CRYPTOSPORIDIUM

PRESENTED BY:
NEETU AMATYA
B.Sc.MLT 3rd YEAR
JFIHS/LACHS
CONTENTS
 HISTORY
 SYSTEMIC CLASSIFICATION
 INTRODUCTION
 HABITAT
 EPIDEMIOLOGY
 MORPHOLOGY
 RESERVIOR, SOURCE & MODE OF TRANSMISSION
 LIFECYCLE
 PATHOGENESIS
 CLINICAL MANIFESTATIONS
 LABORATORY DIAGNOSIS
 TREATMENT & PROPHYLAXIS
 SUMMARY
 HISTORY
 In 1907: Tyzzer firstly described (C.muris) in
the gastric crypts of a laboratory mouse
Ernest Edward Tyzzer

 In 1912:Tyzzer later identified the smaller

parasite C. parvum, in the intestinal villi of
mice, that is now recognized as a major cause
of mammalian cryptosporidiosis.

 In 1976: Human cryptosporidiosis firstly

reported in three year old child in U.S.
 Recognized globally in 1980s and 1990s
 AIDS patients
 Outbreak among veterinary students
 Other receiving immunosuppressive
therapy
 ETYMOLOGY
Cryptosporidium
 From Greek ‘kruptos’ meaning ‘hidden’.

 Oocysts of cryptosporidia lack sporocysts and have ‘naked’ sporozoites;

Cryptosporidium has, therefore, been placed within a new family,
Cryptosporidiidae.
SYSTEMIC CLASSIFICATION

Protozoan
Phylum: Apicomplexa
Class: Sporozoasida
Order: Eucoccidiida
Family: Cryptosporiidae
Genus: Cryptosporidium
Species: parvum, muris, meleagridis, felis, etc.
Family Cryptosporidiae Definition:

 Includes a single genus Cryptosporidium.

 Includes coccidian in which all the sexual and asexual

development take place just the surface membrane or
within the brush border of the host cell.

 Infects the mammals, birds , fishes and reptiles.

 The sporocyst stage is absent.

 INTRODUCTION
 Cryptosporidium is an intestinal coccidian parasite which causes
infection of the small intestine .

 Causes cryptosporidiosis.

 Is smallest oocyst forming coccidian.

 20 species of cryptosporidium are known but only C. parvum

infects human.
 HABITAT
 Intracellular parasite

 Found attached to the surface

epithelial cells of villi or crypts of
the small intestine.

 Less frequently in the stomach,

appendix, colon, rectum and
pulmonary tree.
 EPIDEMIOLOGY
 World wide

 0.6-20% in Western countries and 5-10% in Asia and

Africa.

 Its infection in the immunocompetent hosts has been

described in more than 26 countries.

 In AIDS, the condition has been described with a

prevalence of 3-4% in the US and 50% in Africa and Haiti.
Nepals scenario
 MORPHOLOGY
 The parasite shows 6 distinct morphological forms during its
lifecycle:
A. Oocyst
B. Sporozoite
C. Trophozoite
D. Meront
E. Microgamont
F. Macrogamont

 Infective form of parasite is oocyst.

 OOCYST:
 Smallest oocyst forming coccidian.

 Measures: 4.5-6 um in diameter.

 Spherical or oval, colorless, highly refractile.

 Doesn’t stain with iodine and is weak acid fast.

 Two types of oocyst:

a. Thin walled (80%):causes auto-infection
b. Thick walled (20%):passes in faeces & infect new host
 Both contain 4 cresent shaped, slender
and fusiform sporozoites.

 These sporozoites always remain

parallel to eachother.

 The anterior end of sporozoite is

pointed.

 Posterior end rounded containing a

prominent nucleus.

 Contains 1-8 small granules.

 TROPHOZOITE
 Intracellullar transistional form of parasite.

 Round or oval measuring 2-2.5μm in diameter.

 Consists of a large nucleus with or without a conspicuous

nucleolus.

 Multiply asexually by schizogony producing type I and type

II meronts.
 HOST RANGE
 Wide variety of animals includes man, cattle, rats, dogs
and birds.

 Life cycle completes in one host (both schizogony &

gametogony) i.e. doesn’t require intermediate host.
 RESERVIOR, SOURCE &
TRANSMISSION OF INFECTION
 Oocysts excreted in the faeces are infectious
immediately.

 As low as 10 oocysts to 100 oocysts can cause disease in

humans.

 Man is the key reservoir of infection infection can be

transmitted to human by:
 Human to human transmission
 Animal to human transmission
  Human to human transmission
 Human faeces containing thick-walled oocysts are the
major source of infection (faeco-oral transmission).

 Ingesting food or drinks contaminated with fecal

material.

 Swallowing recreational water contaminated with

Cryptosporidium( cyst unaffected by chlorine water).

 Not washing hands.

 Sexual practices leading to oral exposure with fecal

material.
 Animal to human (zoonose) transmission:
 Infected directly either by ingestion of the oocysts
derived from the faeces of animals (cats, dogs,cattles) or
indirectly by close contact with animals.

OTHERS MODES OF TRANSMISSION:

 Aerosols
 Sexual contact
 Possibly by accidental laboratory infection
 Autoinfection
 LIFECYCLE
 Initially, human acquires infection by ingestion of food and
water contaminated with faeces containing sporulated
oocysts of the parasite.

 These environmentally resistant oocysts are encased in a

durable oocyst wall; a complex protective barrier
consisting of inner and outer oocyst walls composed of a
protein lipid- carbohydrate matrix.

 The infective cycle begins , when an appropriate host

ingests oocysts. As few as 10 oocysts ( infective dose)
have been reported to cause disease.
 C.parvum undergoes both
asexual(schizogony) & sexual
(gametogony)multiplication in a single
host.

 After ingestion, the thick walled

oocysts excyst and 4 sporozoites are
released from each oocyst in the lumen
of the intestine.

 The sporozoites develop into

trophozoites(uninucleate meronts) with
parasitophorous vacuoles (a structure
common to apicomplexans) in the brush
border of intestine.
 The parasitophorous vacuole of Cryptosporidium is unique in
that it remains extracytoplasmic, yet is considered
intracellular as it maintains its position within the host
derived parasitophorous vacuole membrane on top of epithelial
cells.

 These trophozoites undergoe asexual multiplication (schizogony)

and produce type I meronts.

 8 merozoites are released from each type I meront.

 These merozoites enter the neighbouring epithelial cells & repeat

schizogony to form type II meronts.
 4 merozoites are released from each type II meronts.

 The released merozoites enter host epithelial cell to form

the sexual stages, microgamonts & macrogamonts (i.e.
gametogony).

 A microgamonts releases 12-16 microgamates while only

one macrogamete is produced from each macrogamont.
 After fertilization of a macrogamete by microgamete, a thick
walled oocyst is formed.

 This oocyst undergoes sporogony to form sporulated oocyst which

contains 4 sporozoites.

 These oocysts are released into faeces & transmits the infection
from one person to another.

 Some of the oocysts have a thin wall surrounding 4 sporozoites

i.e. thin walled oocysts that causes autoinfection and maintain the
lifecycle of parasite without repeated exposure to thick walled
oocysts present in the environment.
 PATHOGENESIS
 Enters into intestinal lining of the gut
-Goes through Life Cycle.

 Infection begins with the firm attachment of C. parvum to the

mucosal surface of the intestine followed by invasion of epithelial
cells, release cytokines & then activates phagocytes.

 Attracts new leucocytes which inturn releases soluble factors

leading increased intestinal secretion of chloride & water &
inhibition of absorption.
 Associated with damage enterocytes accompanied by
malabsorption & osmotic diarrhea.

 The cholera-like voluminous watery diarrhoea is the key feature

of cryptosporidiosis in the patients with AIDS d/t combination
of secretory & malabsorptive processes.

 Bacterial fermentation of sugars and fatty acids of the

unabsorbed nutrients present in the lumen of intestine cause
offensive and foul smelling stool.
 Pathological changes:
Blunting & loss of villi, lengthening of the crypts & in
filtration of lamina propria by lymphocytes, PMNs
cells & plasma cells.
 HOST IMMUNITY
 Increase in circulating IgM & IgG Abs.

 Possibly reduce severity of subsequent symptomatic

infection but doesn’t confer any protective immunity
against re-infection.

 CMI with circulating Ig probably helps in

immunocompromised patients.

 Little is known about reactivation and reinfection.

CLINICAL MANIFESTATIONS
 Infection vary depending upon the host immune status.

 Causes mainly two distinct clinical entities:

I. A self-limiting diarrhoeal illnesses (1-2 weeks) in
immunocompetent healthy persons, mostly in
children,&
II. Severe prolonged life threatening diarrhea in patients
with AIDS.

 Incubation period: 2-14 days

 CRYPTOSPORIDIOSIS IN
IMMUNOCOMPETENT HOSTS:

 Mild infection with IP 1-2 weeks and recovery is

complete.

 Rarely fatal

 Watery diarrhea, malaise, nausea, fever, crampy

abdominal pain & flu-like illness and in some cases
followed by prostration and weight loss.

 Diarrhoea is foul smelling with 2 - 10 motions/day.

 CRYPTOSPORIDIOSIS IN
IMMUNOCOMPROMISED HOSTS:

 Sever, profuse, watery, mucus rich, chronic cholera-like diarrhea and

70 stools/day and loss of body fluid 17 litre/day; may contain mucus
but rarely blood and leucocytes.

 With CD4 counts <200/µl .

 Duration: 1-48 weeks

 Symptoms: low grade (39°C), fever, nausea,
vomiting ,severe dehydration & crampy abdominal pain,
weight loss.

 In some cases, affects entire GI tract including gall

bladder, bile duct & pancreas & even pharynx and bronchial
tree.
 Vulnerable group of people:
 Patients with AIDS

 Patients with congenital hypogammaglobulinaemia or

SCID symdrome.

 Patients with renal transplantation.

 Persons with IgA deficiency and with severe

malnutrition.
 COMPLICATIONS
 Extraintestinal infections common in
immunocompromised patients like AIDS

 Cholangiopathy, cholecystitis, hepatitis,

respiratory cryptosporidiosis.

 These patients cannot overcome the

infection and the illness become worse.
 PROGNOSIS:

 Prolonged diarrhea of more than 1 month duration and

biliary disease indicate a poor prognosis in patients with
AIDS.

 Mortality rate is higher than 50% with AIDS and death is

d/t severe dehydration caused by prolonged diarrhea.
LABORATORY DIAGNOSIS
SPECIMENS:
 Stool- specimen of choice
 Sputum- in respiratory cryptosporidiosis
 Biopsy- for histopathological examination
 Blood- for detection of Abs
 Bronchial washings & duodenal or jejunal aspirations-
less frequent
 Methods of examination
 Microscopy
 Antigen detection in stool
 Serological test
 Histological findings.
 PCR
 Imaging
 Others
 A. Microscopy
 Wet mount
 Direct wet mount:
Direct wet mount of stool.
For screening of stool specimens for oocysts.
Highly refractile , spherical oocysts that doesn’t stain with
iodine.
Useful with stool specimens containing moderate to high
number of oocysts.
 Stool concentration method :

The formal ethyl acetate oocyst sedimentation technique.

Sheather’s sucrose concentration.

Saturated sodium chloride method

  Staining methods of specimen
 A large number of staining procedure have been
demonstrated.

1.The modified acid-fast, Kinyoun’s (COLD

STAINING METHOD):
 Simple and effective method that uses carbol
fuschin without any heating procedure.

 Red stained acid fast oocyst(2-5um diameter) in the

faeces against blue background.

 Can also be demonstrated in sputum, bronchial

washings and duodenal or jejunal aspirations
 Visualization of Oocytes
 Light pink to dark red(Acid fast).
 Can also visualize sporozoites
 Relatively High Sensitivity and Specificity
 Irregular Staining
 cause “ghost” oocyst

2. Giemsa method:
3. Fluorescene dyes:
 Direct Fluorescent Antibody (DFA) Assay

 Auramine O, auramine-rhodamine, auramine-carbol-fuschin,

acridine orange, 4’,6-diamidino-2-phenylindole(DAPI).

 Fluorescence microscope

 “Gold Standard”
 High sensitivity and specificity

 Requires special equipment

4.Safranin stain
 Oocysts stain a bright red orange
 Not widely used because oocysts may not stain properly

5.Trichrome Stain
 Oocysts may appear unstained
 Lowest sensitivity and specificity among all tests
 Can detect Oocysts, but Cryptosporidium should be
confirmed by diagnostic techniques
6.Others:
 Hot safranin-methylene blue stain

 Modified kohn’s stain

 Modified koster stain

 B. ANTIGEN DETECTION IN STOOL

 ELISA is employed to detect parasitic antigen in stool.

 Used for diagnosis of intestinal infection.

 Monoclonal antibodies against oocyst-wall antigens have

been employed in Cryptosporidium- specific
immunofluoresence antibody tests with fecal specimens.
 Sensitive ELISA methods for detection of cryptosporidial
antigens in fecal samples have been described and commercial
diagnostic ELISA as well as monoclonal antibody kits are
available.

 The immunological techniques are more sensitive than direct

observation or histological staining methods but even more
sensitive detection of parasites has been achieved using PCR.
 C.SEROLOGY
Enzyme Immunoassay (EIA):

 Detects isolated antigens from a patients

sample using antibodies that are tagged with
a color changing enzyme.

 Recently, Western blot using specific 17kDa

& 27kDa sporozoite antigens employed for
epidemiological studies.
 Relatively high Sensitivity and Specificity.

 Does not involve microscopy.

 Screens large numbers of specimens.

 Rapid Immunochromatographic Cartridge
Assays
 Detects isolated antigens from sample
using antibodies. A positive test is
indicated by a colored bar.

 Variable Sensitivity and Specificity

 Some Assays Have Been Recalled

 D. Histopathological examination
 Intestinal biopsy by H&E stain.

 Demonstration of developmental
stage of parasite.

 Cysts 2um to 5um in diameter

arranged in single or clusters in the
intestinal mucosa.

 Other biopsy specimen: jejunum and

occasionally from the rectum
 E. Polymerase Chain Reaction
 Separates DNA fragments based on size.

 435 bp

 High Sensitivity and Specificity

 Several genus-specific PCR restriction fragment

length polymorphism–based genotyping tools have
been developed for detecting and differentiating
Cryptosporidium organisms at the species level.
F. IMAGING METHOD

 Identification of cryptosporidia in the bile by endoscopic

retrograde cholangiopancreatography.

 Confirms the diagnosis

 G. OTHERS

 CD4 Lymphocytes counts helps to know progression of

cryptosporidial disease in patients infected with HIV.

 Diarrhoea resolves spontaneously if CD4 counts are

greater than 200/ul.
 TREATMENT
 Nitazoxanide
 Paromomycin
 Azithroycin
 Individuals with AIDS
 anti-retroviral therapy
 Treatment of HIV infection with highly active
antiretroviral therapy (HAART) has been found to be
highly beneficial in the control of cryptosporidiosis,
probably as a result of recovery of CD4 cellcounts.

 Supportive therapy with replacement of fluid ,

electrolytes and nutrients.
PROPHYLAXIS
 Maintaining health hygiene and avoiding contaminated
water.

 Eating well-cooked food and so on…………

Cryptosporidium
•4-5 mm oocysts
•4 sporozoites
•no sporocysts

Cyclospora
•8-10 mm oocyts
•2 sporocysts
•2 sporozoites each

Isospora belli
•30 x 12 mm oocyts
•2 sporocysts
•4 sporozoites each
 SUMMARY
 In 1907, Tyzzer firstly described (C.muris) in the gastric crypts of a
laboratory mouse.
 Is intestinal coccidian parasite which causes infection of the small
intestine & Causes cryptosporidiosis.
 Infects the mammals, birds , fishes and reptiles and requires single host.
 Worldwide distribution and infects both in immunocompetent &
immunocompromised ( mainly in HIV/AIDS patient)
 Smallest oocyst but absent of sporocyst.
 Thick walled oocyst infect new host and Thin walled oocyst causes auto-
infection.
 Multiply asexually by schizogony producing type I and type II meronts
and sexually by gametogony.
 As low as 10 oocysts to 100 oocysts can cause disease in humans.

 Human to human transmission & Animal to human transmission.

 The cholera-like voluminous watery diarrhoea is the key feature of

cryptosporidiosis in the patients with AIDS d/t combination of
secretory & malabsorptive processes and have low CD4 count.

 DFA: GOLD STANDARD TECHNIQUE.

 PCR: High Sensitivity and Specificity

 DRUG OF CHOICE:Nitazoxanide ,Paromomycin ,Azithroycin

 REFERENCES:
 Bailey & Scott’s Diagnostic Microbiology, 13th E D I T I O N
 District Laboratory Practice in Tropical Countries, Part Second Edition , Monica Cheesbrough
 Parasitology; KD Chatterjee ;13th edition
 Textbook of medical parasitology; Subash Chandra Parija; 2 nd edition
 Medical parasitology ; C P Baveja
 Samendra Sherchan., et al. “Prevalence of Cryptosporidiosis Among School Going Children in
Kathmandu, Nepal”. EC Microbiology 4.1 (2016): 641-646.
 Opportunistic Infection in HIV/AIDS Patients in Nepal,Vol 27 (No. 1) June 2004 THE JOURNAL
OF TROPICAL MEDICINE AND PARASITOLOGY
 Journal of NHRC, Vol 3, No.1, April 2005, The seasonal outbreaks of Cyclospora and
Cryptosporidium in Kathmandu, Nepal
 Internet sources for images