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