Cryptosporidiosis: The Parasite, Its Life Cycle and Lab Diagnosis

The parasite

Cryptosporidiosis is caused by many species and genotypes of Cryptosporidium, a single-celled (apicomplexan) parasite with a wide range of animal hosts. Zoonotic kinds pass from animals to people; others are adapted to one host with no evidence of spread from animals.

• Main human causes: C. parvum (once called C. parvum genotype II) and C. hominis (once genotype I).
• Also in humans: C. meleagridis, C. felis, C. canis, C. ubiquitum, C. cuniculus, C. viatorum, chipmunk genotype I, the mink genotype and C. muris.

Life cycle

The Cryptosporidium life cycle. Diagram: CDC DPDx.

1. Shedding: an infected host passes sporulated oocysts, each holding 4 sporozoites, in feces (and possibly other routes, such as respiratory secretions).
2. Spread: mainly by swallowing fecally contaminated water (drinking or recreational) or food (such as raw milk), or by direct contact with infected animals or people.
3. Invasion: once swallowed (or possibly inhaled), the oocysts open and release sporozoites, which invade epithelial cells of the gut (and possibly the airways), usually in the brush border.
4. Multiplying: they reproduce asexually (merogony), then sexually, forming male microgamonts and female macrogamonts.
5. New oocysts: fertilized macrogamonts become oocysts of two kinds. Thick-walled ones leave the body; thin-walled ones break open inside it and reinfect the host (autoinfection).

Oocysts are infectious as soon as they are passed, so fecal–oral spread can be immediate.

Hosts and range

• Hosts: birds, reptiles and mammals. C. hominis mainly infects people; zoonotic C. parvum is often tied to cattle, especially calves.
• Worldwide: both zoonotic and non-zoonotic kinds occur everywhere, with outbreaks in many countries.
• U.S. outbreaks: linked to swimming pools, water playgrounds and other swimming venues; unpasteurized cider, juice and milk; animal contact; childcare; camps; and ill food handlers.

Illness

• Incubation: about 7 days on average (2–10 days).
• Healthy people: diarrhea that usually clears on its own within 2–3 weeks, sometimes with fever or fatigue.
• Immunocompromised people: severe, even life-threatening malabsorption and wasting.
• Beyond the gut: infections of the lungs or bile ducts, rarely the pancreas, have been reported.

What labs see

Oocysts are round, 4.2 to 5.4 µm across; sporozoites are sometimes visible inside, meaning they have sporulated.

Oocysts (pink arrows) in a wet mount, with a budding yeast (brown arrow). CDC DPDx.

C. parvum oocysts under differential interference contrast (DIC) microscopy. CDC DPDx.

Modified acid-fast stain: oocysts stand out bright red on blue-green. CDC DPDx.

• Trichrome stain: oocysts can be spotted but appear unstained, so this method cannot confirm them.
• Variable staining: as infections resolve, more oocysts fail to take the acid-fast stain — pale "ghosts."

"Ghost" oocysts (red arrows) that did not take up the acid-fast stain; yeasts (yellow arrows) did. CDC DPDx.

Laboratory diagnosis

• Acid-fast staining, with or without concentrating the stool, is the most common clinical method.
• Immunofluorescence microscopy is the most sensitive and specific, followed closely by enzyme immunoassays (EIA).
• PCR and other molecular methods, increasingly used by reference labs, identify the species.

Specimens: stool may be kept in 10% buffered formalin or 2.5% aqueous potassium dichromate. Formalin-based fixatives (formalin, SAF, LV-PVA) harm DNA and are not recommended if molecular testing is planned. Oocyst numbers vary, so test several specimens before calling a result negative. Concentrate first with formalin–ethyl acetate sedimentation; tiny oocysts can get stuck in the ethyl acetate plug, so a faster or longer spin (500 × g, 10 minutes) may help.

Immunoassays: direct fluorescent antibody (DFA), EIA and rapid tests are sold in the U.S., some combined with Giardia and Entamoeba histolytica. DFA is highly sensitive and specific. Microplate EIAs work on fresh, frozen, formalin- or SAF-preserved stool, but not on concentrated or PVA-treated specimens. Watch for false positives with EIA and rapid tests.

Molecular: multipathogen gastrointestinal PCR panels include Cryptosporidium and can catch infections nobody suspected. Only molecular methods, such as those of CDC's CryptoNet, tell species and genotypes apart — for outbreak investigation, not clinical diagnosis.

Laboratory safety

Oocysts are hardy and immediately infectious, a risk to lab workers by accidental ingestion (or possibly aerosols). Beyond standard stool precautions:

• use a biosafety cabinet and/or PPE such as a face shield;
• autoclave or disinfect disposable waste;
• wash reusable items in a lab dishwasher with a chlorine detergent on the sanitize cycle, or soak them about one hour in water at 50°C, then wash;
• for spills: remove organic matter, soak up the bulk, then flood the surface with undiluted 3% hydrogen peroxide, keeping it wet about 30 minutes; blot and let dry 10–30 minutes before use.

Further reading

• Checkley, W., and others, 2015, "A review of the global burden, novel diagnostics, therapeutics, and vaccine targets for Cryptosporidium," The Lancet Infectious Diseases 15(1): 85–94.
• Ryan, U., R. Fayer and L. Xiao, 2014, "Cryptosporidium species in humans and animals: current understanding and research needs," Parasitology 141(13): 1667–1685.

Sources

Based on "Cryptosporidiosis," DPDx — Laboratory Identification of Parasites of Public Health Concern, Centers for Disease Control and Prevention; a work of the United States government in the public domain. The life-cycle diagram, linked from the page, is restored from CDC. Images contributed by the Oregon State Public Health Laboratory and the Kansas Department of Health and Environment are not reproduced.