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5/23/2014

Phylum Apicomplexa
Apical complex, with organelles
involved in Invasion including
Rhoptries,
Micronemes,
Dense granules.

Coccidian Parasites

Plasmodium
Cryptosporidium
Isospora

Subclass:
Coccidiasina

Dr Debasis Biswas

Cyclospora

Sarcocystis

Diarrheal disease

Eimeria

Toxoplasma

Typically asymptomatic;
GI symptoms rare

Overview

Cryptosporidium

Overview

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 <100/mm3)
Fulminanat HIV +ve (CD4 count <50/mm3)

Life Cycle

> 20 species: Initially based on host specificity


C. parvum: Humans; Cattle
C. hominis: Mainly Humans

Single host

Sexual & Asexual phases

Sporulated Oocyst

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Life Cycle

Epidemiological Implications
Sporulated
Oocyst

Readily infectious
Person-to-person transmission
Contrast:
Cyclospora ... unsporulated oocyst (No P2PT)

Water-borne Source: Fecal contaminn.


Transmission Small Size
Resistance to chlorination (rises further with
fecal contamination)
Developing countries: Endemic
vs. Developed countries: Food- & Waterborne outbreaks
Developing countries (5-10%; mostly <5 yrs)
vs. Developed countries (1-3%; mostly
travellers)

Epidemiological Implications
Host
response

Limits duration & severity of infection


Vulnerable hosts:
Children;
Immuno-compromised (HIV/ AIDS: inverse
assocn. with CD4 counts; Transplant
recipients; Diabetes; Cancer)

Proneness to
desiccation

Outbreaks in moist months

Zoonotic
transmission

C. parvum contact with domestic animals


like cattle, sheep, goat, pigs

Pathogenesis
E nt er o c yt e I nf ec t i o n (S m al l i nt est i ne & p r oxi m al co l o n)
Extent of infection associated with immunosuppression
Pref. loss of mature ep cells
Inflamm. Cytokines, e.g. TNF; IL-1;IFN
at villus tips
Chemokines, e.g. IL-8
Increased Epithelial cell turnover
Up-regulation of COX2 by ep. cells
Influx of inflammatory cells
Prodn. of neuropeptides, e.g. Substance P
Na+ malabsorption
Electrogenic Cl- secretion
Increased Intestinal permeability

Villous atrophy
Crypt hyperplasia
Apoptosis, followed by
Necrosis, of ep. cells
Malabsorption:
Vit B12, Bile acids, Fatty acids

Voluminous Watery Diarrhea (resembling Toxin- mediated diarrhea)


Foul-smelling bulky stool (in chronic diarrhea d/t Fat malabsorption)

Clinical manifestations
Immunocompetent hosts
Diarrhea: Median durn. 5-10 d
Acute watery diarrhea
Assoc: Cramps; Nausea; Fever
Vomitting: less common
Course: Recurrence in 40%
pts. after initial resolution
Persistence >14 days in 45% of
children
Chronic: Malabsorption;
Malnutrition; Weight loss

HIV/ AIDS patients


May be asymptomatic/ mild &
self- limiting
Chronic diarrhea: Frequent,
foul- smelling, bulky stool
Often assoc. with weight loss
& malabsorption
Watery diarrhea: Relatively
uncommon

Severe

Cryptosporidiosis

Malnutrition

Cryptosporidium infection causes


acute malnutrition.
Worse consequences: infection in infancy, or
in those with previous malnutrition

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Extra-intestinal manifestations

Diagnosis

Severely immuno-compromised HIV/ AIDS


Biliary Tract

Acalculous cholecystitis
Sclerosing cholangitis
Pancreatitis
Right upper quadrant abd pain
(Intermittent & Colicky)
Enzyme alterations:
(Alk Phos/ AST/ ALT/ Amylase/ Lipase)
USG:
Dilated biliary tracts & Cholecystitis

Respiratory Tract
Asymptomatic
Bilateral Pulm infiltrates

Dyspnea

Diagnosis
Direct IF:
Oocyst-specific monoclonal antibody
Gold standard

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

Demonstration of Oocysts in stool

Antigen detectn:
ELISA
Immunochromatography
PCR:
Increased sensitivity

ZN Stain

Auramine-Rhodamine Immunofluorescence

Intestinal Biopsy

Cyclospora cayetanensis

Cryptosporidium along the surface of intestinal epithelium

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Distinct features cf. Cryptosporidium


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

Unsporulated Oocysts of Cyclospora

Wet mount

ZN Stain

Modified Safranin stain

Diagnosis
Microscopy
Wet mount examn : Oocysts
2 X size of Cryptosporidium oocyst (8 10 )
Differential staining: ZN stain
Auramine-O
Blue autofluorescence under UV epifluorescence
microscopy
Alt. stains : Safranine/ Lactophenol Cotton Blue

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.

Distinct features cf. Cryptosporidium

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
Sporulation

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

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Diagnosis

Unsporulated Oocysts of Isospora

Microscopy
Wet mount examn : Oocysts
Large & Elliptical oocysts (22 33 X 12- 15 )
Differential staining: ZN stain
Auramine-O
Blue autofluorescence under UV epifluorescence
microscopy
Alt. stains : Safranine/ Lactophenol Cotton Blue

Modified Safranin stain


Wet mount

ZN Stain

Autofluorescence

Treatment

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

Immunocompetent
hosts
Fluid replacement.
Anti-motility agents.
C. parvum

Efficacy of anti-parasitic
agents not proven.
Nitazoxanide

PCR:

TMP-SMX (160/800 mg):


BD X 7 days
C. cayetanensis
I. belli

HIV/AIDS pts.
3- drug Anti-retroviral
regimen with protease
inhibitors.
PIs can have anticryptosporidial activity.

TMP-SMX: BD X 7 - 10 days
+ Suppressive therapy thrice
weekly is recommended to
prevent relapse

Phylum Apicomplexa
Plasmodium

Subclass:
Coccidiasina

Cryptosporidium
Isospora

Cyclospora

Sarcocystis

Toxoplasma gondii

Eimeria

Toxoplasma
Congenital Infection

Imm.Compro: T cell deficiency


Imm.Compt: Asymptomatic;
(HIV/AIDS; Transplant; Steroids;
(mostly)
Cytotoxic Drugs; Anti-TNF therapy)

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4 Parasitic stages

3 Infective Stages
Tachyzoites

Tachyzoites

Excreted in
the stool of
Definitive hosts:
Felines

Four
Sporozoites

Invasive form
Rapidly dividing

Macrophage
Sporulated Oocyst

Unsporulated Oocyst
Two
Sporocysts

Tissue Cyst

Tissue Cyst
Environmental form
Survive > 1 yr in moist env. with Bradyzoites
Water, Soil, Vegetable & Fruit
Contamination

Four
Sporozoites

Latent form
Slowly dividing
Long-term survival

Brain tissue (spheroid)


Muscle tissue (elongated)

Sporulated Oocyst

Tachyzoites

Life Cycle of T. gondii


Dissemination form
Invade all vertebrate cell types
Multiply intracellularly
in a parasitophorous vacuole

All infective stages


Crescent- shaped: 5 X 2

Pointed apical end; Rounded posterior end

Apical end: Conoid Cell invasion


Rhoptries, Dense granules, Micronemes .. Secretory organelles

Life Cycle of T. gondii

Sources of infection for humans

Oocyst
Humans:
Int. Hosts

Inf. form for humans

Tachyzoite

Tissue Cyst

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Pathogenesis

Oocysts/ Tissue cysts ingested

Pathogenesis

Oocysts/ Tissue cysts ingested

Release of sporozoiyes/ bradyzoites respectively in intestine

Release of sporozoiyes/ bradyzoites respectively in intestine

Invasion of intestinal epithelium


Intracellular multiplication

Invasion of intestinal epithelium


Intracellular 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

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

Compromised Host Response

Initiation of tissue cyst formation within a week in multiple organs


Effective Host Response (CMI & Humoral)

Compromised Host Response

REACTIVN

Initiation of tissue cyst formation within a week in multiple organs


Effective Host Response (CMI & Humoral)

Killing of tachyzoites; Tissue cysts survive Disruption of Cysts..Multiplicn

Killing of tachyzoites; Tissue cysts survive Disruption of Cysts..Multiplicn

Asymptomatic Chronic Infection;


Inflammn..Necrosis
Occasional cyst disruption & release of bradyzoites
Tissue damage
Minimal tissue damage Clinically silent

Asymptomatic Chronic Infection;


Inflammn..Necrosis
Occasional cyst disruption & release of bradyzoites
Tissue damage
Minimal tissue damage Clinically silent

Clinical features: Immunocompetent Host

Clinical features: Immunocompromised Host

Mostly asymptomatic
May mimic Infectious Mononucleosis (Epstein Barr Virus)
Fever Cervical Lymphadenopathy
Monocytosis in peripheral smear
Rarely: Chorioretinitis
Severity related to genotype of the strain
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.

Clinical features: Congenital Infection

Primary infection in a pregnant mother


Frequency of infection: Efficiency of placental barrier
1st trimester < 2nd trimester < 3rd trimester
<10%
30%
60-70%
Severity of infection (Cong Malformn): Stage of fetal development
1st trimester >2nd trimester >3rd trimester
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
Blindness
Hepatosplenomegaly Pneumonitis
Anemia Thrombocytopenia

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

Diagnosis
A. Parasite- based
Demonstration of parasite:
Tachyzoites in tissue sections/ smears from body fluids
Tissue Cyst in tissues, + surrounding inflammation/ necrosis
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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Diagnosis

Diagnosis

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
Immunoperoxidase staining

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

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
Live tachyzoites + Alk Methylene Blue . Blue stain
Live tachyzoites + Pt serum (Ab) Destruction of parasites
Alk Methylene Blue Pale ghosts

Kinetics of Antibody response

Appear
IgA
IgM
IgG

Plateau

1 week

1 month

4-6 weeks

2-3 months

Disappear
9 months
1 year
Lifelong

Dye Test; IF test : Ab to memb ags Detected early


ELISA: Ab to mixtures of memb & cytosolic ags: Detected later

Dating the infection: Acute vs. Chronic


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

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Dating the infection: Acute vs. Chronic


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

Serology Work-up in a pregnant woman


IgG IgM -

IgG +
IgM -

Distant infection
No Follow/ up

Monthly Follow/ up
Till 2-3 weeks after delivery

Subsequent
Seroconversion

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

+ve

PCR for parasite DNA

T/t switch to
Pyrimethamine + Sulfonamide
Abortion proposed in presence
of echographical signs

-ve

IgG +/IgM +

IgG Avidity

High

Low

Acq >4 mons


earlier

Rising
Titers

Dinagnosis: Immunocompetent
Serologic tests:
To determine the immune status
Pregnant lady in early stage of pregnancy
Uveitis/ Retinochoroiditis w/o h/o congenital infection
Organ Donors/ Transplant recipients
D/D of Fever Lymphadenopathy: CMV/ EBV/ HIV
Parasite isolation/ demonstration: Less common

> 33 wks

Postnatal dg:
PCR: Placenta/ Cord blood
Cord blood serology (IgM/ IgA)
Western Blot of mother-infant
paired serum
Infant serum (IgM/ IgA)

Diagnosis: Immunocompromised
Parasite detection: Cornerstone
Cerebral: CSF/ Blood
Disseminated: Particularly Transplant recipients
Blood/ Bone Marrow Aspirate/ BAL/ CSF
PCR
Isolation: Cell Culture/ Mouse inoculation
Histology
Serologic tests:
To exclude Toxoplasmosis
To monitor disease reactivation
Rising/ Very high IgG titers
Reappearance of IgM

Diagnosis: Retinochoroiditis
Typically Based on:
Ophthalmoscopic exam:
Typical white focal lesions with vitreous
inflammatory reaxn
Seropositivity for Toxoplasma
Response to Anti- Toxoplasma t/t
In case of atypical lesions or inadequate response to t/t:
PCR of AH/ VH
Serologic tests: Local ab production
GWC: Goldmann-Witmer coefficient
Anti- Toxo IgG in AH/ Anti- Toxo IgG in serum
Total IgG in AH/ Total IgG in serum

Western Blot Assay