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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