INTESTINAL PROTOZOAN INFECTIONS

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1 INTESTINAL PROTOZOAN INFECTIONS The protozoa that parasitise the human intestine belong to four groups: amoebae, flagellates, ciliates and coccidia. In general, the intestinal amoebae, flagellates and ciliates have relatively simple life cycles involving asexual multiplication by binary fission, and only two morphological forms: the trophozoite, which is the feeding, growing form, and the cyst stage, which transmits infection from one host to another. The coccidia have much more complex life cycles involving both asexual and sexual multiplication, and several different morphological forms. Of the many protozoa that are known to parasitize the human gut, only the amoeba Entamoeba histolytica, the flagellate Giardia lamblia, and the coccidian Cryptosporidium parvum are important causes of disease in Sri Lanka. AMOEBIASIS The amoebae that parasitize humans include Entamoeba histolytica, E. dispar, E.moshkovski, E. coli, E. hartmanni, Endolimax nana, Iodamoeba butschlii, Dientamoeba fragilis and Entamoeba gingivalis. Of these, only E. histolytica is widely accepted as an important cause of human disease. The most important disease manifestations are amoebic dysentery and amoebic liver abscess. Morphology and life cycle Human beings (and perhaps some non-human primates) are the only natural hosts. The organism has a simple life cycle existing as either the infectious cyst form or the amoeboid trophozoite stage. E. histolytica cysts are round, usually µm in diameter, and surrounded by a refractile wall. They contain 4 nuclei, glycogen and ribosomal assemblies called chromatoid bodies (or bars). They survive the acid of the stomach, travel through the small intestine and, within the terminal ileum or colon, excyst to form the trophozoite stage. Trophozoites are highly motile, and have a pleomorphic (changeable) shape, with a diameter that varies from µm. They ingest bacteria and food particles, reproduce by binary fission, and encyst within the colon, completing the life cycle when infectious cysts are excreted into the environment with faeces. Trophozoites too may exit in faeces (especially when a patient has diarrhoea) but they cannot survive outside the human host. Epidemiology Infection with E. histolytica is found throughout the world, in both tropical and temperate climates, wherever barriers between human faeces and food and water are inadequate. The organism that was first microscopically identified as E. histolytica in the early 1900 s, has now been shown to be two genetically distinct species. This was first done using isoenzyme analyses to identify differences between isolates from asymptomatic individuals and from those with disease. The differentiation was then confirmed with DNA evidence. The 2 species are now designated Entamoeba dispar (the commensal) and Entamoeba histolytica (the pathogen). E. dispar is not a pathogen, and causes no signs of disease or mucosal invasion even in patients with AIDS. E. histolytica is the causative agent of amoebic colitis and all forms of extraintestinal amoebiasis; however, it is also found in many asymptomatic individuals. Most individuals infected with Entamoeba are colonised with E. dispar; but in some regions, the prevalence of E. histolytica is very high in both asymptomatic individuals and in patients with diarrhoea. Amoebic colitis affects children and adults of both sexes. However, amoebic liver abscess mainly affects men between the ages of 18 and 50, in whom rates are 3 20 times higher than in other populations. The reason for this difference is not known; hormonal effects and alcohol are possible factors. T.G.N.C. 1

2 Amoebic disease used to be very common in Sri Lanka, but prevalence has declined since the 1980s. However since 1985 amoebic liver abscess has been a public health problem in Northern Sri Lanka. In rest of the country both forms of disease (amoebic dysentery and amoebic liver abscess) is rarely encountered in clinical practice at present. Transmission Infection is transmitted only by the cyst stage. Infection usually begins with the ingestion of cysts in food or water contaminated with human faeces. More unusual modes of transmission, including oral and anal sex, and contaminated enema apparatus are also possible. Point-source outbreaks of infection have been known to occur when leaking water supply lines are contaminated by leaking sewage lines. Amoebiasis can spread within families, and household contacts of patients with the disease should be screened for infection. Pathophysiology Pathological changes in amoebic colitis can range from mucosal thickening, multiple ulcers separated by regions of colonic mucosa of normal appearance, to diffusely inflamed and oedematous mucosa, and necrosis and perforation of the intestinal wall. The gross findings can strongly resemble those seen in inflammatory bowel disease. Disease begins when E. histolytica trophozoites adhere to colonic epithelial cells, through a specific lectin, which is a complex of 2 disulphide-linked subunits and an associated protein. After adhesion, the trophozoites release peptides known as amoebapores which form pores in the lipid bilayer of the host cell membrane and result in cell lysis. Trophozoites can also kill by inducing apoptosis (programmed cell death). Dying intestinal epithelial cells release cytokines that attract neutrophils and macrophages to the site of infection. Many neutrophils might be killed by contact with trophozoites and release mediators that cause more damage to adjacent intestinal epithelial cells. Macrophages release other mediators which contribute to further infection. Amoebic invasion through the mucosa and into submucosal tissues is the hallmark of amoebic colitis. Lateral extension through submucosal tissues gives rise to the classic flask-shaped ulcer of amoebiasis. The degree of inflammation and resultant tissue damage depends on the hosts immune response. E. histolytica trophozoites (reaching the liver through the portal circulation) create abscesses, which are well demarcated regions of dead hepatocytes, liquefied cells and cellular debris. A rim of connective tissue, with few inflammatory cells and amoebic trophozoites surrounds the lesion. The adjacent liver parenchyma is often completely unaffected. Clinical symptoms and signs Gastrointestinal Patients with amoebic colitis present with bloody diarrhoea and abdominal pain and tenderness. Onset is often gradual, with patients reporting several weeks of symptoms. Multiple, small volume, mucoid stools are common. Even if no blood is to be seen with the naked eye, stools are almost always positive for occult blood. Rectal bleeding without diarrhoea may be seen in children. Fever, weight loss and anorexia may or may not be present. Occasionally patients develop fulminant amoebic colitis with profuse bloody diarrhoea, fever, pronounced leukocytosis, widespread abdominal pain often with peritoneal signs and extensive involvement of the colon. Paralytic ileus, sloughing of the colonic mucosa or intestinal perforation may occur. Amoebomas are localised inflammatory annular masses that develop in the caecum or ascending colon, and cause obstructive symptoms that can be confused with carcinomas. T.G.N.C. 2

3 Liver The most common extra-intestinal manifestation of disease is amoebic liver abscess. This used to be almost invariably fatal, but with rapid diagnosis and effective medical treatment, mortality rates are 1-3%. Some individuals presenting with amoebic liver disease have concurrent amoebic colitis, but more often they have no bowel symptoms, and stool microscopy is usually negative for E. histolytica trophozoites and cysts. Disease should be suspected in anyone with a history of possible exposure (residence in or travel to an endemic area) and fever, right upper quadrant pain, and significant hepatic tenderness. Symptoms are usually acute (<10 days in duration) but can be chronic, with anorexia and weight loss as prominent features. Leukocytosis without eosinophilia, mild anaemia, raised alkaline phosphatase and ESR are the most common laboratory findings. Rarely, amoebic liver abscesses can rupture into the peritoneum, causing sudden onset peritonitis and shock. Other sites Respiratory tract: Atelectasis and transudative pleural effusions are common, especially on the right side and do not require treatment. However, amoebic liver abscesses can rupture through the diaphragm and cause empyema, and sometimes a hepatobronchial fistula. Pleuropulmonary amoebiasis results in cough, pleuritic chest pain, and respiratory distress; while patients with hepatobronchial fistulae can produce large quantities of brown sputum, containing necrotic material and occasionally amoebic trophozoites. Very rarely, a liver abscess may rupture into the pericardium, resulting in pericarditis or cardiac tamponade. Amoebic brain abscesses are also known to occur as a secondary complication in an extremely small number of patients with amoebic liver abscess. Diagnosis Amoebic colitis Diagnosis of amoebic colitis depends on demonstration of E. histolytica in faeces or the colonic mucosa of patients with diarrhoea. This is usually done by stool microscopy, but it cannot distinguish between E. histolytica and E dispar except when trophozoites with ingested red cells can be demonstrated. ELISA assays that identify E histolytica antigens in stool have been developed, but are not widely available in Sri Lanka as yet. In a Sri Lankan patient with bloody diarrhoea, amoebic dysentery needs to be differentiated from other bacterial causes of dysentery. Examination of the diarrhoeal stool with the naked eye may give some indication of the diagnosis: stools in amoebic dysentery tend to have plenty of faecal matter, whereas in bacillary dysentery, there is usually little faecal matter. Other features set out in the following table may also help. Features of stools in amoebic and bacillary dysentery Amoebic dysentery Bacillary dysentery Faecal ph Acidic Alkaline Cellular exudates Very few pus cells, and degenerate red cells Numerous pus cells and varying amounts of blood. Red cells are usually unaltered Bacteria Few Numerous Charcot-Leyden crystals Usually present Usually not present The simplest and most commonly carried out technique for the demonstration of trophozoites and cysts in stools is the examination of a direct faecal smear in saline and / or iodine. Nuclei are more easily seen in an iodine smear than in a saline smear, but chromatoid bars are better seen in a saline smear. However, details of morphology can be seen best in stool smears that are fixed and stained with permanent stains such as iron haematoxylin or trichrome. T.G.N.C. 3

4 To demonstrate trophozoites, the stool sample must be examined within at least 2 hours of being passed, preferably within half an hour. This is because trophozoites are very delicate and fragile and lose their motility quickly. Formed stool from carriers or patients with chronic amoebiasis are more likely to contain cysts than trophozoites. Because the number of cysts varies from day to day, at least 3 stool samples should be examined before excluding infection. The chances of finding cysts may be further increased by the use of a faecal concentration technique such as formol-ether concentration or zinc sulphate flotation. If only a few amoebae are present in a stool sample, it may also be possible to demonstrate them by using culture methods. If a sigmoidoscopy is carried out, this will enable visualization of any lesions that are present. Material may then be aspirated for examination of trophozoites (direct smears, permanent stained smears or culture) or a biopsy may be taken for H&E sections, and the trophozoites may be seen in the edges of the flask-shaped ulcer. The diagnosis of amoebic liver abscess relies on identification of space-occupying lesion of the liver and positive amoebic serology. Ultrasound (or CT scan) is the test of choice. Both methods are very sensitive, but neither is absolutely specific for amoebic liver abscesses. Amoebic serology (demonstration of E. histolytica-specific antibodies in serum, by indirect immunofluorescence, indirect haemagglutination or ELISA) is highly sensitive and specific for the diagnosis of amoebic liver abscess. False-negative serological test results may be obtained early in infection by repeat tests will usually be positive. Treatment Amoebic colitis can be effectively treated with any of the nitroimidazole derivatives (metronidazole, tinidazole and ornidazole), followed by a luminal amoebicide such as diloxanide furoate / Paromomycin / Nitazoxanide / to eradicate organisms in the colonic lumen. Asymptomatic individuals with documented E histolytica infection should be treated with a luminal agent to eradicate infection since they may develop invasive disease, and are also a risk to public health. E dispar infection does not require treatment, but like infection with E coli, it means that the infected person has been exposed to faecally contaminated food or water. Where ever possible, amoebic liver abscesses should be treated with metronidazole, without drainage followed by a luminal agent to eliminate intestinal colonisation. Aspiration under US or CT guidance is recommended for patients in whom the diagnosis is uncertain, where the patient has not responded to metronidazole therapy or has a large left-lobe abscess, or a very large abscess that seems likely to rupture soon. Prevention and control Cysts cannot withstand desiccation, heat of >68 C or freezing at <-10 C for more than 24 hours. At low temperatures (0 5 C), cysts can survive for weeks in liquid media such as water or milk. Ordinary chlorinating of water (3 ppm) is not effective in elimination of cysts. Drinking water may be treated with special preparations of iodine, or with UV rays, for elimination of cysts. Good personal hygiene and environmental sanitation are essential. Personal hygiene involves washing of hands with soap and water after using toilet, before meals and before preparation of foods; drinking boiled water; protection of foods from flies; and disposal of faeces into a safe, well-constructed latrine. In endemic areas, vegetables and fruits that are eaten raw should be washed well in running water. Community measure such as screening of food handlers, sanitary disposal of sewage and provision of purified water and health education of the community are important in controlling the spread of infection. T.G.N.C. 4

5 PATHOGENIC FREE-LIVING AMOEBAE Two genera of free-living amoebae, which normally live in moist soil and water, feeding on bacteria and yeast; may occasionally attack man. Acanthamoeba (Hartmanella) spp. are found in fresh or salt water. It has a trophozoite form, which moves about very slowly by means of spiky pseudopodia (acanthopodia), and a resistant cyst form. Strains of Acanthamoeba can cause various chronic granulomatous diseases, especially in immunocompromised hosts, such as those with AIDS. When it affects the brain, it causes a condition known as Granulomatous Amoebic Encephalitis. Acanthamoeba can also cause keratitis and corneal ulceration, even in immunocompetent individuals. This condition, which is commoner among those who use extended-wear contact lenses, may lead to permanent loss of vision. Naegleria fowleri is a amoeboflagellate that lives in warm water. The trophozoite and the flagellate stage (but not the cyst) can penetrate the olfactory mucosa when inhaled during swimming in contaminated waters. The organism attacks the brain via the olfactory bulb, causing an almost invariably fatal purulent meningitis called Primary Amoebic Meningitis. Prof TGAN Chandrasena Dept. of Parasitology Faculty of Medicine University of Kelaniya T.G.N.C. 5

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