Cryptosporidiosis laboratory tests: Difference between revisions
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====Stained Smears==== | ====Stained Smears==== | ||
Traditional parasitology stains (e.g., [[Giemsa]]) are of limited value. They do not differentiate between | Traditional parasitology stains (e.g., [[Giemsa]]) are of limited value. They do not differentiate between [[oocyst]]s and similarly-sized fecal [[yeast]]s (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. | ||
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Shown below is cryptosporidium oocyst in modified acid fast stain. | Shown below is cryptosporidium oocyst in modified acid fast stain. | ||
[[Image:Cryptosporidium.jpg|250px|center]] | [[Image:Cryptosporidium.jpg|250px|center]] | ||
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====Immunofluorescence Microscopy For Detection Of Oocysts==== | ====Immunofluorescence Microscopy For Detection Of Oocysts==== | ||
Revision as of 14:44, 29 November 2012
Editor-In-Chief: C. Michael Gibson, M.S., M.D. [1] Associate Editor(s)-in-Chief: Kalsang Dolma, M.B.B.S.[2]
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Cryptosporidiosis Microchapters |
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Diagnosis |
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Treatment |
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Case Studies |
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Cryptosporidiosis laboratory tests On the Web |
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American Roentgen Ray Society Images of Cryptosporidiosis laboratory tests |
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Risk calculators and risk factors for Cryptosporidiosis laboratory tests |
Overview
Laboratory Findings
Stool Test
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.
Shown below is cryptosporidium spp. oocysts (pink arrows) in wet mount. A budding yeast (brown arrow) is in the same field.
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.
Shown below is cryptosporidium oocyst in modified acid fast stain.
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.
Shown below is oocysts of C. parvum (upper left) and cysts of Giardia intestinalis (lower right) labeled with immunofluorescent antibodies.
Several Additional Methods For Microscopic Detection Of Oocysts
- 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).
Shown below is oocysts of Cryptosporidium parvum stained with the fluorescent stain auramine-rhodamine.
Enzyme Immunoassays
At least four commercial enzyme immunoassay tests 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 . These tests are
- ProSpecT/ Cryptosporidium
- IDEIA Cryptosporidium
- MeriFluor™ Cryptosporidium/Giardia
- Color Vue Cryptosporidium
- Cryptosporidium Antigen Detection Microwell ELISA
Molecular diagnosis
Conventional PCR
Molecular methods (e.g., polymerase chain reaction – PCR) are increasingly used in reference diagnostic labs, since they can be used to identify Cryptosporidium spp. at the species level. Tests for Cryptosporidium are not routinely done in most laboratories; therefore, health care providers should specifically request testing for this parasite.
Shown below is 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
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.