debris. The nucleus with eccentric and large karyosome. The mature cyst has 8 nuclei. The life cycle is similar to that of E. histolytica, except that the trophozoite in this example doesn’t attack the mucosa of the intestine.

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7. 4. Entamoeba gingivalis It is a parasite of the mouth of man and other mammals, including several species of monkeys, dogs and cats. It is commensal, commonly found in the tartar and debris associated with the gingival tissues of the mouth. It lives in/on the teeth, gum and sometimes tonsils, particularly if there is suppuration (purulence), as in pyorrhoea alveolaris, but it also occurs in apparently hygienic mouths and on dental plates if they are not kept clean. There is little indication that it is pathogenic, it abounds in people with unhealthy oral conditions (i.e., gingivitis or periodontitis). Only trophozoite stage has been described in this parasite. In most respects, it closely resembles E. histolytica. Endocytotic vacuoles are often numerous and may contain oral epithelial cells, leukocytes, occasionally erythrocytes and various microbial organisms although it is not invasive.

8. Transmission is either directly by oral to oral contact (kissing) or indirectly via trophozoite-contaminated food, chewing gum, toothpicks, contaminated drinking utensils, etc.

9. 5. Endolimax nana It is the smallest intestinal amoeba infecting humans. The trophozoite is commensal in the lumen of the colon, caecum and the lower level of the large intestine, and is generally considered nonpathogenic, feeding on the bacteria. Its presence indicates that contaminated material has been ingested. It appears in two stages, trophozoite and cyst. The endoplasm has numerous minute vacuoles (so it has a foggy appearance). The ectoplasm is hyaline and almost transparent. The food vacuoles contain bacteria, vegetable cells and some crystals. The trophozoites multiply rapidly by binary fission. The cysts of E. nana can be identified and distinguished from other cysts by their smaller size, ovoid shape and one to four vesicular nuclei, each usually containing a large, eccentric karyosome. The life cycle is identical to that of other cyst-forming amoebae, with the cyst being the infective stage.

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11. 6. Iodamoeba butschlii The parasite is seldom as common as E. coli and E. nana. It is commensal, lives in the lumen of the large intestine, especially the caecum. It has two stages, trophozoite and cyst. As it is evident from the contents of their food vacuoles, it is feeding on the bacteria and yeast. It is transmitted by the cyst which is very distinctive, facilitating its identification. The cyst is variable in shape, usually irregularly rounded (ovoid) and contains one nucleus. There is a relatively big mass of glycogen that stains deep golden brown with iodine (the cause of the name Ioda.), and also helps in the differentiation of this parasite from other intestinal amebae.

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13. Opportunistic free-living amoebae Free-living amoebae of the genera Naegleria, Acanthamoeba and Balamuthia are facultative parasites of man. They are ubiquitous in nature, found commonly in soil and water (swimming pools, tap water, and heating and air-conditioning units) where they feed on bacteria, but as opportunists, they may produce serious infection of the central nervous system and the eye.Naegleria fowleri It has two stages, motile trophozoites and nonmotile cysts. The trophozoite occurs in two forms amoeboid and flagellate. The amoeboid form is actively motile by means of eruptive, blunt pseudopodia called lobopodia. It has distinctive phagocytic structures known as amoebostomes used for the engulfment and vary

14. in number depending on the strain. Reproduction is by simple binary fission of the amoeboid form. The cysts are uninucleated, spherical and surrounded by a smooth double wall. Cysts and flagellate forms of N. fowleri have never been found in tissues or CSF. The amoeboid form of N. fowleri is the invasive stage of the parasite. Man acquires the infection by nasal contamination during swimming in freshwater lakes, ponds or swimming pools containing the infective form.The infection may also be acquired by inhalation of dust containing infective forms. It is likely that flagellate forms or cysts of N. fowleri could enter the nose. However, since the amoeboid form is the invasive stage of the parasite, it appears that flagellate forms revert to amoeboid forms and the amoeboid form escape from the cysts in the nose.

15. The amoeboid forms invade the nasal mucosa and travel along the olfactory nerves to the brain. They first invade olfactory bulbs and then spread to the more posterior regions of the brain leading to a rapidly fatal infection known as primary amoebic meningoencephalitis (PAM). It occurs in healthy children and young adults with a recent history of swimming in freshwater.Symptoms The patient develops a severe frontal headache, fever (39°-40°C), anorexia, nausea, vomiting and signs of meningeal irritation. Involvement of olfactory lobes may lead to disturbances in smell or taste. The patient may also develop visual disturbances, confusion, irritability, seizures and coma. The disease usually results in death within 72 hours of the onset of symptoms. The period between contact with the organism and the onset of clinical symptoms varies from 2-3 days to as long as 7-15 days. PAM may resemble acute purulent bacterial meningitis, and these conditions may be difficult to differentiate particularly in the early stage.

16. Diagnosis It can be made by microscopic identification of living or stained amoebae in CSF. Amoebae can also be demonstrated by fluorescent antibody staining of the CSF and in the histologic sections of the brain biopsied tissue by immunofluorescence and immunoperoxidase methods. As in the case of fulminating bacterial meningitis, the leucocyte counts (predominantly neutrophils) vary from a few hundreds to more than 20,000 cells/μL. CSF protein content is generally increased and glucose level is low.Treatment At present, there is no satisfactory treatment for PAM. Antibacterial antibiotics and antiamoebic drugs are ineffective. Amphotericin B, a drug of considerable toxicity, is the antinaeglerial agent for which there is evidence of clinical effectiveness.

17. 2. Subphylum Mastigophora (The Flagellates)Pathogenic Flagellates The flagellates are a group of protozoa distinguished by having one to several thread-like extensions from the ectoplasm in their trophozoite stage called flagella (single: flagellum). They are related to Sub-phylum Mastigophora, Class Zoomastigophorea. The flagellate protozoa that parasitize man are primarily divided to:A. The flagellates of the digestive tract and genital organs (Intestinal, oral and genital flagellates).B. Blood and tissue flagellates. The first group of flagellates inhabiting the mouth, intestine and genital tract and are typically lumen parasites.

18. Although no member of the group is a tissue invader, Giardia lamblia in the duodenum and Trichomonas vaginalis in the vagina may produce symptoms.A. Intestinal, oral and genital flagellates1. Giardia lamblia This parasite has a cosmopolitan distribution and its highest prevalence is in both warm and temperate climates (in the tropics and subtropics) and where sanitation is poor, causing a disease called Giardiasis. The infection in children is more frequent than in older people. The parasite appears in two stages, trophozoite and cyst. The trophozoite lives on the epithelial brush border of the upper two thirds of the small intestine (it inhabits the duodenum and the upper part of the jejunum), sticks itself on the epithelial cells, and sometimes enters the bile duct.

19. When the trophozoite is seen from the ventral aspect (view) it appears rounded anteriorly and tapering to a point posteriorly. When viewed from the lateral (profile), it is relatively thin in the anterior half and concave ventrally, forming an adhesive sucking disc acts as an organelle of attachment. The trophozoite has two ovoid nuclei each with a central karyosome and there is two axostyles between the two nuclei extending to the posterior end. In addition, it bears four pairs of flagella extends along the midline.

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21. The cyst is ovoid in shape with a thick wall. It has 4 nuclei founds in one pole of the cysts or distributed as two nuclei in each pole. The cyst has also retracted flagella which appear as stiffly curved fibrils situated in parallel pairs.

22. Life cycleHuman infection occurs by the ingestion of cysts in contaminated water, food, or by the fecal-oral route (hands or fomites).Decystation (or excystation) occurs in the small intestine (specifically in the duodenum) within 30 minutes of ingestion releasing the trophozoites where each cyst produces two trophozoites. The trophozoites then multiply by longitudinal binary fission to form enormous numbers and colonize in the duodenum and upper part of the jejunum, where they may swim freely or attach to the sub-mucosal epithelium via the ventral suction disc. To avoid the acidity of the duodenum, it may localize in the biliary tract. The free trophozoites encyst as they move downstream, and mitosis takes place during the encystment. Cysts are resistant forms and are responsible for the transmission of Giardiasis.

23. Encystation occurs as the parasites transit toward the colon where the intestinal contents lose moisture and the patient starts passing formed stools. Cysts are passed in the stool; the cysts are hardy and can survive several months in cold water. As the cyst matures, the internal structures are doubled, so that when excystation occurs, the cytoplasm divides, thus producing two trophozoites.Both cysts and trophozoites can be found in the feces, the cyst is the stage found most commonly in nondiarrheal feces. In victims of giardiasis, massive infection is common. The presence of up to several billion trophozoites in a single diarrheic stool sample is not unusual. Man is the primary host although beavers, pigs and monkeys are also infected and serve as reservoirs. Because the cysts are infectious when passed in the stool or shortly afterwards, person-to-person transmission is possible. Ingestion of 100 or more cysts is considered infective.

24. PathogenicityThe infection is common in children 6 to 10 years of age but is also seen often in older children and adults. Outbreaks are frequent in daycare nurseries and other institutions where sanitation may be inadequate. Since Giardia has not been known to produce toxins, it appears that symptoms result from combined mechanical and chemical factors. The presence of the parasite on the surface of the epithelial cells (on the brush border) causes mechanical complication leads to malabsorption of nutrients and digested food because it forms a pavement like sheet covering and damaging the mucosa. In addition, it causes an inability of the small intestine to absorb such essential fat-soluble substances and vitamins such as carotene, vitamin B12 and folate.

25. These absorptive abnormalities may be accompanied by reduced secretion of a number of small-intestinal digestive enzymes, such as disaccharidase. The parasite also causes heavy mucus secretion and feed on this mucus, the amino acids and the other substances passes through the intestine which lead to malnutrition and vitamin deficiency. In addition, attachment of the trophozoite to the mucosal surface causes shortening of the villi of the small intestine, inflammation of the crypts and lamina propria, lesions on the mucosal cells and occasionally, the trophozoites penetrate the mucosa, but this is rare. Occasionally, bile duct and gall bladder involvement may produce jaundice and colic. These symptoms may become evident as early as 3 to 25 days (the average 10 days) after ingestion of cysts.

26. Symptoms1. Early symptoms include Epigastric pain, abdominal cramps and discomfort expressed as flatulence, abdominal distension, nausea and foul-smelling bulky, explosive, and often watery diarrhea. The stool contains excessive lipids but very rarely any blood or necrotic tissue.2. The large quantity of mucus and the presence of fats with the stool leads to continuous diarrhea with a large quantity of mucus. The aggregation of fats result from the malabsorption of it. Sometimes constipation or steatorrhea also may occur.3. Weight loss.4. The more chronic stage is associated with vitamin B12 malabsorption, disaccharides deficiency and lactose intolerance, fat soluble vitamin deficiency.

27. Diagnosis The General Stool Examination (GSE) is used primarily for this purpose. The diagnosis is based on the detection of the typical cyst in the formed stool, and the trophozoite and cyst of the parasite in diarrheal stools using normal saline and iodine preparation or using iron hematoxylin stain as in the case of E. histolytica. Trophozoites must be distinguished from the non-pathogenic flagellate Trichomonas hominis, which is an asymmetrical flagellate with an undulating membrane. The development of a stool enzyme-linked immunosorbent assay (ELISA) has been shown to be both a specific and sensitive rapid diagnostic tool.Treatment Giardiasis may be disappearing spontaneously but usually is eradicated following therapy with Quinacrine hydrochloride (Atabrine) or Metronidazole. Tinidazole can also be used for a 1-day treatment. Paromomycin (Humatin) may be useful in pregnancy. Furazolidone is often used for treating children.

28. 2. Chilomastix mesnili It is a common flagellate living as a harmless commensal in the caecum and the colon of man. It has well-defined trophozoite and cyst stages. The trophozoite has a single nucleus lies near the center and a large conspicuous cytostome (mouth) is seen on one side of the nucleus. The cytoplasm contains numerous food vacuoles. The cyst is lemon-shaped with a small projection at the anterior end and surrounded by a thick tough cyst wall. The trophozoites feed on enteric bacteria and multiply by binary fission. In freshly passed liquid stools, only trophozoites are seen, in semi-formed stools, both trophozoites and cysts may be observed, and in well-formed stools, only cysts are present.

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30. Transmission of the parasite from one person to another takes place by ingestion of food or water contaminated with the cysts stage. C. mesnili does not produce any symptoms. The diagnosis can be made by the detection of the trophozoites and cysts in the fecal smear.

31. 3. Trichomonas vaginalis The parasite has a cosmopolitan distribution, causes a disease called Trichomoniasis. This species is found only in a trophozoite stage. It is with a higher prevalence among persons with multiple sexual partners or other venereal diseases. The parasite has a large nucleus, and four anterior flagella of equal length, a fifth one lies on the margin of a relatively short undulating membrane which does not extend beyond the posterior margin of the membrane (the flagellum). There is also a delicate axostyle protruding for a considerable distance beyond the posterior tip of the organism. Multiplication by longitudinal binary fission.

32. The normal habitat of the parasite is the vagina and urethra of women and the urethra, seminal vesicles and prostate of men. It may also be found in the urinary bladder in females. Mostly, non-pathogenic for males but sometimes causes urethritis. Transmission of the infection is principally by sexual intercourse, or by the infected mucus in the W.Cs. or underwear. Damp washcloths and similar items are also sources of infection among children and adults.Pathogenicity and Symptoms Human trichomoniasis is a widely prevalent sexually transmitted disease (STD) of worldwide importance. The parasite lives on the mucosa feeding on the bacteria and the leucocytes. T. vaginalis is an obligate parasite, it cannot live without close association with the vaginal, urethral or prostatic tissues. The organism is responsible for a mild vaginitis with discharge. Vaginal discharge contains a large number of parasites and leucocytes and is liquid, greenish or yellow.

33. 1. The infection in women is frequently symptomatic. The symptoms vary from mild to severe. While about 15% of women with trichomoniasis complain of symptoms, altered vaginal secretions are evident in many more. Vaginitis with a purulent discharge is the prominent symptom, and can be accompanied by vulvar and cervical lesions, abdominal pain, dysuria and dyspareunia. It also leads to heavy, acidic, mucoid secretion, excessive discharge together with genital sprays may produce urticaria and acute vulvitis. The incubation period is 5 to 28 days.2. In men, the infection is frequently asymptomatic. Symptoms are much less noticeable, although occasionally, urethritis, epididymitis, prostatitis can occur and swelling of the prostate gland.

34. DiagnosisMicroscopic examination of wet mounts may establish the diagnosis by detecting the actively motile trophozoites. For the diagnosis of male infections, smears of urethral and prostatic discharges stained with Giemsa stain is useful, whereas vaginal and urethral discharges in the females are the useful samples. Examination of the urine of both sexes and examination of prostate secretions of the male following prostate massage are also helpful diagnostic procedures. The parasite may also be detected by fluorescent microscopy by staining with fluorescein-labelled monoclonal antibody.In difficult cases, cultivation of a swab sample in Diamond's medium can be used, but the results are not available before 3 to 7 days. Several types of ELISA have been developed either to measure antibodies or to detect the antigens of the parasite in clinical samples. Polymerase chain reaction (PCR) for the diagnosis of trichomoniasis has also been developed.

35. Treatment Metronidazole is the most effective drug. Restoration of the normal pH of the vagina by periodic douches with a dilute solution of vinegar is an effective preventive method and can control mild infections. It is recommended that sexual partners be treated at the same time. The prescription can take the following manner:Metronidazole orally (250mg, 3 times daily for 7 days) or 2 gm orally as a single dose although it is contraindicated in pregnant patients. In this situation, topical therapy with clotrimazole 100mg daily for seven days is recommended.Vaginal inserts (suppositories) of 500mg Metronidazole daily concurrently with the oral regimen provide increased efficacy in resistant infection.

36. There are two other species related to genus Trichomonas:3. Trichomonas tenax It is a harmless commensal of the human mouth, living in the tartar around the teeth, in the cavities of the carious teeth, in necrotic mucosal cells in the gingival margins of gums, in the pus pockets, tonsillar follicles, as well as in the nasopharyngeal region. Transmission is necessarily by direct contact, usually kissing or using contaminated eating tools, salivary droplets and fomites. Diagnosis can be made by demonstration of T. tenax in the tartar by microscopy. This organism is non-pathogenic and can be avoided through proper oral hygiene. Like Entamoeba gingivalis, it tends to flourish in unhealthy environments. Its presence is associated with gum diseases. No therapy is indicated, the better oral hygiene will rapidly eliminate the infection.

37. 4. Trichomonas hominis This organism is smaller than T. vaginalis, generally considered a nonpathogen. It inhabits the colon and caecum of man and several other primate species and feeds on enteric bacteria. It does not invade the intestinal mucosa. It can infect the dogs, cats, mice and other rodents, so, these animals act as reservoir hosts to human infection. Identification of trophozoites in fresh fecal preparations provides the most accurate means of diagnosis.