Cryptosporidium parvum

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Cryptosporidium parvum
Small in size 5x4.5 µm C. Parvum oocyst
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Cryptosporidium parvum

• Enteric parasite
• One of the three most common diarrhea-causing
  pathogens in the world

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Prevalence

• Found in most parts of the world
• Most prevalent in Asia, Africa, Australia, South
  America
• Antibody prevalence in Peru and Venezuela 64
• 32 in Peace Corps workers
• More prevalent in rural areas of U.S.

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Transmission

• Fecal-oral route
• Fomites
• Water
• Drinking water (even after treatment)
• Swimming pools
• Unpasteurized Apple Cider
• Animal contact
• Food

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Infectivity

• Can be infected by just one oocyst
• 10 billion oocysts per gram infected feces

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Life Cycle
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Life Cycle
Life cycle
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Oocyst

• Double walled
• Resistant to chlorine, drying, progressive
  freezing, salt water
• Only stage in life cycle that can live ex vivo
• Imbeds itself in gut epithelium and releases
  sporozoites
• Reproduction continues sexually and asexually

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Clinical Characteristics

• Secretory diarrhea (some mucous, but no blood)
• Slight fever, fatigue, myalgia
• Oocysts may infect the lungs and trachea,
  resulting in cough
• Dehydration and extreme weight loss in
  immunocompromised

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Laboratory diagnosis of cryptosporidiosis
Cryptosporidium spp.

• Basic guidelines
• A. Multiple stool specimens (at least 3) should
  be tested before a negative result is reported.
• B. To maximize recovery of oocysts, stool
  specimens in formalin, or other fixatives, should
  be concentrated prior to microscopic examination
  (e.g.,10 min at 500 g when using the formalin

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• ethyl-acetate concentration procedure).
  Exception Specimens to be used for rapid
  car-tridge assays should NOT be concentrated
  because antigens are lost during the procedure!
• C. Choice of diagnostic techniques depends on
  available equipment and reagents, experience,
• and considerations of time and cost.

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1. Wet mount

• In bright-field microscopy using differential
  interference contrast (DIC), oocysts appear as
  small round structures (4 to 6 µm) similar to
  yeasts. They do not auto fluoresce.

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2. Modified acid-fast stain

• Oocysts (4 to 6 µm) often have distinct oocyst
  walls and stain from light pink to bright red.
  However, staining may be variable. In particular,
  infections that are resolving can have colorless
  oocyst ghosts. Mature oocysts may have
  discernible sporozoites (up to 4).

Modified acid-fast oocyst stain
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3. Direct fluorescent antibody (DFA) assay

• This technique offers the highest combination of
  sensitivity and specificity and is considered the
  gold standard by many laboratories. However, it
  does not provide a stained slide that can be
  archived. It requires special equipment
  (fluorescence microscope) and commercially
  available test kits.

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Sensitivity and specificity

• Sensitivity and specificity are statistical
  measures of the performance of a binary
  classification test. The sensitivity (also called
  recall rate in some fields) measures the
  proportion of actual positives which are
  correctly identified as such (i.e. the percentage
  of sick people who are identified as having the
  condition) and the specificity measures the
  proportion of negatives which are correctly
  identified (i.e. the percentage of well people
  who are identified as not having the condition.

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Definitions Imagine a scenario where people are
tested for a disease. The test outcome can be
positive (sick) or negative (healthy), while the
actual health status of the persons may be
different. In that setting -True positive Sick
people correctly diagnosed as sick -False
positive Healthy people wrongly identified as
sick -True negative Healthy people correctly
identified as healthy -False negative Sick
people wrongly identified as healthy
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Other methods for detecting Cryptosporidium in
stool.
6. Enzyme immunoassay (EIA) The EIA does not
rely on microscopy skills, is highly sensitive
and specific, and is useful for screening large
numbers of specimens.
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Cell-free culture ofCryptosporidium parvum

• Simple technique for culturing Cryptosporidium
  without host cell interference.
• The potential of improving routine water and
  other environmental monitoring for
  Cryptosporidium.
• Contribute much to our understanding of the
  developmental and evolutionary biology of
  Cryptosporidium.

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Amplification of oocysts

• In Vivo
• - Large animal Lambs
• - Small animal Neonatal mice
• In Vitro
• - Cell cuture

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In Vitro Cultivation

• Oocysts preparation
• - neonatal mice
• - Inoculation with 100,000-120,000 oocysts per
  mouse by gastic tube
• After 7 day post infection, mice will be
• killed and collected intestinal part.
• Extraction and purification

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In Vitro Cultivation

• Media preparation
• Excystation media
• Sterile Acidic water (pH2.5-3.0) Trypsin
  EDTA(0.5) 45 µl

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• Cell free cultivation
• Put 350,000 oocysts from stock into 9 ml of
  excystation media.
• Incubate 30 min at 37 C with shaking every 5
  min.
• spin down at 3,500 rpm 10 min
• Aspirate excystation media
• Resuspend pellet with 10 ml of maintenance media
  and mix well
• Add 10 ml of maintenance media into 25cm2 flask
• Add 5 ml of oocysts in maintenance media into the
  flask
• Leaving amount of Cryptosporidium oocysts
  maintenance
• for checking excystation rate
• Incubate at 37 C with 5 CO2

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24 hrs after culture
trophozoite
empty shell
Sporozoite
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48-72 hrs after culture
Meront type 1
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48-72 hrs after culture
Meront type II
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6 day after culture
Macrogamete and Microgamete
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7-8 after culture

• Fertilization and Zygote

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After 21 day culture
Sporulated oocysts
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Treatment

• Nitazoxanide
• Prevents parasite replication
• Immunocompetent
• C. parvum will usually pass on its own
• Immunocompromised
• AIDS patients treat with antiretrovirals and
  strengthen immune system, no cure

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Prevention

• Water filtration
• Filters must be lt1 um to filter oocyst
• Swimming pools
• Must be drained if infected fecal accident
• Pasteurization
• Hand washing
• Particularly in daycares

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Bottled Watersnot all are created equal

• Water so labeled has been processed by method
  effective against crypto
• Reverse osmosis treated
• Distilled
• Micro-filtered
• Chlorinated
• Ozone-treated
• Ultraviolet light-treated

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Outbreaks

• New York 1996 unpasteurized apple cider

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Summary

• Cryptosporidiosis caused by cryptosporidium
  parvum
• Transmitted via fecal-oral route
• Oocyst stage in life cycle is resilient
• Oocyst imbeds itself in gut epithelium
• Infection usually occurs from tainted water, even
  if it has been treated
• No cure in immunocompromised

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My home work

• Look deeply about
• PCR , PROCESSURE AND TYPES

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Put your trust in allah , but keep your powder dry