Amoebae.ppt

Intestinal Protozoa

Amoebae and ciliates

Dr. Devika Iddawela

Department of Parasitology 2008/2009 Batch

OBJECTIVES

1. Name the common intestinal amoebae &ciliates that infect humans

2. Of the intestinal amoebae, name the organisms that are pathogenic to human

3. Outline the life cycle of Entamoeba histolytica /Balantidium coli indicating the stages that cause pathogenic effects and are of diagnostic importance in the above

4. Identify points in Life cycle where preventive measures are applicable

5. Describe the mechanism of pathogenesis

6. Describe the pathogenesis and clinical features of these stages

7. Describe the mode(s) of transmission, prevention and control of amoebiasis

8. Describe the laboratory methods of diagnosis of these organisms

Intestinal protozoan

• 1.Amoebae – moves by means of pseudopodia• 2. Ciliates – are propelled by rows of cilia that

beat with a wave like motion

3. Flagellates- move by long whip like flagellae

4. Coccidia: lack the specialized organelles of motility

Phylum protozoa is classified into 4 subdivisions based on methods of locomotion

Amoebae

Unicellular organisms

Characterized by possessing pseudopodia by which these organisms move and engulf food particles

such as bacteria, red blood cells

• Asexual reproduction – binary fission

Most are free living

can exist as trophozoite (growing stage) or cyst

( dormant stage)

Differentiate on morphological features of either trophozoite or cyst

Differentiating features of trophozoite:

Size,

Type of motility – directional or non- directional

fast or sluggish

character of pseudopodia

,Cytoplasmic inclusion bodies : Red blood cells, food vacuoles containing bacteria, yeast

Differentiating features of cyst :

size

shape

number of nuclei, structure of nuclei

presence of glycogen mass

Chromatoid body or bar - coalesced RNA within the cytoplasm

number of nuclei, arrangement of peripheral chromatin, position of the karyosome

Nuclear structure:

Chromatin ; Nuclear DNA present as peripheral chromatin

Karyosome: small condensed mass of chromatin within the nuclear space

Peripheral chromatin – chromatin adhering to nuclear membrane

Genus : Entamoeba

Parasites of alimentary tract - man, monkeys vertebrates and invertebrates

Characteristics of this genus :

Nucleus more or less spherical

Nuclear membrane line with chromatin granules

Small karyosome situated at or near the centre

Trophozoite has single nucleus

Endolimax and Iodamoeba

peripheralchromatin

Large karyozome

Entamoeba

karyosome

Genus:

Genus:

Grouped according to the number of nuclei in the mature cyst (1,4,8)

Amoebae that parasitize humans

Intestinal amoebae: ( inhabit the large intestine)

Entamoeba histolytica

E.dispar

E.coli

E.hartmani

Endolimax nana

Iodamoeba butschlii

Dientamoeba fragillisOral cavity : Entamoeba gingivalis

There are two stages in the life cycle of these amoebae.

1.Trophozoite:mortile and feeding stage. Multiply by binary fission 2. Cyst : Inactive, non motile and infective stage

No cyst stages in D.fragilis & E.gingivalis

Of several species of amoebae live in the alimentary tract of human MAJORITY are commensals ONLY Entamoeba histolytica is pathogenic D.fragilis and I.butschlii, may cause intestinal infection

ENTAMOEBA HISTOLYTICA

• cosmopolitan distribution

• worldwide incidence: 0.2-50%• highest prevalence in areas with poor sanitation

• no animal reservoirs

•estimated 50 million cases/year100,000 deaths/year

Entamoeba histolytica

Disease: amoebiasis

Blood and mucous diarrhoea

Pathogenic organism parasitize large intestine of man

E. dispar identical morphology but not

Invasive ( non-pathogenic)

RBCs

Nucleus

20-40 m, motility-active, progressive, directionalPseudopodia- finger like, hyaline, very rapidly extrudedInclusions- red blood cells (invasive forms)Nucleus- single, fine central kayosome, regular peripheral chromatin

Trophozoite

Cyst – spherical, 10-20 m (E. hartmanni <10 m)Nuclei: 1-4, structure like in trophozoiteChromatoid bodies: thick, 1-2 stain like chromatin, disappear as cyst matures (does not stain with Iodine)

E. dispar identical morphology

Life cycle

Infective stage

• Ingestion of mature cysts

• Excysts in small intestine

• Each cyst give rise immature trophozoites

• Maturation takes place in caecum•Trophozoites feed grow and divide causing pathological effects

Amoebiasis

Pathogenesis - Infection with E.histolytica does not necessarily lead to disease. The outcome depends on :

•Host factors•Parasite factors

Host Factor Contributions

• Physico-chemical environment of the gut influenced by bacterial flora, mucus secretion & gut motility

• Degree of immunological resistance

Important virulence factors of E.histolytica

• Adhesion molecules ( N- acetyl-D-galactosamine inhibitable lectine Gal/GalNac) – adhesion to colonic mucine and host cells• induce contact dependent cytolysis,

• Channel-forming peptides(Amoebapores): Stored in cytoplasmic granules & release following target cell contact, forms iron exchanging channels in plasma membrane – lysing the target cells

Parasitic factors

3. Cystein protinases – Aid in penetration of host tissue by digesting extracellular matrix, cleaving collagen, elastin,fibrinoge in extracellular matrix by stimulating host cell proteolytic cascade

Resistance to host response • complement resistance-inactivates the inactivates the complement factors complement factors and are thus resistant and are thus resistant to Complement mediated lysis. to Complement mediated lysis.

• Limit the effectiveness of humoral response by degrading both IgA and IgG

4. Species/strain differences; E. dispar non invasive, Pathogenic zymodemes =E.histolytica

Pathology

Intestinal Amoebiasis –LARGE INTESTINE

• Penetration of mucus layer• contact-dependent killing of epithelium• breakdown of tissues (extracellular matrix)• contact-dependent killing of neutrophils, leukocytes, etc. initially produce focal and superficial erosions in large

intestine with unaffected mucosa in between

Adhere to colonic mucin and host clls

Amoebic ulcerationwith unaffected mucosa in between

•Trophozoites advance laterally and downward into the submucosa producing a 'flask-shaped' ulcer ( typical appearance of intestinal amoebiasis)

Flask shaped ulcers -Base in submucosa and small opening on the mucosal surface

• Trophozoite penetrates the intestinal epithelium and then the muscularis mucosa & enter in to submucosa

Trophozoites penetrate the muscle

and serous layers leading to

intestinal perforations ,peritonitis

Rarely involvement of blood vessels at the base of the ulcer may produce profuse bleeding

Amoeboma - Amoebic granuloma

An inflammatory thickening of the intestinal wall, due to repeated invasion of colon by E histolytica

common sites- ascending colon & caecum

Haematogenous spread to other organs

Tissue invasion:Initial lesion – large intestine, caecum, ascending colon, sigmoidorectal region.

Extraintestinal Amebiasis

• primarily liver (portal vein)• other sites less frequent

Blood stream and lymphatic spread causeextra-intestinal amoebiasis(liver, skin, brain, heart)

Hepatic amoebiasis

Single abscess- Rt. Lobe (commonest)

predisposing factors: alcohol

Spread to other sites- direct

-blood stream

Hepatic abscess ( common site is right lobe)

Pulmonary Amoebiasis

• rarely primary• rupture of liver abscess

through diaphragm• fever, cough, dyspnea,

pain,

Cutaneous Amoebiasis

• intestinal or hepatic fistula

• perianal ulcers• urogenital (eg, labia,

vagina, penis)

Clinical features

Intestinal disease Majority of infections are asymptomatic[cyst passers are infective carriers]

asymptomatic cyst passer

• Amoebic colitis

Gradual onset ( symptoms presenting over 1-2 weeks)

abdominal pain, tenesmus , watery or bloody diarrhoea, anorexia, loss of weight. Fever only 10- 30%

Rectal bleeding without diarrhea can occur, especially in children

•fulminant colitis- Rare complication• abrupt onset ofprofuse bloody diarrhoea, high fever,dehydration ,wide spread abdominal pain+ perforation (peritonitis)

•amoeboma (amoebic granuloma)- painful abdominal mass

• perianal ulceration

Extraintestinal Disease _ sings & symptoms depend on the organ affected

liver abscess –

Frequently affect adults than children, Male>female

60-70% of patients with amebic liver abscess do not have concomitant colitis, a history of dysentery within the previous year

hepatomegaly, liver tenderness, pain in the upper abdomen, High fever and anorexia, Weight loss, vomiting, fatigue

Diagnosis of Amoebiasis

Trophozoites

Direct wet faecal smears in saline can demonstrate motile trophozoite. Fresh sample of faeces ( preferably with in 30 min) should be examine to visualize live trophozoite.

confirmed on a permanently stained smear to identify morphological features of nucleus

Eg; Trichrome or Iron haematoxylin

• Biochemical Methods: Culture and Isoenzyme analysis to differentiate E.dispar from E.histolytica

Entamoeba histolytica

Sigmoidoscopy:Visualize characteristic ulcersLook for trophozoites in mucosal aspirateBiopsy can be taken from the edge of ulcer stained with H &E

Cyst

Wet faecal smear ( saline or iodine)

If cysts are few to be present in direct smear, cysts can be concentrated either by floatation ( Zinc sulphate centrifugal floatation) or by sedimentation ( Formal-Ether )

Faecal concentration methods

Trophozoite4.Mature cyst with 4 nuclei

Immature cyst

Immature cyst

E.Coli cyst

Size – 10 -20 µm, >4nuclei

Nucleus ; eccentric karyosome with irregular coarse chromatin

Chromatoid bodies infrequent ,needle shape when present

Differentiation of E.Hislolytica from other non-pathogenic intestinal protozoa is very important

Iodamoeba butshclii cyst

7 -15 µm, , glycogen mass is large, dark brown with iodine

Acute dysentery- predominant form trophozoites

saline, stained smear, cultureColitis – cysts - saline, iodine, concentration methods

Faecal examination: minimum of 3 samples in 7 dayswet/permanent/culture

Diagnosis – Intestinal amoebiasisDefinitive diagnosis [GOLD STANDARD]– demonstrate parasite in stools/rectal smearsSTOOLFULL REPORT= SFRTrophozoites with ingested red blood cells indicate invasive amoebiasisPresence of cysts does not indicate active disease but infective carriers (cysts are infective)

Without the specific presenceof ingested RBCs in the cytoplasm the pathogen, E. histolytica & the non pathogen, E. disparAre morphologicaly identical BUT Biochemically different

Detection of E.histolytic specific antibodiesBy Enzyme linked immunosorbent assy(ELISA)Useful in non-endemic areas where E. histolytica infection is not common

Antigen Detection in stool• Antigen-based ELISA sAdvantages

Differentiate E. histolytica from E. dispar; (ii) they have excellent sensitivity and specificity;

Immunodiagnosis

Emerging methods in Diagnosis

• These are considered the most useful tests for detecting E. histolytica. They test directly for the parasite itself by exposing some stool to a strip of paper coated with antibodies. The parasites will stick to the antibodies on the paper. The test distinguishes E. histolytica from other parasites.

• Disadvantage : costly

Molecular Biology-Based Diagnostic Tests - PCR

• Detection of parasite DNA in faeces by PCR• Provide high sensitivity and specificity for the

diagnosis of intestinal amoebiasis

•WBC/DC –leucocytosis >10,000/mm3

• immunodiagnosis :

•serology - Serum antibody detection –ELISA

•Serum antigen detection by ELISA

Extraintestinal -Hepatic

•abscess aspiration

only selected cases

reddish brown liquid

trophozoites at the abscess wall

•imaging

X –ray, CT, MRI, ultrasound

•Abscess fluid Ag detection (ELISA)

Typical aspirate- chocolate syrup

Trophozoites are found on marginal wallCommonly found in the last portions of aspirated material

Peters & Gilles. Atlas of Tropical Medicine and Parasitology- 4 th Ed. Mosby-Wolfe 1995

CT scan of abscess in R lobe

X ray showing fluid level

amoebic hepatic abscess causing a raised right diaphragm with pleural effusion

Normal chest X ray

TestColitis Liver abscess

Sensitivity Specificity SensitivityMicroscopy (stool)

<60% 10-50% <10%

Microscopy (abscess fluid)

NAb NA <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)

NA NA ∼100% (before treatment)

PCR (stool) >70% >90% Not doneSerum antibody detection (ELISA)

>90% >85% 70-80% (acute), >90% (convalescent)

sensitivity and specificity of tests of diagnosis for amoebiasisa

TransmissionThrough cysts

Sources of infection:

Food and water contaminated with infected faeces.

Food handlers excreting cysts are an important source of contamination of foods

Houseflies also act as a mechanical vectors contaminating food

Sexual transmission

• Direct – hand to mouth• Indirect- contamination of food/water

Man is only reservoir host

Because of the protection conferred by cyst wall , cyst can survive days and weeks in external environment

Cyst Can be killed: Boiling- Above 68 ° CIodine (200 ppm)/acetic acid 5-10%Remove from water by sand filtrationOrdinary chlorination does not kill cysts

Epidemiology

Amoebiasis is cosmopolitan but no correlationbetween infection and disease

Generally in developed countries asymptomaticIn tropics/low socio-economic standardsHigh pathogenicity

High risk groups: travelers, institutional inmates homosexuals,

immunocompromised individuals, children in day care centers

Prevention

Reduce environmental contamination: detecting and treating infected persons

Improve environmental sanitation

Avoid ingestion of infected cyst by personal protection

Food safety• Thoroughly cook all raw foods. • * Thoroughly wash raw

vegetables and fruits before eating.

• * Reheat food until the internal temperature of the food reaches at least 167º.

• Wash your hands before preparing food, before eating, after going to the toilet or changing diapers

CiliatesCiliates

What are ciliates ?

Protozoa with cilia

Cilia -

Hair like structures used for locomotion and feeding.

Shorter than flagella and more in number

• Use cilia for movement or feeding

• Can have more than one nucleus (macronucleus, micronucleus)

• Feed through a “mouth” like structure (oral groove,

Ciliophora – ciliates

Generally larger than other protozoa

Reproduce by binary fission

ONLY ciliate that is known to parasitize man is Balantidium coli

Balantidium coli

Largest protozoan parasite of man

A common parasite of pigs

Pig the main reservoir

Human infection is less frequent

Parasitize distal ileum and colon

Invade the mucosa and causes blood and mucous diarrheoa

It is a zoonotic infection

C/f similar to amoebic dysentery but no extra-intestinal spread

Pathogenic to man as it invade the intestinal tissue

Two morphological forms

Trophozoite

Cyst

Trophozoit Cyst

EM view

Cilia

Life cycle

Diagnosis

Detection of cysts and trophozoits in faecal smears.

Regarding E. histolytica

A. Cyst is the infective stage

B. Does not attach to intestinal mucosa

C. Inhabits the human large intestine

D. Extaintestinal spread is possible

E. Nucleus has a central karyosome

Regarding amoeba

A. E. gingivalis has cyst stage in their life cycle

B. Can differentiated by their characteristic movements

C. E. dispar is a human pathogen

D. E. coli and E. histolytica are morphologically identical

Regarding Balantidium coli

A. It is not pathogenic to human

B. Trophozoite has only one nucleus

C. It is a zoonotic parasite

D. Cyst is covered with cilia

E. Trophozoite is the infective stage to human

True /false E.histolytica

Inhabits human large intestine

E. Histolytica cyst is a infective stage to human

Transmitted by faeco-oral route

E.Histolytica trophozoite is morphologically identical to E. dispar

True or false

Genus Entamoeba has large katyosome in side the nucleus

E. Histolytica trophozoite moves sluggishly

E. Histolytica trophozoite has single nucleus with centrally placed karyosome

E. Gingivalis has trophozoite and cyst in their life cycle

Acute amoebic dysentery, predominant form is cyst in stools

Flask shaped ulcers are typical lesion in intestinal amoebiasis

Trophozoites in faecal samples is a commonly associated with hepatic amoebiasis

In amoebic colitis, predominant form in the faeces is trophozoite

E histolytica and E dispar cysts cannot differentiate microscopically

Fever is a common clinical feature of amoebic colitis.

Abscess fluid microscopy is useful in the diagnosis of amoebic liver abscess