Intestinal Protozoa

INTESTINAL PROTOZOA PRESENTATION BY OKELLO EMMANUEL ONEN TOPICS: 1. CRYPTOSPORODIASIS 2. CYCLOSPOROSIS 3. TOXOPLASMOSIS 4. ISOSPOROSIS

CLASSIFICATION

• Phylum : Apicomplexa • Class : coccidea • Genus : Isospora, Toxoplasma, cryptosporidium, cyclospora X-TICS OF COCCIDEA: • Both sexual and asexual cycles take place in the same host • Invade intestinal epithelium • Grow in the host cells

CRYPTOSPORODIASIS

Source: Through ingesting of Oocysts in contaminated food and water. Caused by Cryptosporidium parvum Life cycle in man: • Oocysts are ingested in contaminated food or water by man to the intestine • The oocysts will release the sporoziotes and infect tthe intestinal wall • Sporoziotes multiply into the trophozoites by asexual means (schizogony) • The trophozoites will release again merozoites • Merozoites will develop into female and male

Cont.... • Male will fertilise the female to form zygotes • Zygotes will now mature to form the oocyts which can now be released in Stool

Life cycle flow

Laboratory diagnosis

sample: • Blood • Stool • Gastric washing • Duodenal Biopsy • CSF Test to be done: • Wet preparation for ooocysts using Iodine, eosin and saline • Uv light fluorescence • Stool Concentration method for parasites ie.

test cont....

• Monoclonal antibody test • Modified Zn staining or auramine • Molecular test i.e PCR test for the DNA of the parasite images of oocyst:

Toxoplasma • causative agent is the Toxoplasma gondii • It causes an opportunistic infection in human called toxoplasmosis when the immune system is too low to prevent infections like that

Sources of infection • Ingestion of cyst-containing meat (carnivores) • Ingestion of oocysts (environmental

contamination) • Congenital infection from mother to foetus • Transfusion (rare) • Raw goat’s milk, lambing, transplantation Definitive host: • Cats of the felidae family Intermediate host: • Birds • Rodents • Pigs and man being an opportunistic host

Symptomatology : Toxoplasma • Most infections benign • Rarely severe – hepatitis, encephalomyelitis, myocarditis

• Few cases of retinochoroiditis which can progress to blindness • At risk groups are children

Life cycle of Toxoplasma

life cycle

• Unsporulated oocysts are released by cats to the environment in feaces • The oocysts take 1-5days to sporulate in the soil becoming infective • Intermediate host in nature become infected after ingesting soil, water or plants contaminated with th oocysts • The oocysts transform into tachyzoitte shortly after ingestion by the intermediate host • The tachyzoites localise in neural and muscle tissue then develop into tissue cysts

life cycle cont.......

• Cats being the definitive host get infected on eating the intermediate host containing the tissue cysts or by directly ingesting sporulated oocysts • Human can become infected after ingesting the sporulated oocysts directly from contaminated water and food or the tissue cysts in other animals meat • Human can also get it through blood transfusion and transplacentally from mother to foetus Congenitally

Diagnostic tests for Toxoplasma

• Sabin-Feldman dye test (DT)

• Enzyme immunoassay for T. gondii • • •

• •

specific IgM (EIA) Immunsorbent agglutination assay (ISAGA) Enzyme immunoassay for IgG avidity Isolation and culture of parasite in blood and tissues then intraperitoneal inoculation into mice's peritoneal then after six days tested for toxoplasmosis Direct detection by microscopy using fluorescent microscope PCR for the parasite DNA

Images of the Toxoplasma gondii

A. B. D. A: Tachyzoites B: sporulated oocysts C: Unsporulated oocysts

C.

Differential Diagnosis • Immunocompetent adults (DT, IgM EIA) • Pregnant women (maternal serum DT,

IgM EIA, IgG avidity; Amniotic fluid culture or PCR)

• Neonates (DT, EIA, ISAGA for IgM, IgA) • Organ transplantation (DT, IgM EIA) • Immunodeficiency (serum and CSF : DT,

EIA, ISAGA for IgM and IgA; PCR, culture, microscopy)

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Avoid consumption of raw or undercooked meat

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Litterpans should be changed daily

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Wash hands after handling raw meat, litter pans & soil

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Pregnant women should avoid contact with cats

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Issue of prenatal

Prevention and control

• • • •

Treatment

Only accepted treatment pyrimethamine with trisulfapyrimines for 1 month Intravenous clindamycin used to treat encephalitis in AIDS patients In France spiramycin has been used to treat toxoplasmosis in pregnancy Spiramycin is available in the US on a case-by-case basis In other positive for toxoplasmosis

Cyclospora

• the causative agent is the Cyclospora cayetenensis • It causes a disease Cyclosporosis Signs and symptoms After 7 days the following symptoms will appear: • watery diarrhea • Loss of appertite • Weight loss • Cramps in the stomach • Blotting

Life cycle

• Ingestion of the sporulated oocysts by man • The oocysts will excyst while in the GIT and release sporoziotes • The sporoziotes will attack the intestinal epithelium cells inwhich sexual development and asexual maturation occurs leading to Unsporulated oocysts formation. • The Unsporulated oocysts will be shed off in stool to the environment • While in the environment the oocyts will sporulate after some days to weeks at 22-32 degrees Celsius to form two sporocyst each with elongated

Laboratory diagnosis Samples: • Duodenal biopsies • Bowel biopsy • Blood • Stool TEST DONE: uWet mount: here we deal with 3 samples and it's by light microscopy, UV fluorescence, and differentialinterference contract (DIC ) to look for sporocyst in

images of wet mount

A. B. A: Wet mount light microscopy B: UV light fluorescence C: DIC microscopy

C.

Concentration technique

• this is in cases of low parasitemia and in cyclospora oocysts conditions which release few oocytes at a time whjch might be missed so Concentration is advised before other steps are done. STAINED SMEAR : uModified ZN staining: we use 1% acid aalchol decolourizing agent, carbol fuchsin for staining, 3% malachite green or methylene blue counter stain and absolute methanol for fixing the smear but no heat. The results will be pinkish red colour on a green or blue background.

Images of Modified Zn

uModified Safranin: the reagents are: acid alchol for fixing, safranin heated to boiling, malachite green or methylene blue to counter stain. we use heated safranin anx dip the well fixed smear in it. The oocysts will appear as reddish orange on a blue or green background

images of Modified Safranin

uMolecular diagnosis: we do PCR on the sample to . get the rRNA and DNA using agarose gel 2% uSerology

• • • • • • •

Prevention and control avoid under cooked food Give prophylactic treatment to a at risk population Monitor and surveillance Proper diagnosis of each condition Screening of epidemic populations Sensitisation of the locals Boosting the immune system of the immune suppressed in order to avoid opportunistic infections

ISOSPOROSIS • caused by Isospora belli in human. • It causes coccidiosis or isosporosis in human host Clinical signs: ü Small Bowel mucosal atrophy ü Watery diarrhea ü Weight loss ü Cholecytitis mostly in immuno suppressed people

life cycle • ingestion of the oocyst in contaminated food and water • Oocysts will travel to the small intestine on the ceacum • Oocysts develop to sporoziotes within the gut • The oocysts will rapture to release the sporoziotes • Sporoziotes will penetrate the villi cells and foem the trophozoites hence schizogony • Trophozoites will form schizonts • Schizonts will develop merozoites •

cont.....

• The merozoites will Invade further villi cells and others will develop and mature to form the motile microgamatocytes (male) and macrogametocytes (female ) • The male will fertilise the female to form the zygotes • The zygotes will develop cyst walls and form oocysts hence sporogony • The oocysts divide at the nucleus to form two sporoblast • Eacb sporoblast will develop cysts walls and form the sporocyst

Cont.... • The oocysts formed mature or immature will now be passed out in Stool and contaminates the environment and water where it will infect man when ingested continuing the lifecycle

life cycle

laboratory diagnosis

sample: ü Stool ü Bow biopsy Gastric washing ü Blood Test: • Wet preparation for motile parasites using iodine, eosin and normal saline • Formal ether Concentration method to concentrate the parasites in low parasitemia • Modified ZN staining to identify the parasite by staining character

laboratory diagnosis cont.. • Perform PCR Annalysis to identify the genetic content of the parasite Morphology of the oocyst : ü Shape being oval and thick walled ü Mature oocysts seen with 2 sporocysts and each sporocyst containing 4 sporoziotes making a total of 8 sporoziotes in a mature oocysts ü If oocysts are ruptured, sporocyst are seen singly or paired together

images of Isospora belli in Safranin

wet mount of Isospora oocysts

References • www.cdc.gov/parasites • Atlas of Medical Helminthology and Protozoology, intestinal protozoa, 4th edition 2001 • Monica Cheesbrough, District laboratory practice in tropical countries, part 1, chap 5, parasitological test, pg 200-208