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INTESTINAL & TISSUE FLAGELLATES, INTESTINAL

COCCIDIA

Dr. Rumala MorelDepartment of Parasitology2007/8 BatchMay 2010

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Intestinal Flagellates - Giardia lamblia (intestinalis)

Intestinal Coccidia- Cryptosporidium, Cyclospora

Microsporidia

Tissue Flagellate - Trichomonas vaginalis

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Giardia lamblia (intestinalis)Giardiasis

Flagellate inSmall intestine

Trophozoite 8-16 m pear shape 2 nuclei4 pairs of flagellae bilaterally symmetricalConvex dorsally ventral adhesive disc

Cyst: 8-14 m , oval, 4 nuclei,remnants of flagellae etc.

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Pathogenicity

Duodenum & upper jejunumTrophozoites attached NOT invasive

Can ascend bile duct ? cholecystis

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Pathogenicity

disrupts mucosal structure & function luminal factors- contribute to diarrhoea, malabsorption

(1). Trophozoitedamages microvilli affects brush -border enzymes eg. disaccaridases, cytopathic substances - proteases, surface lectins

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Pathogenicity2. Immune mediated A). T-cell activity villous atrophy

associated crypt hypertrophyB). anti-Giardia sIgA

3. Luminal factors: causing STEATORRHOEABacterial overgrowth - deconjugation of BS

damage mucosal cell membranesBile salts in lumen– increased uptake by Giardia

DUAL ROLE of BILE SALTS – Giardia need BS fora). growth in SI b). encystation in LI

Pancreatic enzyme inhibition – trypsin, lipase activity

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Transmission

Cysts- killed at 62° C, unaffected by chlorinationsurvives 3 months in water Zoonotic transmission +

Diagnosis

Trophozoites/cystsCyst excretion inconsistent – take several samples

at intervals Duodenal aspirates/samples (string test) - reliable

for trophozoites

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COCCIDIAIntracellular - alternation of asexual & sexual

cyclesDevelopment in epithelial cells (usually gut) of

the definitive hostTISSUE COCCIDIA

• Toxoplasma gondii• Sarcocystis spp

INTESTINAL COCCIDIACryptosporidium –

C.parvum C.hominis Cyclospora cayetanensis Isospora belli Sarcocystis spp.

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EPIDEMIOLOGY - COCCIDIACryptosporidiosis- Asia/Africa 5-10%Isospora and Cyclospora - endemic in many

parts of Africa, Asia, and South America.

Severity depends on CD4(+) cell counts, If count : > severe disease, > atypical disease, > risk of disseminated disease.

EPIDEMIOLOGY OF INTESTINAL PROTOZOA IN IMMUNESUPPRESSED

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COCCIDIAN LIFE CYCLE

sporozoiteschizont

merozoites

male & female gametocytesmale & female gametesZygote

Oocyst(sporocyst) (sporozoites)

SCHIZOGONY(MEROGONY)

SPOROGONY

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OPPORTUNISTIC INFECTIONInitially recognized in immunocompromised hostsSevere, chronic fatal diarrhoea

Cryptosporidium parvum

Cryptosporidiosisanimal pathogen- 1907human cases- 1976

Immunocompetent - Self limiting diarrhoea (15-40 d)moderate – severe Water bourne outbreaksTraveller’s diarroeaChildhood diarrhoea – day care centres

ZOONOSISParasite of man & vertebratesC.parvum incattle,buffaloes,goats, cats etc

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Widespread in many animals2 important species• associated with human infection

1. C. parvum - commonest2. C. hominis

• primarily animal (cattle-bovine spp.) but ability to cause cross-infection in humans

• Genomes sequenced

Cryptosporidium

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CryptosporidiumLife cycle:classified as a Sporozoaas oocyst releases4 sporozoites

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Distinct phases in LC• Excystation of orally ingested oocyst with

release of 4 sporozoites• Invasion of intestinal epithelial cells via apical end of

sporozoite; vacuole formed of both host and parasite membranes

• Intitiation of asexual multiplication -merogony• Differentiation of micro and macro gametes - gametogony• Fertilization initiating sexual replication• Development of oocyst 4 sporozoites form within oocyst BEFORE excretion.Complete LC on epithelial cells (intestinal/respiratory) of

ONE host – intracellular but extracytoplasmicasexual & sexual development in same host

= monoxenous

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CRYPTOSPORIDIOSIS LIFE CYCLE - CDC80%

20%

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CryptosporidiumClosely associated to the apicalplasma membrane inPARASITOPHOROUS VACUOLE

enterocytefeeder organelle

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Cryptosporidium infecting enterocytes

TEM

SEM

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Lack of tissue specificityInfects biliary tract and respiratory system

(1) Sporozoites released from the oocystadhere to the epithelial mucosal surface

(2). Release cytokines activate phagocytes which release soluble factors

intestinal secretion of water and chloride and inhibiting absorption

Eg. histamine, serotonin, adenosine, prostaglandin, leukotrienes, platelet-activating factor

PATHOGENESIS

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(3). Epithelial cell death due toA). direct result of parasite invasion,multiplicationB). through T cell mediated inflammation

villus atrophy and crypt hyperplasia

NUTRIENT MALABSORPTIONDIARRHOEA

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Diagnosis1. Modified acid-fast stain +/- stool concentration

–most labs

2. METHOD OF CHOICE – most sensitive & specificImmunofluorescence microscopy 3.Enzyme immunoassaysRESEARCH - genetic techniques using PCR / DNA based antigen detection

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immunocompetent - moderate to severe diarrhoea (15-40 d) but self limiting

Bile acids enhances infectivity

immunocompromisedChronic diarrhoea in AIDS patientsCD4+ count <100/mm2 - life threateningAntiviral treatment improves

Cryptosporidium parvum CLINICAL FEATURES & TREATMENT

TREATMENT – only in immunesuppressed.Drug of Choice = NITAZOXANIDESignificant oocyst clearance.

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Transmission

- minimum 10 oocysts needed to infect - 50% infective dose is only 132 oocystsfor healthy person (DuPont et al. 1995)

(1) Waterbourne diarrhoea outbreaks -highly resistant to chlorination

- associated with surface water sources1993 - Milwaukee outbreak 400,000 affected

(2) Food (rare) do not survive cooking- contaminated cold foods/eaten raw – filter feeders

OOCYST- faeco oral route

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Zoonotic transmissionAnimal-person: calves, goats, lambs 50% calves shed oocysts ? Genotype

Person-person: CHILDHOOD DIARRHOEApeak age 1-5yrs high frequency in day care centers, day care employees at risk

Nosocomial infection+

Carrier- rate not known

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Cyclospora cayetanensis

Case reports- 1980’sIn immunocompromised/Immunocompetent host- traveller’s diarrhoeafood borne outbreaks

Oocysts – 8-10 m, unsporulated when voided2 sporozoitesstains with acid- fast stains

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Cyclospora cayetanensis Oocysts in faeces

Cryptosporidium

8-10m

3-5 m

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Isospora belliin intestine

Oocysts in faeces

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Microsporidia

Very small (1µm) obligate intracellular protozoan parasites with spores - unicellularInfection via single amoeboid germ (contained in spore) discharged by tubular filament

Parasites (enteric + tissue)

of Vertebrates

& Invertebrates

Pathogenic inImmunocompromised host

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MicrosporidaLife Cycle

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Microsporidia- immunofluorescenceshowing extruded polar tubules

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Gram-positive microsporidial spores of Enterocytozoan bieneusi- patient with AIDS

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Thin section from small intestine of patient with AIDS infected with E bieneusi

Developing microsporidial spores

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Intestinal Protozoa

Amoebae- Entamoeba histolyticaFlagellates- Giardia lamblia

(intestinalis)

Ciliates- Balantidium coliCoccidia- Cryptosporidium, Cyclospora Microsporidia-

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FAECAL SPECIMEN COLLECTION

Sample: Size: 1-2 table spoonsContainer: Clean, wide mouthed with lid

eg. yoghurt cupContaminants (interfering substances):

urine, water- destroys trophozoitesDirt- interferes with examination could introduce free living organisms

Oil, barium, kaolin, bismuth- artifactsAntibiotics- reduce no. organisms

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DIAGNOSIS OF INTESTINAL PROTOZOA

A). Examine 3 stool samples - 85-95% sensitivity

B). Direct Wet Smear – see motile trophozoites

C). Concentration methods - maximize recovery of cysts

eg. Formalin-ethyl-acetate concentrationZinc sulphate floatation

Basic guidelines – Ref. CDC/Entamoeba bench aid

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Preparations- for microscopic examinationSampled directly or after concentration

Temporary - wet mounts:Normal SalineIodine (Lugol’s iodine)

Permanent mounts:

Trichrome, Iron- haematoxylene,

Acid – fast stain =Modified Ziehl

Neelson for Cryptosporidium

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PREVENTION OF INTESTINAL INFECTIONSHEALTH EDUCATION

• Wash hands with soap and water after using the toilet and before handling food.

• Boil drinking water• Avoid eating from unhygienic places. • Traveller’s diarrhoea - avoid drinking unboiled tap water and

avoid uncooked foods washed with unboiled tap water. Bottled or canned carbonated beverages, pasteurized fruit drinks, and steaming hot coffee and tea are safe to drink.

• child care center - wash hands with of soap water after every diaper change, even if you wear gloves.

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Trichomonas - intestinal & uro-genitalflagellates

3 species in Man Trichomonas tenax T hominis T vaginalis - pathogenic

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General features of Trichomonas

NO cystic stagePear shaped4-5 anterior flagellaUndulating membraneNucleus - singleAxostyle- central rodCytostome +/- mouth

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T hominisT vaginalis T tenax

15-20 m x10 mcytostome – short undulating membraneVagina,urethra,bladder,prostate

7-8 mIntestinecommensalcytostome +

7 m

oral cavity

cytostome +

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Vagina: stratified squamous epitheliumIn the superficial layer epithelial cellglycogen anaerobically metabolized by commensal floraDodderlein’s bacilli lactic acidpH 4 -5 inhibits organisms

healthy adult female

female child/post menapause /pregnanacy:favourable for T.vaginalispH alkaline, secretions scanty

Trichomonas vaginalis -PATHOGENESIS

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Trichomonas vaginalis -PATHOGENESIS

No cyst – only transmitted sexually

NOT invasive adheres to vaginal squamous epithelium /

free in vaginaNot columnar epithelium – does not infect

endocervix of uterus

Local IgA highVaginal pH > 4.5 (loss of acidity)

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Trichomonas vaginalisTRICHOMONIASISAnnual worldwide Incidence= 180 million

In STD clinics : 7-32%

X3 higher risk of HIV infection

Frequently co-exists with other STI’s-candidiasis-gonorrhoea-syphilis-HIV

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Trichomonas vaginalisLife cycle

Asymptomatic malecarries infection to female sexual partners

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Women - vulvo – vaginitisIP = 3-28 days50-90% of infected women symptomaticVaginal discharge – wateryfrothy, fishy odour,greenish yellow50% dyspareunia

Trichomoniasis- Clinical features

Most men – asymptomaticFew Non Gonococcal Urethritis [NGU]

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Trichomoniasis - diagnosis

• Specimen Collection - WOMEN:Self Obtained Low Vaginal Swabs [SOLV] as sensitive as clinician obtained High Vaginal Swabs [HVS]

• BEST immediate wet smear – see ‘TWITCHING’ motility

• fluorescent stains/culture

HVS collection

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Trichomonal vaginitis: appearance throughvaginal speculum-creamy frothy dischargeoften 11ry infected with Candida albicans

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Living trophozoites of Trichomonas vaginalisfound in vaginal discharge-wet smear

BEST WAY TODIAGNOSETrichomoniasisINWOMEN

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Trichomoniasis - diagnosis

MEN:• Urethral discharge – wet

mount NOT sensitive

• Culture - Urethral discharge or- Urine sediment after prostatic

massage

CultureSensitivity >95%Anaerobic @ 37 0 C

Giemsa stained T. vaginalis

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PAP TEST – Papanicolau stain x400Sensitivity < wet mount

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Trichomoniasis

• PREVENTION & CONTROL• As for all STIs• Monogamous relationship

with a single known person!• Contact tracing & treatment

of all partners – asymptomatic males

• Condoms• Education of sex workers

TREATMENTMetronidazole

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ReferencesWEBSITES/linkshttp://www.cdc.gov/ncidod/dpd/parasites/

BOOKSManson’s Tropical Diseases – 22nd Edition.

Cook & ZumlaWorms and Human Disease - Ralph Muller,

Derek Wakelin 2002A colour atlas of tropical medicine and

parasitology – W. Peters & HM Gilles