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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