13 December 2006
1 PHLN SUMMARY LABORATORY DEFINITION
1.1.1 Definitive Criteria
a Detection of typically stained oocysts, 4 to 6 m m using Modified Kinyoun acid-fast stain or direct fluorescent antigen (DFA);
b Positive immunodiagnostic detection result on faeces; OR
c Positive polymerase chain reaction (PCR).
1.1.2 Suggestive Criteria
is a coccidian parasite belonging to the family Cryptosporidiae. Originally only one species Cryptosporidium parvum,
was recognized divided into different genotypes which were host adapted e.g. tohumans(genotype 1), cattle (genotype 2) and dogs.1
taxonomy has been reviewed and many of the the host-adapted genotypes have been given species status based on at least four basic components: oocyst
morphology, natural host specificity, genetic characterizations,
and compliance with the International Committee for Zoological Nomenclature. Altogether, 13 Cryptosporidium
spp. are currently recognized: C. muris, C. andersoni, C. parvum, C. hominis, C. wrairi, C. felis,
and C. canis
in mammals; C. baïleyi, C. meleagridis,
and C. galli
in birds; C. serpentis
and C. saurophilum
in reptiles; and C. molnari
in fish. The clarification of Cryptosporidium
is useful for understanding the biology of Cryptosporidium
spp., assessing the public health significance of Cryptosporidium
spp. in animals and the environment, characterising transmission
dynamics, and tracking infection and contamination sources. 2
Both C. hominis
and C. parvum
are commonly detected in humans although there is some evidence that infections with C. hominis
infections tend to result in greater shedding of oocysts. Contact with cattle is not necessary for all C. parvum
infections and many have been shown to be spread from human-to-human. In addition to C. parvum
and C. hominis, C. felis, C. canis
and C. meleagridis
have been detected in AIDS patients.3
All life stages of the parasite are intracellular. At the time of excretion, the oocysts contain four infectious sporozoites. After ingestion and excystation by the host, the anterior end of each excysted sporozoite adheres to the luminal surface of an epithelial cell until microvilli surround it, making it intracellular but extracytoplasmic. Once established, it begins the infective process, producing new oocysts. Each oocyst measures 4 to 6 m m in diameter, and is shed as two distinct types. Approximately 20% of excreted oocysts are thin-walled (meronts), environmentally sensitive, and excyst endogenously, resulting in auto-infection of the host. The rest are thick-walled oocysts that are environmentally resistant, and are shed in the faeces or sputum, and are immediately infectious to other hosts. Cryptosporidium
infection is highly associated with travelling, exposure to farm animals, and person to person transmission in settings such as day care centres, swimming pools and medical institutions. There have been a number of notable waterborne outbreaks, most specifically the Milwaukee outbreak, which resulted in more than 400,000 cases after a breakdown in the water treatment system.4
Foodborne transmission has also been demonstrated, usually as a result of poor hygiene with infected food handlers.5 Cryptosporidium
is of particular concern for four reasons:
(1) The oocyst is extremely resistant to disinfection and cannot be killed with routine water-disinfection procedures;
(2) The disease is not treatable with antibiotics;
(3) The mortality from infection in severely immunocompromised patients can be as high as 50-60%; and
(4) Animal and human faecal wastes are associated with transmission of the disease to humans.
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3.1 Direct Microscopy Cryptosporidium
cannot be cultured in vitro
by methods that are practicable for use in a diagnostic laboratory.
It can be detected quite easily in clinical specimens using stained preparations.
3.1.1 Suitable specimens
Generally fresh stools are submitted for laboratory analysis, but faecal specimens stored in 10% formalin, 5% formalin or SAF are preferred since the Cryptosporidium
oocysts are immediately infective on passage. Frequently, one stool may not be sufficient to make a diagnosis, particularly if the patient is in the process of recovery.
3.1.2 Test details – stained preparations Cryptosporidium
oocysts, because of their small size (4 to 6 m m) and similarity to yeasts, are easily missed in direct faecal specimen wet mounts. They are best visualised using modified Kinyoun Acid-Fast stain11
or fluorescent monoclonal antibody (FA) staining reagents. Typically, more oocysts will be seen in watery stools, whereas they become increasingly difficult to find in normal stools. The FA stains have a high specificity but are also more expensive to use.
3.1.3 Test sensitivity
Staining sensitivity is very dependent on the quality of the specimen. Formed stools contain larger numbers of artifacts, making interpretation more difficult, particularly when low numbers of oocysts are detected. Direct FA test sensitivity is very high and approaches 100%.9,10
3.1.4 Test specificity
Some acid-fast stains also stain yeast cells to produce red-coloured oval cells of the same size range. The modified Kinyoun stain provides the most clear-cut staining result. The direct FA stain is 100% specific.
3.1.5 Predictive values
A negative diagnosis from a Modified Kinyoun-stained preparation does not preclude the presence of Cryptosporidium.
At least three consecutive specimens may be required.
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3.1.6 Suitable test acceptance criteria
Either : a) Modified Kinyoun stain: red staining oocysts showing internal sporozoites around the
cell wall rim (crescent moon appearance) or whole oocysts brightly red coloured.
b) Fluorescent antibody stain: brightly fluorescent oocysts of the appropriate size range
after direct FA under a fluorescent microscope.
3.1.7 Suitable internal controls
Positive control material should be stained with each staining run to assess the reliability of the staining reagents.
3.1.8 Suitable test validation criteria
Typical oocyst morphology using modified Kinyoun or direct FA compared to positive control.
3.1.9 Suitable external quality control (QC) program
Royal College of Pathologists of Australasia (RCPA).
3.1.10 Special considerations
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3.2 Antigen Detection
3.2.1 Test details
Direct Antigen detection of oocysts in faecal specimens using commercially available kits is now the preferred screening method of many laboratories, particularly following recent amendments to the Medical Benefits Schedule, which now reimburses laboratories for this test. Positive specimens are usually examined by microscopy to confirm the presence of typical oocysts. The earliest kits were enzyme immunoassays, using microtitre plates,6
Recently, simple, rapid, dipstick – like chromatographic immunoassays have become available, several using monoclonal antibodies to detect both Giardia
in a single step.
3.2.2 Suitable specimens
Fresh or frozen stool specimens or stool specimens preserved in formalin or sodium acetate-acetic acid –formalin (SAF).
3.2.3 Test sensitivity
Enzyme immunoassay (EIA) kits are reported to have sensitivities ranging from 93 to 97%.10
3.2.4 Test specificity
EIA assays may give false positive results, especially with blood stained faeces. Therefore, the presence of oocysts should be confirmed by microscopy.
3.2.5 Predictive values
Negative predictive values approaches 100% for all immunodiagnostic detection tests. Positive predictive value approaches 100% for rapid dipstick type tests but may be lower for EIA’s
3.2.6 Suitable test acceptance criteria
A positive immunodiagnostic detection test result as defined by the kit manufacturer.
Positives should be confirmed by another technique.
3.2.7 Suitable internal controls
Kit internal controls
3.2.8 Suitable test validation criteria
3.2.9 Suitable external quality control (QC) program
None available for immunodiagnostic assays.
3.2.10 Special considerations
Concentrated or polyvinyl alcohol – treated (PVA) samples are unsuitable for testing with available EIA kits.
3.3 Nucleic acid detection
PCR tests have been developed, but they are not in routine use in laboratories,7,8.
3.4 Serological tests
Serum antibodies produced in response to Cryptosporidium
infection have been detected by EIA about 10 days post-infection11.
A 27-kDa antigen seems to be recognised by hyperimmune sera and is consistent in Cryptosporidium
This band recognition peaks and remains until 4 to 5 weeks postinfection. Although dot blots and Western blots have been developed in some research laboratories, no commercial kits have been released for general use as yet.12
Serological tests are useful for epidemiological studies and outbreak investigations, but are rarely useful for diagnosis of infection in individuals.
4 TYPING AND SUBTYPING METHODS
Genotyping methods have recently been developed to further characterise Cryptosporidium
oocysts from different sources. The tests are only available at present in a few specialised laboratories.10
5 LABORATORY NOMENCLATURE FOR NATIONAL DATA DICTIONARY
5.1 Organism name(s) listCryptosporidium parvum
5.2 Typing/subtyping nomenclature list(s)
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1. Caccio, S., Homan, W., Camilli, R., Traldi, G., Kortbeek, T., and Pozio, E. 2000. A microsatellite marker reveals heterogeneity within human and animal genotypes of Cryptosporidium parvum.
Parasitology. 120(Part 3):237-244.
2. Xiao, L, Fayer, R, Ryan, U, Upton, SJ. 2004. Cryptosporidium
taxonomy : recent advances and implications for public health. Clin Microbiol Rev 17:72-97
3. Morgan, U., Weber, R., Xiao, L.H., Sulaiman, I., Thompson, R.C.A., Ndiritu, W., Lal, A., Moore, A. and Deplazes, P. 2000. Molecular characterisation of Cryptosporidium isolates obtained from human immunodeficiency virus-infected individuals living in Switzerland, Kenya and the United States. J. Clin Microbiol. 38(3):1180-1183.
4. Garcia, L.S., and Bruckner, D.A. 1997 Diagnostic Medical Parasitology, 3rd
. ed., p54-83. ASM Press, Washington, D.C.
5. Quiroz, E.S., Bern, C., MacArthur, J.R., Xiao, L.H., Fletcher, M., Arrowood, M.J., Shay, D.K., Levy, M.E., Glass, R.I. and Lal, A. 2000. An Outbreak of Cryptosporidiosis linked to a food handler. J. Infect Dis. 181(2):695-700.
6. MacPherson, D.W., and McQueen, R. 1993. Cryptosporidiosis: multiattribute evaluation of six diagnostic methods. J. Clin. Microbiol. 31:198-202
7. Morgan, U.N., Constantine, C., Forbes, D.A., and Thompson, R.C.A. 1997. Differentiation between human and animal isolates of Cryptosporidium parvum
using rDNA sequencing and direct PCR analysis. J. Parasitol. 83:825-830.
8. Balatbat, A.B., Jordan, G.W., Tang, Y.J. and Silva, J. Jr. 1996 Detection of Cryptosporidium parvum
DNA in human feces by nested PCR. J. Clin. Microbiol. 34:1769-1772.
9. Garcia, L.S., Shum, A.C., and Bruckner, D.A. 1992. Evaluation of a new monoclonal antibody combination reagent for direct fluorescent detection of Giardia
cysts and Cryptosporidium
oocysts in human fecal specimens. J. Clin. Microbiol. 30:3255-3257.
10. Siddons, C.A., Chapman, P.A. and Rush, B.A. 1992. Evaluation of an enzyme immunoassay kit for detecting Cryptosporidium
in faeces and environmental samples. J. Clin. Microbiol. 45:479-482.
11. Morgan UM, Pallant L, Dwyer BW, Forbes DA, Rich G, Thompson RCA. 1998. Comparison of PCR and Microscopy for Detection of Cryptosporidium parvum
in Human Faecal Specimens: Clinical Trial J. Clin. Microbiol. 36:995-998.
12. Rodriguez, Hernandez, J., Canut Blasco, A., and Martin Sanchez, A.M. p 330-333. In Murray, P.R., Baron, E.J., Pfaller, M.A., Tenover, F.C. and Yolken, R.H. (Ed). 1999. Manual of Clinical Microbiology. 7th
Edition. American Society for Microbiology.
13. Arrowood, M.J. 1997. Diagnosis, p. 43-64. In R. Fayer (ed.), Cryptosporidium
and Cryptosporidiosis. CRC Press, Inc., Boca Raton, Fla.
14. National Pathology Accreditation Advisory Council. Draft Laboratory Accreditation Standards for Nucleic Acid Detection Techniques. Consultation Draft - September 1999.
15. Further references concerning assay characteristics for commercial kits may be found in the package inserts.