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PROTOZOAN Protozoans are classified according to the organ of

locomotion:o Amoeba- E. histolyticao Flagellates- G. lamblia, D. fragiliso Ciliate- B. colio Sporozoans (not discussed in this lecture)

Phylum SarcomastigophoraSubphylum Sarcodina – with pseudopods/ amoeba

Entamoeba histolyticaSubphylum Mastigophora – with flagellates

Giardia lambliaDientamoeba fragilis

Phylum Apicomplexa Phylum Ciliophora – with ciliates

Balantidum coli Phylum Microspora

A. INTESTINAL AMOEBA General Characteristics of Different Stages:

o Trophozoiteo Motile, rapid, unidirectionalo reproducingo feedingo lives in the lower GIT

o Cysto non-motileo non-feedingo infective for humans

Transmission: o ingestion of fecally contaminated food or watero ingested cysts pass to the lower intestine, where

they excyst and multiply as trophozoites Amoeba Parasites:

1. Nonpathogenic amoeba - commensals- has to be correctly identified to ensure proper

therapy

Entamoeba hartmanni Entamoeba coli Iodamoeba butschlii Endolimax nana Blastocystis hominia

2. Opportunistic Free Living Amoeba - potentially pathogenic to man- cause CNS infections

Naegleria fowleri Acanthamoeba sp.

3. Pathogenic Amoeba Entamoeba histolytica

ENTAMOEBA HISTOLYTICA- named by Schaudin in 1903- only amoeba with potential for tissue invasion- 3rd most common cause of morbidity/mortality due to

a parasiteDiseases:- Amoebiasis- Amoebic dysentery- Amoebic hepatitis (if liver is involved)Physiology- grows best under reduced oxygen tension- dependent upon bacterial flora for growth - trophozoites easily destroyed than cysts- strains cannot be differentiated in the basis of

morphologyReproduction:- Binary fission- Cyst formation (8 amoebulae are produced by the

metacystic amoebae after excystation)Morphology- Morphologically the same as Entamoeba dispar

o non-pathogenic (NP) variety of the parasiteo can’t be distinguished from E. histolytica(without

engulfed RBC) microscopically; requires PCR and other special laboratory exam

Subject: MicrobiologyTopic: Intestinal Parasitism 1: ProtozoansLecturer: Date of Lecture: Transcriptionist: kekie Editor: I <3 demseyPages: 10

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Stages:o TrophozoitePrecystYoung CystMature Cyst

Stages Trophozoite Precyst CystGeneral Description

- feeding or vegetative stage- the only form seen in extra-

intestinal tissue- 10-60 u

- Colorless- round or oval

cells- smaller than

the trophozoite but larger than the cyst

Young Cyst Mature cyst- round or oval- 10 to 20 u in diameter- smooth, refractile, non-staining, 0.5 u thick- contains vacuoles with

glycogen and sausage-shaped chromatoidal bodies

Cytoplasm 2 zones:1. Ectoplasm

- Clear hyaline outer margin2. Endoplasm- Granular inner region that

contains nucleus and RBC(pathognomonic)

- devoid of food inclusions

- contain vacuoles with glycogen- contains vacuoles with

glycogen and sausage-shaped chromatoidal bodies

- do not have chromatoidal bodies

Nucleus - single eccentric nucleus- nuclear membrane is lined in

the inner surface with uniformly distributed chromatin granules

- centrally located karyosome

- single nucleus - 4 small nuclei

Motility - sluggish pseudopodial action

- no progressive movement

- nonmotile

*sorry mabibitin kasi ung life cycle kaya sa nxt page n lang..o.O

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Life Cycle

From CDC: Cysts and trophozoites are passed in feces . Cysts are typically found in formed stool, whereas trophozoites are typically

found in diarrheal stool. Infection by Entamoeba histolytica occurs by ingestion of mature cysts in fecally contaminated food, water,

or hands. Excystation occurs in the small intestine and trophozoites are released, which migrate to the large intestine. The

trophozoites multiply by binary fission and produce cysts , and both stages are passed in the feces . Because of the protection conferred by their walls, the cysts can survive days to weeks in the external environment and are responsible for transmission. Trophozoites passed in the stool are rapidly destroyed once outside the body, and if ingested would not survive exposure to the gastric

environment. In many cases, the trophozoites remain confined to the intestinal lumen ( : noninvasive infection) of individuals who are

asymptomatic carriers, passing cysts in their stool. In some patients the trophozoites invade the intestinal mucosa ( : intestinal

disease), or, through the bloodstream, extraintestinal sites such as the liver, brain, and lungs ( : extraintestinal disease), with resultant pathologic manifestations. It has been established that the invasive and noninvasive forms represent two separate species, respectively E. histolytica and E. dispar. These two species are morphologically indistinguishable unless E. histolytica is observed with ingested red blood cells (erythrophagocystosis). Transmission can also occur through exposure to fecal matter during sexual contact (in which case not only cysts, but also trophozoites could prove infective).

KEKIE

INGESTEDTROPHOZOITES EXCYST IN LOWER ILEUMTROPHOZOITES MULTIPLY BY BINARY FISSIONTROPHOZOITES ENCYST IN LARGE INTESTINECYST AND TROPHOZOITES IN LUMEN OF LARGE BOWELREMAIN IN THE LUMEN OF COLON AND MULTIPLYTROPHOZOITES AND CYSTS IN THE FECESIMMATURE CYST (1 NUCLEUS)IMMATURE CYST (2 NUCLEI)MATURE CYST (4 NUCLEI) *infective stage

EXTERNAL ENVIRONMENT

EXTRAINTESTINAL ABSCESS

MAN

INVADE WALL OF LUMEN & MULTIPLY

RETURN TO THE LUMEN

TROPHOZOITE DISINTEGRATES

CIRCULATION

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Diagnostic Stage:- cyst and trophozoiteInfective Stage:- mature cystHabitat:- duodenum, proximal jejunum and large intestine- excystation(to emerge from cyst): duodenum- encystation(to form cyst or be enclosed by capsule):

large intestineMain source of infection:- Cyst-passing chronic patient- Asymptomatic carrierMode of transmission:- Ingestion of water and vegetables contaminated with

infected feces- Food contaminated by flies or the hands of infected

food handlers- Direct transmission by cyst carriers- Sexual transmissionPathology:Lesions:1. INTESTINAL

- primary 2. EXTRAINTESTINAL

- Secondary- in patients with clinical dysentery or in those with

mild or latent infections- most commonly involved organ is the liver - other involved tissue: lung, brain, skin(ex.

perianal)Character of lesion:- can cause ulceration: Flask-shaped cavity- Cavity contains cells, mucus and amoebae- Signs of inflammation are usually absent unless with

secondary bacterial infection Affected Sites:

- Most frequent – CECAL AREA- Less frequent – ascending colon, rectosigmoid

Factors influencing pathogenic activities : - Host resistance- Virulence and invasiveness of the amoebic

strain- Condition of the intestinal tract

Symptomatology- Clinical response is exceedingly variable depending

upon the location and severity of the infection:1. Asymptomatic infection

- most common2. Acute intestinal amoebiasis

- incubation period: 1-14 weeks- severe dysentery with numerous small stools

containing blood, mucus, and shreds of necrotic mucosa

- acute abdominal pain and tenderness- fever, dehydration, toxemia, and prostration

may be marked

3. Chronic amoebiasis -recurrent shreds of dysentery with intervening

periods of mild or moderate gastrointestinal disturbances and constipation localized abdominal tenderness liver may be enlarged

4. Hepatic amoebiasis -most common and grave complication on

intestinal amoebiasis5. Pulmonary amoebiasis 6. Amoebic infection of the brain 7. Skin infection

DiagnosisRecovery and identification of the parasite in the feces or tissues1. Feces

- Direct Fecal Smearo At least 3 fresh stool specimens should be

examined.o A permanently stained smear and a saline

mount of each specimen should be prepared and examined

o Note: Fecalysis/ Direct Fecal Smear generally requires 3 fresh stool specimens to be examined for the presence of parasites. At least one specimen with parasite is considered positive. Absence of the parasite is assumed when 3/3 stool specimens are negative.

- Culture of Stool- Immunologic tests for detection of E. histolytica in

the stooo ELISAo IFA using soecific monoclonal antibodies

2. Tissue- Serologic studies are most useful in extraintestinal

diseaseso IHAo ELISA

Clinical diagnosis - History of travel to or residence in an endemic area- Signs and symptoms on PE, sigmoidoscopy, and

roentgenologyTreatment- Metronidazole (Flagyl)

o 750 mg t.i.d. orally for 5 to 10 dayso DOC

- Surgical drainage or close aspiration for large abscessesPrevention and Control- Treat cases- Personal hygiene- Environmental sanitation- Boiling of water or treatment of water where water is

not potable- Protection of food from contamination- Wash uncooked vegetables thoroughly- Health education of the public regarding methods pf

avoiding infection

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Possible courses of Amoebiasis

Differentiation of E. histolytica from other amoebae contaminating fecesTROPHOZOITE CYST OTHERS

Entamoeba histolytica Active, progressive unidirectional movements

Sharply defined ectoplasm

Blade-like hyaline pseudopodia

Indistinct nucleus Ingested RBCs

1-4 nuclei Diffuse glycogen mass Large, bar-shaped

chromatoidal bodies

Pathogenic

Entamoeba coli More granular cytoplasm

Narrower, less differentiated ectoplasm

Blunter and broader pseudopodia

More sluggish, indeterminate movements

Heavier, irregular peripheral chromatin

Large, eccentric karyosome in the nucleus

Larger More granular

cytoplasm Slender, splinter-like

chromatoidal bodies As many as 8 nuclei

Of medical importance only because it may be mistaken for E. histolytica

Intestinal commensal

Endolimax nana Large, irregular karyosome

6 to 12 um Oval Up to 4 nuclei Large, irregular

karyosome No peripheral

chromatin

Intestinal commensal

Iodameoba butschlii Large, irregular karyosome

8 to 10 um 1 nucleus Large, irregular

karyosome No peripheral

chromatin Large glycogen vacuole

(stains brown with iodine)

Intestinal commensal Differentiated from Endolimax

nana only by its size

Entamoeba gingivalis Large number of food vacuoles

Dark-staining bodies derived from the nuclear fragments of WBCs

NONE Intestinal commensal Only species known to ingest

WBCs ~10% in persons with healthy

gums; ~95% diseased teeth and gums

Dientamoeba fragilis Binucleated Moves by means of

pseudopodia (reason why it is classified as an amoeba)

NONE Intestinal commensal Classified among trichomonads

but considered by mosty parasitologists to be an amoeba

B. INTESTINAL FLAGELLATES Introduction

MAN CONSUMES CYSTS OF E.

HYSTOLYTICA

NOT INFECTED INFECTED

CARRIER

REMAIN ASSYMPTOMATIC

MILD AMOEBIC HEPATITIS

DYSENTERY

LIVER ABSCESS

MAY DIE IF NOT TREATED

MAY DIE IF NOT TREATED

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Subphylum Mastigophora1. Requires blood-sucking invertebrates as

biological vector- W. bancrofti, B. malayi2. Species transmitted from person to person

without biological vector2.1. intestinal flagellates

2.1.1. Pathogenic1. Giardia lamblia – small intestine2. Dientamoeba fragilis - colon

2.1.2. Non-Pathogenic1. Chilomastix mesnili – small

intestine2. Trichomonas hominis – colon3. Enteromonas hominis4. Retortomonas intestinalis5. Trichomonas tenax – mouth

2.2. Genital flagellates1. Trichomonas vaginalis

NOTE: Organisms mentioned above must be differentiated from pathogens to avoid misdiagnosis and mistreatmentFlagellates other than Dientamoeba are easily recognized by their rapid motility

GIARDIA LAMBLIA (G. duodenale or G. intestinale)Diseases- Giardiasis- Lambliasis- Traveller’s Diarrhea- Leningrad’s Disease

Epidemiology- Worldwide distribution- Man is the natural host- Infection more common in children (6-10 y/o)

- More frequent among population groups practicing oral-anal intercourse than in the general population

Physiology- Habitat: Duodenum and Upper Jejunum and at times

possibly the bile duct and gall bladder- Food is absorbed from the intestinal contents but may

possibly obtain nourishment from the epithelial cells through the SUCKING DISK

- Doesn’t penetrate mucosa- Factors that favor multiplication:

o Alkaline environmento Increased achlorhydiao Rich carbohydrate diet

MorphologyStages:1. Trophozoite

- Bilaterally symmetrical- Pear-shaped/ tear drop shaped- 12-15 um- Broad, rounded anterior and tapering posterior

extremity- Dorsal surface is convex- Ventral surface : 3/4 ovoid, concave sucking disk- 2 nuclei with large central karyosomes- 2 axonemes- 2 blepharoplasts- 2 parabasal bodies- deeply staining bars- 4 pairs of flagella- Rapid, jerky, twisting motion

2. Cyst - Ellipsoidal- 9-12 um- Smooth, well-defined wall- 2 to 4 nuclei

Life Cycle

Cysts are resistant forms and are responsible for transmission of giardiasis. Both cysts and trophozoites can be found in the feces

(diagnostic stages) . The cysts are hardy and can survive several months in cold water. Infection occurs by the ingestion of cysts in

contaminated water, food, or by the fecal-oral route (hands or fomites) . In the small intestine, excystation releases trophozoites (each

cyst produces two trophozoites) . Trophozoites multiply by longitudinal binary fission, remaining in the lumen of the proximal small

bowel where they can be free or attached to the mucosa by a ventral sucking disk . Encystation occurs as the parasites transit toward

the colon. The cyst is the stage found most commonly in nondiarrheal feces . Because the cysts are infectious when passed in the stool or shortly afterward, person-to-person transmission is possible. While animals are infected with Giardia, their importance as a reservoir is unclear.Diagnostic Stage:- cyst and trophozoite

Infective Stage:- mature cyst

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Habitat:- duodenum and upper jejenum; bile duct and gall

bladder- excystation: duodenum- encystation: large intestineTransmission:- Food and water contaminated by sewage, flies or food

handlers- Hand-to-mouth- Sexual contactPathology and Symptomatology- Incubation period: 12- 20 days- CRP136

Cysteine-rich surface protein; potential gardia toxin similar to snake toxin

- Derangement of normal villous architecture- Shortening of villi

- Mast cell-mediated reactions contribute to the inflammation and edema seen in the mucosa and lamina propria

- Mucosal invasion NOT necessary to produce malabsorption syndrome

- Malabsorption syndrome Steatorrhea and impaired absorption of carotene,

folate, and vitamin B12 in patients with giradial diarrhea

Production of disaccharidases and other mucosal enzymes can also be reduced

Uptake of bile salts by the protozoa may inhibit normal biologic activity of pancreatic lipase

Symptoms: flatulence, abdominal distension, nausea, anorexia, passage of foul-smelling and bulk stools, and eventual weight loss (stool does not contain blood or increase in PMNs)

Explosive watery foul smelling diarrhea with increase amount of fats and mucus

Diagnosis- Demonstration of cysts in formed stools- Demonstration of trophozoites and cysts in diarrheic

stools- Immunologic tests fir detection of antigen in the stool- String test

Examination of duodenal contents for trophozoites

- Demonstration of organisms in small bowel biopsiesTreatment- Quinacrine HCl

100 mg t.i.d. for 5-7 days children: 6 mg/kg daily for 5 days

- Metronidazole 250 mg t.i.d. for 7 days

Prevention and Control- Treat cases- Personal hygiene- Environmental sanitation- Boiling of water or treatment of water where water is

not potable

- Protection of food from contamination- Wash uncooked vegetables thoroughly- Health education of the public regarding methods of

avoiding infection

DIENTAMOEBA FRAGILISEpidemiology- Worldwide distribution- Method of transmission is uncertainMorphology- Found only as trophozoite

Has no observable flagella but is classified as a trichomonad even though it moves by means of pseudopodia rather than flagella when seen in feces (classification was based on DNA analysis)

6 to 20 um Exhibits sluggish condirectional motility Binucleated, granulated and vacuolated cytoplasm Recognized only in fresh liquid or soft stools May ingest RBC

Life Cycle of Dientamoeba fragilis

The complete life cycle of this parasite has not yet been determined, but assumptions were made based on clinical data. To date, the cyst

stage has not been identified in D. fragilis life cycle, and the trophozoite is the only stage found in stools of infected individuals . D.

fragilis is probably transmitted by fecal-oral route and transmission via helminth eggs (e.g., Ascaris, Enterobius spp.) has been

postulated . Trophozoites of D. fragilis have characteristically one or two nuclei ( , ), and it is found in children complaining of intestinal (e.g., intermittent diarrhea, abdominal pain) and other symptoms (e.g., nausea, anorexia, fatigue, malaise, poor weight gain).Diagnostic Stage:- trophozoiteInfective stage

- trophozoiteHabitat- cecum and large intestine

TROPHOZOITES IN THE

CECUM AND THE LARGE INTESTINEMULTIPLYS ASEXUALLY BY BINARY FISSION

DIAGNOSTIC AND

INFECTIVE STAGE:

BINUCLEATED

TROPHOZOITES PASSED

IN FECES

METHOD OF INFECTION: UNCERTAIN, BUT

PROBABLY HAND/MOUTH TRANSFER FROM

INFECTIVE INDIVIDUALS OR FROM FOCALLY

CONTAMINATED SOURCES

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Transmission- Fecal-oral route- Transmission associated with helminthic infection:

ascariasis and enterobiasisSymptomatology- Usually asymptomatic but may produce moderate,

persistent diarrhea and GI symptoms in some individuals

Diagnosis- identification of trophozoites in feces- At least 3 stool specimens should be examined- A permanently stained smear for each specimen

should be prepared and examined especially because there is no cyst stageo Stain: Trichrome stain

- Trophozoites can be found in formed stoolsTreatment- Iodoquinol

650 mg t.i.d. for 10 days DOC

- Tetracycline 250 mg q.i.d. for 7 days

NONPATHOGENIC INTESTINAL FLAGELLATES- important to distinguish these flagellates from

pathogenic flagellates by noting their morphologic difference

TROPHOZOITE CYSTChilomastix mesnili

-4 anterior flagella-1 nucleus-Cytosomal groove-Curved posterior

-Clear knob on the anterior-Cytosome-1 nucleus-Lemon-shaped

Trichomonas hominis

-4 anterior flagella-Costa-1 nucleus-Undulating membrane-Trailing flagellum-Axostyle

NONE

C. INTESTINAL CILLIATEBALANTIDIUM COLI

Diseases- Balantidiases- Balantidosis- Balantidial dysenteryEpidemiology- Worldwide distribution, especially in the tropics- Incidence in humans is very low- High incidence in hogs/pigs(reservoir host)- Hogs, however, are not important source of human

infectionMorphology- Largest intestinal protozoa and the only ciliated

protozoa to infect humans- Stages: Trophozoite, Cyst

1. Trophozoite Able to invade mucosa and cause

extraintestinal infection, however, this is rare Ovoid or bean-shaped 60u to 45um Shaped like a sac Enclosed in a delicate protective pellicle

covered with spiral longitudinal rows of cilia Anterior end has indentation forming the

cytostome or primitive mouth Posterior end has a cytopyge or excretory

pore Has 2 nuclei

o Macronucleus – large, bean-shaped/kidney-shaped

o Micronucleus – small, in the concavity of the macronucleus

With vacuoles and inclusions in the cytoplasm2. Cyst

Round or oval 52-55 um Covered with thick cell wall May show only the macronucleus, vacuoles

and cilia

Life Cycle of B. coli

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Cysts are the parasite stage responsible for transmission of balantidiasis . The host most often acquires the cyst through ingestion of contaminated food or water . Following ingestion, excystation occurs in the small intestine, and the trophozoites colonize the large intestine . The trophozoites reside in the lumen of the large intestine of humans and animals, where they replicate by binary fission, during which conjugation may occur . Trophozoites undergo encystation to produce infective cysts . Some trophozoites invade the wall of the colon and multiply. Some return to the lumen and disintegrate. Mature cysts are passed with feces .

Diagnostic Stage:- Trophozoite and cystInfective Stage- Mature cystHabitat:- large intestine- excystation: small intestine Mode of Transmission:- hand to mouth route- ingestion of contaminated food and water

Pathology and Symptomatology- Many infections are asymptomatic- Invade the mucosa and submucosa of the large intestines and produce ulcers- Humans have a high natural resistance; healthy persons less likely to develop illness

1. Acute infection 6-15 liquid stools/day With mucus, blood and pus

2. Chronic infection Intermittent diarrhea alternating with constipation Tender colon Anemia Cachexia

Diagnosis- Identification of trophozoites or cysts in feces or intestinal mucosa

Wet mounts of fresh or concentrated specimens are best Organisms stain very darkly on permanently stained preparations

- Sigmoidoscopy Useful in obtaining material for examination in patients with sigmoidorectal infection

Treatment1. Tetracycline-DOC2. Iodoquinol

CYST WILL PASS TO SMALL INTESTINE (EXCYSTATION OCCURS)TROPHOZOITES IN LARGE INTESTINEMULTIPLY ASEXUALLY BY BINARY FISSIONTROPHOZOITES INVADE MUCOSA CAUSING ULCERS (EXTRAINTESTINAL SPREAD IS RARE)ENCYSTATIONCYST (INFECTIVE) PASSED IN FECESINGESTION OF INFECTIVE CYST: TRANSMITTED BY FECES, FINGERS, FOOD, FOMITES AND FLIES

TROPHOZOITE (NON-INFECTIVE) MAY BE FOUND MORE COMMONLY IN OFT OR WATER STOOL

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3. MetronidazolePrevention and control- Treat cases- Personal hygiene- Environmental sanitation- Boiling water or treatment of water where water is not potable- Protection of food from contamination- Wash uncooked vegetables thoroughly- Health education of the public regarding methods of avoiding infection

ENTAMOEBA HISTOLYTICA

GIARDIA LAMBLIA DIENTAMOEBA FRAGILIS

BALANTIDIUM COLI

DISEASES AmoebiasisAmoebic dysenteryAmoebic hepatitis

GiardiasisLambliasisTraveller’s Diarrhea

BalantiadiasisBalantidosisBalantidial dysentery

MORPHOLOGYTROPHOZOITE Bull’s eye karyosome Old man’s face

Pear shapedAnterior end: cytostomePosterior end: cytopyge

NUCLEUS Single, eccentric Binucleated Binucleate Macronucleus : Bean shapedMicronucleus

KARYOSOME Center CenterMOVEMENT Unidirectional

Pseudopodia4 flagellaTwisting movement

NondirectionalPseudopodia

OTHERS Easily destroyed Ventral sucking disk2 parabasal bodies2 axonemes2 blepharoplast

No flagella Surrounded by cilliCystosomeCytopyge

CYST Cigar-chromatid bodies1-4 nucleiRound or oval

Ellipsoidal2-4 nuclei

none Thick cyst wallRound or oval

LIFE CYCLEHabitat Large intestine Duodenum and upper

jejunumLarge intestine Large intestine

Infective Stage Mature cyst Mature cyst Trophozoite Mature cystDiagnostic Stage Trophozoite and cyst Cyst in formed stool

Cyst and trophozoite in diarrheic stool

Trophozoite Trophozoite and cyst

Transmission Ingestion Ingestion Uncertain IngestionDIAGNOSIS

Diagnostic Stage Cyst and trophozoite in feces

Cyst and trophozoites Binucleate trophozoites Cyst and trophozoites

Methods Recovery and identification of parasite in the feces or tissueImmunologic testSerologic studiesClinical diagnosis

String or enterocapsule testDemo in stool or biopsyImmunologic tests

Identification of trophozoite in feces

ID of trophozoite in feces

Pathology Flask-shaped ulcer Malabsorption, derangement of villi, mast cell mediated inflammation and edema of mucosa and Lamina Propria

Usually asymptomatic Can also produce ulcer but usually asymptomatic

Drug of Choice Metronidazole Metronidazole Iodoquinol Tetracycline

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Woot woot! 1 tranx down..ELKIE!!!! THANK YOU SO MUCH!!! Ang ayos tlga!!