coccidian parasites
DESCRIPTION
coccidian parasitesTRANSCRIPT
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Coccidian Parasites
Dr Debasis Biswas
Phylum Apicomplexa
Plasmodium
Cryptosporidium
CyclosporaIsospora
Sarcocystis
Toxoplasma
EimeriaSubclass:
Coccidiasina
Apical complex, with organelles involved in Invasion including
Rhoptries, Micronemes, Dense granules.
Diarrheal disease
Typically asymptomatic;GI symptoms rare
Cryptosporidium
Ubiquitous environmental distribution: water bodies
Resistant to chlorination
Prolonged survival in moist conditions
Low infectious dose Recreational exposure (Swimming)
Intracellular life cycle: Intestinal epithelium
Infection --- Asymptomatic
--- Diarrhea
Self limiting . Normal hosts
Acute -onset; Persistent (>14 days). Children in developing countries
Chronic .. HIV +ve (CD4 count
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Life CycleSporulatedOocyst
Readily infectiousPerson-to-person transmissionContrast:Cyclospora ... unsporulated oocyst (No P2PT)
Epidemiological Implications
Water-borne Transmission
Source: Fecal contaminn.Small SizeResistance to chlorination (rises further withfecal contamination)Developing countries: Endemicvs. Developed countries: Food- & Water-borne outbreaksDeveloping countries (5-10%; mostly 14 days in 45% of children
Chronic: Malabsorption; Malnutrition; Weight loss
May be asymptomatic/ mild & self- limiting
Chronic diarrhea: Frequent, foul- smelling, bulky stool
Often assoc. with weight loss & malabsorption
Watery diarrhea: Relatively uncommon
Clinical manifestations
Immunocompetent hosts HIV/ AIDS patients
Cryptosporidiosis Malnutrition
Cryptosporidium infection causes acute malnutrition.
Severe
Worse consequences: infection in infancy, orin those with previous malnutrition
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Severely immuno-compromised HIV/ AIDS
Biliary Tract
Asymptomatic Bilateral Pulm infiltrates
Right upper quadrant abd pain(Intermittent & Colicky)
Enzyme alterations: (Alk Phos/ AST/ ALT/ Amylase/ Lipase)
USG:Dilated biliary tracts & Cholecystitis
Extra-intestinal manifestations
Respiratory Tract
Acalculous cholecystitisSclerosing cholangitis
Pancreatitis
Dyspnea
Diagnosis
Microscopy
Wet mount examn : Oocysts
4 6 diam: Yeast forms
Differential staining: ZN stain
Auramine-O
Stool concn technique: Increases sensitivity
Sedimentn: Formalin ether/ Formalin ethyl acetate
Floatn: Sheathers Sucrose/ Saline
Diagnosis Direct IF:
Oocyst-specific monoclonal antibody
Gold standard
Antigen detectn:
ELISA
Immunochromatography
PCR:
Increased sensitivityZN Stain Auramine-Rhodamine Immunofluorescence
Demonstration of Oocysts in stool
Cryptosporidium along the surface of intestinal epithelium
Intestinal Biopsy
Cyclosporacayetanensis
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C. cayetanensis: Humans are the only hosts
Not a zoonotic infection
Unsporulated oocysts excreted by infected individuals: Sporulation requires 7 days of maturation in external environment: No P2PT
Sporulation Oocyst with 2 sporocyst;
Each having 2 sporozoites
Infection --- Asymptomatic
--- Diarrhea
Flulike illness may precede diarrhea
Diarrhea associated with fever in approx. 25% of cases
Diarrhea may be cyclic or relapsing.
Extra-intestinal complicns: Reiter Syn; Guillain-Barre Syn
Distinct features cf. Cryptosporidium Diagnosis
Microscopy
Wet mount examn : Oocysts
2 X size of Cryptosporidium oocyst (8 10 )
Differential staining: ZN stain
Auramine-O
Blue autofluorescence under UV epifluorescencemicroscopy
Alt. stains : Safranine/ Lactophenol Cotton Blue
Unsporulated Oocysts of Cyclospora
ZN StainWet mount Modified Safranin stain
Diagnosis Histopathology / EM of Jejunal aspirates/ Biopsy:
Villous atrophy
Ac/ Chr inflammn in lamina propria
Cyclospora: supranuclear locn within cytoplasm of enterocytes, cf . Cryptosporidium: surface of enterocytes
PCR:
Flow cytometry:Sensitive for low parasitic concns.
Isospora belli
I. belli: Humans are the only hosts
Not a zoonotic infection
Unsporulated oocysts excreted by infected individuals: Sporulation requires 1-2 days of maturation in external environment: No P2PT
Sporulation hindered at temp. < 200C and > 400C
Oocyst with 1 sporoblast Oocyst with 2 sporocysts
Each sporocyst having 4 sporozoites
Rarely, some sporozoites may migrate to various tissues and form tissue cysts in LN, liver, spleen, etc
Distinct features cf. Cryptosporidium
Sporulation
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Diagnosis
Microscopy
Wet mount examn : Oocysts
Large & Elliptical oocysts (22 33 X 12- 15 )
Differential staining: ZN stain
Auramine-O
Blue autofluorescence under UV epifluorescencemicroscopy
Alt. stains : Safranine/ Lactophenol Cotton Blue
Unsporulated Oocysts of Isospora
ZN Stain
Wet mount
Modified Safranin stain
Autofluorescence
Diagnosis Histopathology / EM of Jejunal aspirates/ Biopsy:
Villous atrophy
Infiltration of inflammatory cells, particularly eosinophils, in lamina propria
Crypt hyperplasia
Isospora: parasitophorous vacuoles of enterocytes
PCR:
Treatment
Immunocompetenthosts
HIV/AIDS pts.
C. parvum
Fluid replacement.Anti-motility agents.
Efficacy of anti-parasitic agents not proven.Nitazoxanide
3- drug Anti-retroviralregimen with protease inhibitors. PIs can have anti-cryptosporidial activity.
C. cayetanensisI. belli
TMP-SMX (160/800 mg): BD X 7 days
TMP-SMX: BD X 7 - 10 days + Suppressive therapy thrice weekly is recommended to prevent relapse
Phylum Apicomplexa
Subclass: Coccidiasina
Plasmodium
Cryptosporidium
CyclosporaIsospora
Sarcocystis
Toxoplasma
Eimeria
Imm.Compt: Asymptomatic;(mostly)
Imm.Compro: T cell deficiency(HIV/AIDS; Transplant; Steroids;
CytotoxicDrugs; Anti-TNF therapy)
Congenital Infection
Toxoplasma gondii
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Unsporulated Oocyst
Sporulated Oocyst
FourSporozoites
TwoSporocysts
Tachyzoites
Macrophage
4 Parasitic stages
Tissue Cyst
Brain tissue (spheroid)Muscle tissue (elongated)
Excreted in the stool ofDefinitive hosts:Felines
Sporulated Oocyst
Tachyzoites
Invasive formRapidly dividing
3 Infective Stages
Tissue Cyst with Bradyzoites
Latent formSlowly dividing
Long-term survival
FourSporozoites
Environmental formSurvive > 1 yr in moist env.
Water, Soil, Vegetable & FruitContamination
Tachyzoites
Crescent- shaped: 5 X 2 Pointed apical end; Rounded posterior end
Apical end: Conoid Cell invasionRhoptries, Dense granules, Micronemes .. Secretory organelles
All infective stages
Dissemination form Invade all vertebrate cell types Multiply intracellularly
in a parasitophorous vacuole
Life Cycle of T. gondii
Life Cycle of T. gondii
Humans: Int. Hosts
Inf. form for humans
Sources of infection for humans
Oocyst
Tissue CystTachyzoite
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Oocysts/ Tissue cysts ingested
Release of sporozoiyes/ bradyzoites respectively in intestine
Invasion of intestinal epitheliumIntracellular multiplication
Mesenteric Lymph nodes Lymphatics.. Blood. Distant Organs .. Brain/ Eye/ Cardiac Muscle/ Skeletal Muscle/ Lungs/ Placenta
During this process, tachyzoites can invade all cell types.. Survive in parasitophorous vacuoles... Evade killing mechanisms in macrophages
Effective Host Response (CMI & Humoral)
Initiation of tissue cyst formation within a week in multiple organs
Pathogenesis
Killing of tachyzoites; Tissue cysts survive
Asymptomatic Chronic Infection; Occasional cyst disruption & release of bradyzoites
Minimal tissue damage Clinically silent
Compromised Host Response
Disruption of Cysts..Multiplicn
Inflammn..NecrosisTissue damage
Oocysts/ Tissue cysts ingested
Release of sporozoiyes/ bradyzoites respectively in intestine
Invasion of intestinal epitheliumIntracellular multiplication
Mesenteric Lymph nodes Lymphatics.. Blood. Distant Organs .. Brain/ Eye/ Cardiac Muscle/ Skeletal Muscle/ Lungs/ Placenta
During this process, tachyzoites can invade all cell types.. Survive in parasitophorous vacuoles... Evade killing mechanisms in macrophages
Effective Host Response (CMI & Humoral)
Initiation of tissue cyst formation within a week in multiple organs
Killing of tachyzoites; Tissue cysts survive
Asymptomatic Chronic Infection; Occasional cyst disruption & release of bradyzoites
Minimal tissue damage Clinically silent
Compromised Host Response
Disruption of Cysts..Multiplicn
Inflammn..NecrosisTissue damageR
EA
CT
IVN
Pathogenesis
Mostly asymptomatic
May mimic Infectious Mononucleosis (Epstein Barr Virus)
Fever Cervical Lymphadenopathy
Monocytosis in peripheral smear
Clinical features: Immunocompetent Host
Type II:
Western Europe & North America Less severe manifestns
Other genotypes:
Africa & South America . Higher incidence of Chorioretinitis
or other serious manifestns like meningoencephalitis,
pneumonitis, myocarditis, etc.
Rarely: Chorioretinitis
Severity related to genotype of the strain
HIV/ AIDS; Recipient of Immunosuppressive T/t:
Reactivation >> Re-infection
Reactivation of latent infection depdt. on duration & degree
of immunosuppression
Transplant Recipient: Infection from a cyst-containing graft
Commonest: Heart Transplant
Life- threatening (cf. Immunocompetent: Virulence depends on genotype)
Predominant manifestn: Toxoplasma Encephalitis
Other sites involved:
Lungs: More common in transplant recipients.
Leads to massive dissemination
Eyes: Chorioretinitis
Heart: Myocarditis
Clinical features: Immunocompromised Host
Primary infection in a pregnant mother
Frequency of infection: Efficiency of placental barrier
1st trimester < 2nd trimester < 3rd trimester
2nd trimester >3rd trimester
Clinical features: Congenital Infection
Necrosis foci & strong inflammation in fetal brain & eye tissues:
Hydrocephalus Mental Retardn Seizures Microcephaly
Deafness Cerebral Calcification
Microphthalmia Cataract Strabismus Increased IOP
Optic neuritis Retinal necrosis Uveitis Chorioretinitis
BlindnessHepatosplenomegaly Pneumonitis
Anemia Thrombocytopenia
A. Parasite- based
Demonstration of parasite:
Tachyzoites in tissue sections/ smears from body fluids
Tissue Cyst in tissues, + surrounding inflammation/ necrosis
Diagnosis
Isolation of parasite:
Mouse inoculation
Cell culture
Demonstration of parasitic DNA:
PCR of blood/ body fluids/ tissues
B. Host response- based
Antibody to parasite: Serologic tests
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A. Parasite- based
Demonstration of parasite:
Tachyzoites in tissue sections/ body fluids (CSF/ Amn fl/ BAL)
Tissue Cyst in tissues, + surrounding inflammation/ necrosis
Encephalitis/ Lymphadenitis/ Pneumonitis/ Myocarditis
Wright- Giemsa stain: Cheap; Quick; Less sensitive
Fluorescent Antibody staining
Immunoperoxidasestaining
Diagnosis A. Parasite- based
Isolation of parasite:
Blood/ Body Fluids: Suggests acute infection
Placenta/ Fetal tissues: Suggests congenital infection
Mouse inoculation: More sensitive
Cell culture: Quicker .. 3-6 days
Demonstrn of parasite- laden cells: Plaques
containing tachyzoites
Diagnosis
A. Parasite- based
Demonstration of parasitic DNA:
PCR: targeting multi copy B1 gene or REP-529 gene
Increased sensitivity before or within the 1st week of t/t.
Whole Blood/ Buffy Coat: Disseminated Toxoplasmosis
Amniotic Fluid: Intrauterine Infection
Placenta/ Fetal tissues: Congenital Toxoplasmosis
CSF: Toxoplasma Encephalitis
Diagnosis B. Host response- based
Serologic tests
Primary method of dg
Acute or chronic? .. Battery of tests:
IgM/ IgA/ IgG ELISA
IgG Avidity ELISA
Sabin- Feldman Dye test (IgG): Used to be the gold std
Diagnosis
Live tachyzoites + Alk Methylene Blue . Blue stain
Live tachyzoites + Pt serum (Ab) Destruction of parasitesAlk Methylene Blue Pale ghosts
Kinetics of Antibody response
Appear Plateau Disappear
IgA1 week 1 month
9 months
IgM 1 year
IgG 4-6 weeks 2-3 months Lifelong
Dye Test; IF test : Ab to memb ags Detected earlyELISA: Ab to mixtures of memb & cytosolic ags: Detected later
IgM detection
Most ELISA tests detect IgM for months/ years post-infection
Not necessarily a marker of acute infection, unless in high titers
Need to confirm acute infection with a 2nd technique
IgG Avidity ELISA
Affinity of ab response rises during the course of infection
Low- affinity IgG ab: marker of recent infection
Strength of ab binding measured by introducing a washing
step using a dissociating buffer (usually urea)
Ratio of IgG titers between treated and untreated samples
increases with increasing avidity
High avidity IgG excludes infection acq. in the past 4 mons.
Thus excludes infectn acqd in pregnancy, if done in 1st trimestr
Low/ int avidity IgG: Ambivalent; unless very low
Dating the infection: Acute vs. Chronic
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Rising IgG/ IgM titer detection
Serum samples drawn 3 weeks apart
Rising titer Infection acqd < 2 months before earlier sample
Caveat: T/t may reduce or abolish the rise in IgG titer
Dating the infection: Acute vs. Chronic Serology Work-up in a pregnant woman
IgG +IgM -
IgG -IgM -
IgG +/-IgM +
Monthly Follow/ up Till 2-3 weeks after delivery
Subsequent Seroconversion
IgG Avidity
High Low
Acq >4 monsearlier
RisingTiters
Distant infectionNo Follow/ up
Diagnosis: Congenital Infection Confirmation of maternal infection in pregnancy
Treatment of mother with Spiramycin & monthly USG
Amniotic fluid puncture after 16 weeks of gestation and 4 weeks after infection
PCR for parasite DNA
T/t switch to Pyrimethamine + Sulfonamide
Postnatal dg:PCR: Placenta/ Cord blood
Cord blood serology (IgM/ IgA)Western Blot of mother-infant
paired serumInfant serum (IgM/ IgA)
Abortion proposed in presenceof echographical signs
+ve -ve > 33 wks
Serologic tests:
To determine the immune status
Pregnant lady in early stage of pregnancy
Uveitis/ Retinochoroiditisw/o h/o congenital infection
Organ Donors/ Transplant recipients
D/D of Fever Lymphadenopathy: CMV/ EBV/ HIV
Parasite isolation/ demonstration: Less common
Dinagnosis: Immunocompetent
Parasite detection: Cornerstone
Cerebral: CSF/ Blood
Disseminated: Particularly Transplant recipients
Blood/ Bone Marrow Aspirate/ BAL/ CSF
PCR
Isolation: Cell Culture/ Mouse inoculation
Histology
Diagnosis: Immunocompromised
Serologic tests:
To exclude Toxoplasmosis
To monitor disease reactivation
Rising/ Very high IgG titers
Reappearance of IgM
Typically Based on:
Ophthalmoscopic exam:
Typical white focal lesions with vitreous
inflammatory reaxn
Seropositivity for Toxoplasma
Response to Anti- Toxoplasma t/t
Diagnosis: Retinochoroiditis
Western Blot Assay
In case of atypical lesions or inadequate response to t/t:
PCR of AH/ VH
Serologic tests: Local ab production
GWC: Goldmann-WitmercoefficientAnti- Toxo IgG in AH/ Anti- Toxo IgG in serum
Total IgG in AH/ Total IgG in serum