Handout No. 4Prof.J.S.EdirisingheMBBS (Cey), MSc (Lond), PhD (Lond), MD (Col)Faculty of Medicine & Allied Sciences,Rajarata University of Sri Lanka.Saliyapura.January 2009

INTESTINAL PROTOZOAN INFECTIONS

ContentsIntroductionIntestinal amoebaeIntestinal flagellatesIntestinal ciliatesIntestinal coccidia

Geographical distributionMorphologyBasic PathologyClinical aspectsDiagnosisTransmissionPrevention and controlEpidemiology

Broad objectivesOn completion of the learning activity, the students should possess the basic knowledge of the aetiology, pathogenesis, clinical features, treatment, epidemiology, prevention and control of intestinal protozoan infections of humans

Specific objectivesAmoebiasis, giardiasis Be able tolist the common intestinal protozoan parasites of humans and indicate their locationsdraw and label the two morphological forms of Entamoeba histolytica, Entamoeba dispar, Giardia lamblia recognize these forms on saline, iodine and permanently stained faecal smearsdescribe the clinical consequences of the presence of these parasites in the locations indicated in (a)describe the life cycle of E.histolytica and G.lamblia describe the mode of infectiondescribe the laboratory diagnosis of amoebiasis and giardiasisexplain the collection of a specimen of feces for laboratory diagnosis of acute and chronic amoebiasisdescribe the preventive measures applicable amoebiasis and giardiasisname the drugs used in the treatment of these infections

Practical skillsa) Identify common intestinal protozoa of humans in laboratory specimensb) Make faecal smears in saline and iodine for the diagnosis of these infectionsc) Examine such smears under the light microscope and identify the parasitesd) Follow safe laboratory procedures during practical classes

Intestinal Protozoans of humans are described under five groups

Amoebae Flagellates CiliatesEntamoeba histolytica Giardia lamblia (intestinalis) Balantidium coliEntamoeba dispar Trichomonas tenax (oral cavity)Entamoeba coli Trichmonas hominisEntamoeba gingivalis

Iodamoeba butschlii

Coccidia Other intestinal protozoaCryptosporidium spp. Blastocystis hominisIsospora belliCyclospora spp.

INTESTINAL AMOEBAEOf the amoebae listed above, only E.histolytica is pathogenic to humans. I.butschlii has a potential pathogenic role. In general there are two main stages in the life cycle of these parasites. These are the motile, feeding forms known as trophozoites and the non motile, non-feeding, resistant forms, the cysts. The trophozoites multiply by binary fission while cysts are dormant and non-multiplying. E.gingivalis has no known cyst stage. The cyst stage is important in transmission except in E.gingivalis. The distinctive morphological features of these two stages help to differentiate the two stages. Distinctive features of the trophozoites –size, motility, shape of the pseudopodia, inclusion bodies in the cytoplasm, nucleus, nuclear membrane, arrangement of chromatin in the nucleus and the position of the karyosome.Distinctive features of the cysts – size, presence of glycogen mass, nuclear membrane, arrangement of chromatin in the nucleus, the position of the karyosome and the shape of the chromotoid bodies.

Morphological features of the trophozoitesFeatures E.histolytica E.coli I.butschlii

Size (µm) 10 - 40 10- 40 9 – 20Motility Active Slow slowPseudopodia Finger like, rapidly

protrudedShort, blunt, slowly protruded

Short, blunt, slowly protruded

Inclusions RBCs in invasive forms Bacteria and debris No RBCsNo. of nuclei One One One Karyosome Central, small Eccentric and large Large and granular

Peripheral chromatin Small, regularly arranged on the inner side of the nuclear membrane

Coarse and irregularly arranged

Usually absent

Morphological features of the cystsFeatures E.histolytica E.coli I.butschlii

Size (µm) 10 – 15 10 – 20 7 – 15Shape Round Round VariableGlycogen mass as stained with iodine

Diffuse, seen in immature cysts

Diffuse, seen in immature cysts

Large, dark brown, outlined sharply

Nuclei 1 – 4 1 - 8 1Chromatin and karyosome

Like in trophozoites Like in trophozoites Like in trophozoites

Chromotoid bars Rods with rounded ends Like needles when present

Absent

AMOEBIASISAmoebiasis is the common name of the infection caused by E.histolytica. The

infection has a worldwide distribution but the infection rates are much higher in environments where hygiene and sanitation are poor. The main outcome of the infection is amoebic dysentery, following the mucosal invasion of the colon by the trophozoites stage of the parasite. Spread of the infection may occur locally or to remote sites via blood stream. Recent studies have shown the existence of two species within the species originally recognized as E.histolytica. It is now known that E.histolytica is the species that have the capacity to invade tissue while the other species, designated as E.dispar is unable to invade. It is now believed that the original global prevalence worked out might include a large proportion of E.dispar, thus, resulting in wrong statistics. The two species however, are morphologically indistinguishable. Sophisticated techniques such as DNA or isoenzyme patterns are needed to differentiate the two speciesLife cycle – Infection results following ingestion of the cyst. Excystation occurs in the lower part of the small intestine. Each cyst gives rise to 8 young trophozoites. They move down to the large intestine. There they feed, grow and multiply by binary fission. At some point they stop feeding, round up, and secrete a wall round each of them forming cysts. The nucleus of each cyst divides resulting in 4 nuclei that is seen in mature cysts. Eventually the cysts are voided in the faces. The contaminate soil, water, vegetables, fruits, fingers etc. and enter the human gut directly or indirectly. Trophozoites are fragile and short-lived outside the human gut and are destroyed by environmental temperature, chemicals, and even urine. They do not take part in transmission of the infection. For all intents and purposes humans are the only hostsPathogenesis – Infection with E.histolytica does not necessarily mean clinical disease. Disease or amoebic dysentery starts with the invasion of the mucosa of the colon by the parasite. Non-invasive parasites remain in the lumen of the colon. There are several factors that promote or stimulate invasion of tissue. Host factors include: gut bacteria that influence the physico-chemical environment of the colon, mucus secretion, gut motility, and the immunological status. Parasite factors include: isoenzyme patterns, ability to resist lysis by human serum.Tissue invasion – Initially a breach in the superficial mucosa is made the aid of lytic enzymes. The amoebae than enter the sub mucosa through the muscularis mucosa. Here they multiply and spread laterally forming the typical ‘flask’ shaped ulcers. Ulcer formation stimulates the goblet cells in the neighbouring tissue to secrete excess mucus. In the lytic

process small blood vessels are also lysed leading to bleeding. From the sub mucosal location the trophozoites may spread to other organs via the blood stream. There is no cyst formation in the tissues. Invading (pathogenic) trophozoites are distinguished from the non-invading luminal trophozoites by the presence of RBCs in their cytoplasm. Clinical features – Large number of infected persons are asymptomatic. Others get blood and mucous diarrhoea. Parasites carried via the blood stream may settle in various organs causing pathology. Right lobe of the liver is a common site. Other extra-intestinal sites include pericardium, brain, bone, and rarely skin. A massive liver abscess may rupture into the pleural cavity through the diaphragm. What ever the organ the trophozoites start multiplying by binary fission and feed on the surrounding tissue and blood making use of the lytic enzymes.Diagnosis – The diagnosis of amoebic dysentery is by the demonstration of live, motile trophozoites with ingested RBCs in the patient’s faeces. If no RBCs are found in the cytoplasm of the parasites they may be commensal amoebae from the colonic lumen. Other material from which parasites could be demonstrated are in mucus from sygmoidoscopy, biopsies and aspirates of liver abscesses. Indirect evidence of the infection may be obtained by serological tests. Indirect tests are useful in diagnosing extra-intestinal amoebic infections. In suspected amoebic dysentery trophozoites with ingested RBCs should be looked for. A minimum of three consecutive samples of faeces using a concentration technique and permanent staining should be carried out. Faecal samples or mucus could also be cultured in the laboratory. In wet saline mounts motile trophozoites with ingested RBCs could be seen. Demonstration of cysts only or trophozoites with no ingested RBCs rules out amoebic dysentery. Kits using immunological techniques are commercially available. Epidemiology – There is no correlation between the prevalence of infection and clinical disease. In countries with poor sanitary facilities and minimal hygiene, the disease is common. In developed countries infection is common. Faecal contamination of food and water is the major method of transmission and therefore the factors that lead to such situations have to be determined.Transmission – Ingestion of the mature cyst is responsible for the infection. Contamination of food and water is major mode of transmission. Drinking water as well as recreational and irrigation water are important. Cyst passing food handlers are an important source of infection. Flies can transmit the cysts mechanically from faeces to food. Cyts are sensitive to desiccation, heat (96º C for one minute) and freezing (<10° C for over 24 hours). Cysts survive in liquid media such as water, milk for weeks particularly at low temperatures. Standard chlorination is not effective in killing the cysts.Treatment – Invasive disease or amoebic dysentery have to be treated vigorously with appropriate drugs. One school of thought refrained from treating luminal amoebiasis where RBC ingested trophozoites could not be detected or when only cysts were seen. Harboring any form of the parasite indicates that faecal contamination of food or water had taken place. Most physicians however treat all patients who pass trophozoites or cysts. Prevention - Use of sanitary latrine, washing hands with soap and water after using the latrine and keeping finger nails short are good hygienic practices. All food handlers have to be examined to detect carrier states. Thorough washing of vegetable s and fruits before consumption should be practiced. Drinking water should be boiled and cooled. When cannot be sure of the cleanliness or the freshness, it is safer to avoid salads and fruits.

Current status of amoebiasis in Sri LankaAmoebic dysentery and amoebic liver abscesses were common in this country up to the late sixties. There has been a gradual decline in the prevalence up to present times. The exact reasons for this decline are not known.

FlagellatesGiardiasis or Giardia infection

Giardiasis is caused by the parasite Giardia intestinalis (lamblia). The disease is considered as the commonest intestinal parasitic infection. This is a flagellate parasite and inhabits the duodenum and the upper jejunum.

Giardia exists in forms, the trophozoites and the cyst. The trophozoite is 12 – 15 µm long and 5 – 9 µm wide. It has two nuclei and four flagella starting from basal bodies at the anterior pole of the nuclei. In the centre, lying more posteriorly are the curved median bodies. The dorsal surface of the trophozoites is convex and the ventral concave surface has the sucking discs. Cysts are oval in shape and are 7 – 10 µm in length. The cyst contains two to four nuclei, median bodies and the cytoskeletal remnants.Location in host – They are found attached to the mucosa of the upper small intestine (duodenum, jejunum). They are rare in the ileum except in massive infectionsPathogenesis – Giardia does not invade the intestinal mucosa but live attached to the mucosa with their sucking discs. Feeding activities of the parasite on the enterocyte surface cause irritation. There is damage and flattening of the villi due to the mechanical effect as well as due to inflammation. This interferes with micro absorption... In heavy infections sheets of attached Giardia may cover the mucosal surface, interfering mechanically with absorption. Due to the location of in the upper small intestine, fats and fat-soluble vitamin absorption is impaired.Clinical features – Giardiasis results in acute diarrhea. It is described as ‘explosive’ diarrhoea, worse in the mornings. Faeces become offensive, bulky and pale. Patients often observe ‘floating’ faeces, indicating the presence of fat. Chronic infection leads to frank steatorrhoea and weight loss.Diagnosis - Diagnosis is by demonstrating trophozoites or cysts in the diarrhoeic stools. Cysts are passed irregularly and therefore several samples nave to be examined to confirm the diagnosis. ‘Entero-test’ available commercially can be used. Treatment - Epidemiology and transmission – Contamination of water with cysts is the main mode of transmission. Water reservoirs, swimming pools and irrigation water may get contaminated due to poor sewage disposal methods.

CiliatesBalantidium coli infection - Balantidiosis

Balantidium coli are the largest of the protozoan parasites that infect humans. It is commonly a parasite of pigs and monkeys. The location of infection in the host is the colon... Occasionally it can cause human infections resulting in colitis.

Balantidium exists in two forms. The trophozoite is large (60 – 70 µm in length and the outer surface is covered with short cilia. Inside the cytoplasm is the characteristic

macronucleus which is kidney shaped. A round, a much smaller micronucleus is also present located more posteriorly. Towards the anterior end, a cytostome is present and at the lower end a cytopyge. Numerous food vacuoles may be seen in the cytoplasm. The cyst is large and rounded (50 – 60 µm) and cilia may still be visible. Cyst is the infective stage.

The trophozoites invade the colonic mucosa, and while multiplying spreads to the sub mucosa. In stained histological sections clusters of parasites could be seen forming ‘nests’. The major clinical feature is blood and mucus diarrhea. Extra-intestinal spread is known to occur, particularly in immuno-compromised persons.

Intestinal coccidiosesThe human alimentary tract may be parasitized by several species of coccidian. In

immuno-competent persons the infection is generally self-limiting. They may cause serious disease in the immuno-compromised persons.

Cryptosporidium parvum infects a wide range of animals (mostly livestock) and humans. Both sexual and asexual reproduction takes place in the same host. The parasites are found intra-cellularly but on the surface of the small intestinal epithelial cells. In heavy infections other epithelia such as the respiratory system may be affected. Transmission of the infection is by the sporulated oocysts that are passed in the infected person’s faeces. Special stains such as acid-fast stain have to be used to demonstrate the oocysts in the feacal samples.

Blastocystis hominis – BlastocystosisThe taxonomic position of the parasite is still not certain although it is currently

classed as a protozoan. There is increasing evidence to suggest its pathogenic role in causing diarrhoea. It is recommended that the presence of the organism in stool samples be routinely reported.

The commonly seen form the rounded body of varying size from 6 – 40 µm with a characteristic large central body composed of CHO and lipids. This body appears as a vacuole as it is usually unstained. On the rim of cytoplasm are several nuclei.Transmission is probably via contaminated food and water.

Trichmonas vaginalis infection –Trichmonal vaginitisTrichomonas vaginalis is not an

intestinal parasite, but as the species name suggests, it parasitizes the human vagina. It is highly specific to this site but may infect the male genital tract as well. In males the infection may be asymptomatic or may cause a urethritis. The infection generally co-exists with other genital infections. Morphology – Only the trophozoites stage is found. It is ovoid in shape, 10 – 20 µm in length. It has four anterior flagella and another along the undulating membrane that runs down 2/3rd of the length of the body. An axostyle runs along the middle of the body. It is actively phagocytic and highly motile. Only the trophozoites stage is found

as there is no cyst formation. It has a worldwide distribution and is a common genital infection in Sri Lanka. Pathogenesis – The infection involves squamous epithelium of the vagina. In males urethra is mostly infected. The inflammation is accompanied by the production of large number of polymorphonuclear neutrophils and macrophages. These changes results in a profuse vaginal discharge. The parasite does not invade tissue. Clinical features - The infection results in a vulvo-vaginitis accompanied by a profuse vaginal discharge. In the male urethritis may be accompanied by a urethral discharge.Diagnosis - Demonstration of the actively motile trophozoites in fresh vaginal discharge smears. Characteristic motility can be demonstrated by a bed side test in the clinic. The organism can be cultured in the laboratory Transmission - Transmission is through sexual intercourse. Treatment - Metronidazole Prevention – All methods used for the prevention of STD should be applied including the use of condoms. Health education is important.

Collection of specimens for the diagnosis of amoebiasis, giardiasis, balantidiosis, intestinal coccidioses, and trichomonal vaginitis

Amoebiasis – The trophozoites of E.histolytica are fragile and delicate. They are extremely susceptible to changes in temperature, pH and chemicals in the external environment. Outside the human body the trapohozoits live only for a few minutes. First they round up and may be indistinguishable from macrophages to untrained eyes. Hence collection of faecal samples of suspected patients should be carried out meticulously, taking all precautions to keep the amoebae alive.

1. Fresh samples of faeces (mainly blood and mucus) have to be examined (ie. within ½ hour of passing faeces) when an acute infection is suspected. A clean container (need not be sterile) without traces of antiseptics have to be used. A wide mouthed bottle (easily cleaned and sterilized) or plastic containers such as yoghurt cups (to incinerated) are good. Instruct the patient to pass faeces in to a toilet where there are no antiseptics. The sample should not be mixed with urine. Give instructions according to the situation (water seal latrine, pit latrines etc.). Portion of faeces with mucus should be selected. Ask patients to give about a table spoonful of the sample (not less, not more). Examine for motile trophozoites immediately in a saline smear. Acute infection is confirmed by demonstrating actively motile trophozoites with ingested RBCs in the cytoplasm. Cysts being the resistant form can be detected in faecal samples taken without the above precautions. For both forms several samples on 3 – 5 consecutive days have to be examined.

2. For detecting trophozoites in drained liver abscess pus the last part of the aspirate coming from the walls of the abscess has to be examined.

3. These samples may be cultured in suitable media in the laboratory.

Giardiasis – The ‘explosive’ type of diarrhoea is more in the mornings and the samples are best taken during before noon. The trophozoites are fragile and not commonly seen in faecal samples. Unlike with amoebiasis, the presence of cyst is diagnostic of the infection. No special precautions are necessary for collecting faecal samples. Several samples have to be

examined. Material obtained by the ‘Enerotest’ or by endoscopy has to be examined in saline and iodine smears as soon as possible.Balantidiosis – The trophozoites form cysts as soon as they reach the external environment and are easily detected in saline and iodine smears. No special precautions are necessary.

Intestinal coccidioses – For the diagnosis of Cryptosporidium parvum infections the oocysts passed in the faeces have to be demonstrated. They cannot be seen in saline or iodine smears. Special stains such as Zeil-Neilson’s or acid - fast stains have to be used. With these stains the oocysts appear as small magenta coloured bodies.

Trichomonal vaginitis – Vaginal discharge can be taken into a cotton wool swab and then placed on a drop of saline and examined immediately (bed-side test). The organism’s motility is characteristic with the anterior flagella rotating as in a helicopter. Permanently stained slides could be made with these smears. Material from the specula used in vaginal examination could also be used for the diagnosis. In males 24 hour urine should be collected and a specimen centrifuged to detect organism in the deposit.

JSESaliyapuraAnuradhapura21st January 2009