CRYPTOSPORIDIOSIS

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Cryptosporidium spp.

Causal Agent:
Many species of Cryptosporidium exist that infect humans and a wide range of
animals. Although Cryptosporidium parvum and Cryptosporidium hominis
(formerly known as C. parvum anthroponotic genotype or genotype 1) are the
most prevalent species causing disease in humans, infections by C. felis, C.
meleagridis, C. canis, and C. muris have also been reported.

Life Cycle:
Life cycle of Cryptosporidium parvum and C. hominis.
Sporulated oocysts, containing 4 sporozoites, are excreted by the infected host
through feces and possibly other routes such as respiratory secretions .
Transmission of Cryptosporidium parvum and C. hominis occurs mainly through
contact with contaminated water (e.g., drinking or recreational water).
Occasionally food sources, such as chicken salad, may serve as vehicles for
transmission. Many outbreaks in the United States have occurred in
waterparks, community swimming pools, and day care centers. Zoonotic and
anthroponotic transmission of C. parvum and anthroponotic transmission of C.
hominis occur through exposure to infected animals or exposure to water
contaminated by feces of infected animals . Following ingestion (and possibly
inhalation) by a suitable host , excystation occurs. The sporozoites are
released and parasitize epithelial cells ( , ) of the gastrointestinal tract or
other tissues such as the respiratory tract. In these cells, the parasites undergo
asexual multiplication (schizogony or merogony) ( , , ) and then sexual
multiplication (gametogony) producing microgamonts (male) and
macrogamonts (female) . Upon fertilization of the macrogamonts by the
microgametes ( ), oocysts ( , ) develop that sporulate in the infected host.
Two different types of oocysts are produced, the thick-walled, which is
commonly excreted from the host , and the thin-walled oocyst , which is
primarily involved in autoinfection. Oocysts are infective upon excretion, thus
permitting direct and immediate fecal-oral transmission.
Note that oocysts of Cyclospora cayetanensis, another important coccidian
parasite, are unsporulated at the time of excretion and do not become infective
until sporulation is completed.

Geographic Distribution:
Since the first reports of human cases in 1976, Cryptosporidium has been found
worldwide. Outbreaks of cryptosporidiosis have been reported in several
countries, the most remarkable being a waterborne outbreak in Milwaukee
(Wisconsin) in 1993, that affected more than 400,000 people.

Clinical Features:
Infection with Cryptosporidium sp. results in a wide range of manifestations,
from asymptomatic infections to severe, life-threatening illness; incubation
period is an average of 7 days (but can range from 2 to 10 days). Watery
diarrhea is the most frequent symptom, and can be accompanied by
dehydration, weight loss, abdominal pain, fever, nausea and vomiting. In
immunocompetent persons, symptoms are usually short lived (1 to 2 weeks);
they can be chronic and more severe in immunocompromised patients,
especially those with CD4 counts <200/µl. While the small intestine is the site
most commonly affected, symptomatic Cryptosporidium infections have also
been found in other organs including other digestive tract organs, the lungs, and
possibly conjunctiva.

Laboratory Diagnosis:
Acid-fast staining methods, with or without stool concentration, are most
frequently used in clinical laboratories. For greatest sensitivity and specificity,
immunofluorescence microscopy is the method of choice (followed closely by
enzyme immunoassays). Molecular methods are mainly a research tool.
Safety
Oocysts in stool specimens (fresh or in storage media) remain infective for
extended periods. Thus stool specimens should be preserved in 10% buffered
formalin or sodium acetate-acetic acid-formalin (SAF) to render oocysts
nonviable. (Contact time with formalin necessary to kill oocysts is not clear; we
suggest at least 18 to 24 hours). In addition, the usual safety measures for
handling potentially infectious material should be adopted.

Specimen processing
Stool specimens may be submitted fresh, preserved in 10% buffered formalin
(see above, “Safety”), or suspended in a storage medium composed of aqueous
potassium dichromate (2.5% w/v, final concentration). The use of mercuric
chloride-containing preservatives (e.g.,polyvinyl alcohol, PVA) is not
recommended due to incompatibilities with some methodologies and the
environmental hazards posed by the disposal of mercury-containing
compounds. Oocyst numbers can be quite variable, even in liquid stools.
Multiple stool samples should be tested before a negative diagnostic
interpretation is reported. To maximize recovery of oocysts, stool samples
should be concentrated prior to microscopic examination. Formalin-ethyl
acetate sedimentation is the recommended stool concentration method for
clinical laboratories. Two potential shortcomings of oocyst concentration
techniques are:

Sedimentation methods are generally performed using low speed centrifugation.

Given their small size and mass, cryptosporidial oocysts may become trapped
in the ether or ethyl acetate plug and fail to sediment properly. Increased
centrifugation speed or time (500 × g, 10 minutes) may be warranted when
attempting to recover cryptosporidial oocysts.
Resolution of cryptosporidial infections is accompanied by increasing numbers
of non-acid-fast, oocyst “ghosts.” Such oocysts may not float or sediment as
expected, giving rise to false-negative results.

Diagnostic findings:
 Microscopy.
 Enzyme immunoassays.
 Molecular methods.
 Antibody detection: There are currently no commercially available
serologic assays for the detection of Cryptosporidium-specific antibodies.
However, immunoblots for detecting the 17 and 27 kDa sporozoite
antigens associated with recent infection may be useful for epidemiologic
investigations.

Treatment:
Rapid loss of fluids because of diarrhea can be managed by fluid and
electrolyte replacement. Infection in healthy, immunocompetent persons is self-
limited. Nitazoxanide has been approved for treatment of diarrhea caused by
Cryptosporidium in immunocompetent patients. Immunocompromised persons
and those in poor health are at highest risk for severe illness. The effectiveness
of nitazoxanide in immunosuppressed persons is unclear. For persons with
AIDS, anti-retroviral therapy, which improves immune status, will also reduce
oocyst excretion and decrease diarrhea associated with cryptosporidiosis.

Microscopy
1) Wet mounts:

Wet mount examination (with iodine) is used mainly for screening, and is
especially useful with specimens containing moderate to high numbers of
oocysts. However, it should be combined with a more sensitive confirmatory
stain or assay. Fresh or concentrated fecal specimens can be examined, using
either conventional bright light, phase contrast or differential interference
contrast (or Nomarsky) microscopy.

Oocysts of Cryptosporidium parvum,

in wet mount, seen with differential
interference contrast (DIC)
microscopy.
The oocysts are rounded, 4.2 to 5.4
µm in diameter. Sporozoites are
visible inside the oocysts, indicating
that sporulation has occurred. (In
comparison, oocysts of Cyclospora
cayetanensis, another important
coccidian parasite of humans, are
twice larger and upon excretion are
not sporulated, i.e., do not contain
sporocysts.)

2) Stained smears:

Traditional parasitology stains (e.g., Giemsa) are of limited value. They do not
differentiate between oocysts and similarly-sized fecal yeasts (the main differential
diagnosis of Cryptosporidium in microscopy) and other debris. Modified acid-fast
staining technique is a simple and effective method: the oocysts stain bright red against
a background of blue-green fecal debris and yeasts. The acid-fast staining technique has
been modified and improved, including: hot and cold modified acid-fast stains;
incorporation of dimethyl sulfoxide (DMSO); and incorporation of the detergent
tergitol.
 Oocysts of
Cryptosporidium parvum
stained by the modified
acid-fast method. Against
a blue-green background,
the oocysts stand out in a
bright red stain.
Sporozoites are visible
inside the two oocysts to
the right.

Oocysts of Cryptosporidium parvum

stained by the modified acid-fast
method. This image shows that the
staining can be variable. In particular,
infections that are resolving can be
accompanied by increasing numbers
of non-acid-fast oocysts “ghosts”.

3) Immunofluorescence microscopy for detection of oocysts:

This method offers increased sensitivity and specificity compared to staining

techniques. It has found widespread application in research and clinical
laboratories as well as for monitoring oocyst presence in environmental
samples. The assays generally work well with fresh or preserved stools
(formalin, potassium dichromate), but some fixatives can cause problems (e.g.
MIF). Several commercial IFA products are presently available, including
MeriFluor™ Cryptosporidium/Giardia (Meridian Diagnostics Inc., Cincinnati, OH,
45244, USA); Detect IF Cryptosporidium (Shield Diagnostics, Ltd., Dundee DD1
1 SW, Scotland, UK); and Crypto IF Kit (TechLab, Blacksburg, VA, 24060,
USA). These assays exhibit broad reactivity with C. parvum and other
Cryptosporidium species, so they should be applicable to human and veterinary
specimens.
 Oocysts of C. parvum (upper left) and
cysts of Giardia intestinalis (lower
right) labeled with immunofluorescent
antibodies.

4) Several additional methods for microscopic detection of oocysts

include:

Alternate bright-field stains (e.g., hot safranin-methylene blue stain, modified

Kohn’s stain, modified Koster stain, aniline-carbol-methyl violet and tartrazine)
negative stains fluorescent stains (including auramine O, auramine-rhodamine,
auramine-carbol-fuchsin, acridine orange, mepacrine, and 4’,6-diamidino-2-
phenylindole (DAPI) and propidium iodide staining)
These exhibit potentially higher sensitivities but, like all nonspecific chemical
staining methods, yield false-positives and may leave some oocysts unstained;
these methods may be useful for screening samples, but identification should
be confirmed with more specific assays (IFA, EIA).

Oocysts of Cryptosporidium parvum

stained with the fluorescent stain
auramine-rhodamine.

Enzyme immunoassays
At least four commercial EIA tests (see Table below) have been introduced for
the detection of cryptosporidial antigens in stool samples. These kits are
reportedly superior to conventional microscopic examination (especially acid-
fast staining methods) and show good correlation with the monoclonal antibody-
based immunofluorescence assays. Kit sensitivities and specificities ranged
from 66.3% to 100% and 93% to 100%, respectively.
Molecular diagnosis
Conventional PCR

Agarose gel (2%) analysis of a PCR diagnostic test for detection of

Cryptosporidium parvum DNA. PCR was performed using primers CPBDIAGF
and CPBDIAGR.1

Lane S:
Molecular base pair standard (100-bp ladder). Black arrows show the size of
standard bands.
Lane 1:
C. parvum positive fecal specimen. The red arrow shows the diagnostic band
of 435 bp for zoonotic Cryptosporidium parvum.

Real-Time PCR

TaqMan-based real-time PCR

A TaqMan-based real-time PCR assay for detection and speciation of Cryptosporidium
parvum (bovine genotype) and Cryptosporidium hominis (human genotype) has been
developed and validated at CDC.2 The assay combines the detection of two genomic
targets: the 18S rRNA gene to achieve a sensitive detection of Cryptosporidium spp.
and a gene with unknown function to provide species differentiation. Each DNA
sample is run in two parallel reactions. The first consists of the highly sensitive
detection of the Cryptosporidium 18S rRNA gene and the species-specific detection of
C. parvum in a duplex format. The other reaction detects C. hominis on the species
level.