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{{Cryptosporidiosis}}
==Overview==
==Overview==
Diagnosis of cryptosporidiosis is made by microscopic identification of the oocysts in stool or tissue with acid-fast staining or direct immunofluorescence.


==Laboratory Findings==
==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 [[oocyst]]s. 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.
[[Image:WET MOUNT.jpg|250px|center]]
----
====Stained Smears====
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.
;Shown below is cryptosporidium oocyst in modified acid fast stain.
[[Image:Cryptosporidium.jpg|250px|center]]
----


Diagnosis of cryptosporidiosis is made by examination of stool samples. Because detection of Cryptosporidium can be difficult, patients may be asked to submit several stool samples over several days. Most often, stool specimens are examined microscopically using different techniques (e.g., Acid fast stain|acid-fast staining, direct fluorescent antibody DFA , and/or enzyme immunoassays for detection of Cryptosporidium sp. antigens).
====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.
[[Image:Crypto Giardia IFA.JPG|250px|center]]
----
 
====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 stain]]s
* 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 [[oocyst]]s 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.
[[Image:DPDxCrypto oo Aura.JPG|250px|center]]
----
 
===Enzyme Immunoassays===
 
At least four commercial [[enzyme immunoassay test]]s 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 assay]]s.  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.
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.
[[Image:PCR.JPG|300px|center]]
----
====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.
==Gallery==
<gallery>
Image: Cryptosporidiosis10.jpeg| Histopathology of cryptosporidiosis, intestine. <SMALL><SMALL>''[http://phil.cdc.gov/phil/home.asp From Public Health Image Library (PHIL).] ''<ref name=PHIL> {{Cite web | title = Public Health Image Library (PHIL) | url = http://phil.cdc.gov/phil/home.asp}}</ref></SMALL></SMALL>
Image: Cryptosporidiosis09.jpeg| Histopathology of cryptosporidiosis, gallbladder. <SMALL><SMALL>''[http://phil.cdc.gov/phil/home.asp From Public Health Image Library (PHIL).] ''<ref name=PHIL> {{Cite web | title = Public Health Image Library (PHIL) | url = http://phil.cdc.gov/phil/home.asp}}</ref></SMALL></SMALL>
Image: Cryptosporidiosis08.jpeg| Cryptosporidiosis of gallbladder in AIDS. <SMALL><SMALL>''[http://phil.cdc.gov/phil/home.asp From Public Health Image Library (PHIL).] ''<ref name=PHIL> {{Cite web | title = Public Health Image Library (PHIL) | url = http://phil.cdc.gov/phil/home.asp}}</ref></SMALL></SMALL>
Image: Cryptosporidiosis06.jpeg|Stool smear micrograph revealing Cryptosporidium parvum as the cause of this patient’s Cryptosporidiosis. <SMALL><SMALL>''[http://phil.cdc.gov/phil/home.asp From Public Health Image Library (PHIL).] ''<ref name=PHIL> {{Cite web | title = Public Health Image Library (PHIL) | url = http://phil.cdc.gov/phil/home.asp}}</ref></SMALL></SMALL>
Image: Cryptosporidiosis05.jpeg|Micrograph of a direct fecal smear is stained to detect Cryptosporidium sp., an intracellular protozoan parasite. <SMALL><SMALL>''[http://phil.cdc.gov/phil/home.asp From Public Health Image Library (PHIL).] ''<ref name=PHIL> {{Cite web | title = Public Health Image Library (PHIL) | url = http://phil.cdc.gov/phil/home.asp}}</ref></SMALL></SMALL>
Image: Cryptosporidiosis04.jpeg|Morphologic details of Cryptosporidium parvum oocysts, i.e.,encapsulated zygotes, stained using the modified acid-fast method. <SMALL><SMALL>''[http://phil.cdc.gov/phil/home.asp From Public Health Image Library (PHIL).] ''<ref name=PHIL> {{Cite web | title = Public Health Image Library (PHIL) | url = http://phil.cdc.gov/phil/home.asp}}</ref></SMALL></SMALL>
Image: Cryptosporidiosis01.jpeg|TEM reveals ultrastructural pathology seen in a tissue sample in a case of cryptosporidiosis. <SMALL><SMALL>''[http://phil.cdc.gov/phil/home.asp From Public Health Image Library (PHIL).] ''<ref name=PHIL> {{Cite web | title = Public Health Image Library (PHIL) | url = http://phil.cdc.gov/phil/home.asp}}</ref></SMALL></SMALL>
</gallery>
==Gallery==
<gallery>
Image: Cryptococcosis11.jpeg| Encapsulated yeast Cryptococcus neoformans. <SMALL><SMALL>''[http://phil.cdc.gov/phil/home.asp From Public Health Image Library (PHIL).] ''<ref name=PHIL> {{Cite web | title = Public Health Image Library (PHIL) | url = http://phil.cdc.gov/phil/home.asp}}</ref></SMALL></SMALL>
Image: Cryptococcosis10.jpeg| SABHI agar slant culture is growing Cryptococcus neoforman grown at 37°C. <SMALL><SMALL>''[http://phil.cdc.gov/phil/home.asp From Public Health Image Library (PHIL).] ''<ref name=PHIL> {{Cite web | title = Public Health Image Library (PHIL) | url = http://phil.cdc.gov/phil/home.asp}}</ref></SMALL></SMALL>
Image: Cryptococcosis08.jpeg| Test tube slant culture, which had been colonized by Cryptococcus neoformans and Histoplasma capsulatum. <SMALL><SMALL>''[http://phil.cdc.gov/phil/home.asp From Public Health Image Library (PHIL).] ''<ref name=PHIL> {{Cite web | title = Public Health Image Library (PHIL) | url = http://phil.cdc.gov/phil/home.asp}}</ref></SMALL></SMALL>
Image: Cryptococcosis02.jpeg| Photomicrograph revealed some of the ultrastructural details displayed by numerous Cryptococcus neoformans fungal organisms (475x mag). <SMALL><SMALL>''[http://phil.cdc.gov/phil/home.asp From Public Health Image Library (PHIL).] ''<ref name=PHIL> {{Cite web | title = Public Health Image Library (PHIL) | url = http://phil.cdc.gov/phil/home.asp}}</ref></SMALL></SMALL>
Image: Cryptococcosis01.jpeg| Selective growth medium containing thistle seed extract inoculated with a fungal organism. <SMALL><SMALL>''[http://phil.cdc.gov/phil/home.asp From Public Health Image Library (PHIL).] ''<ref name=PHIL> {{Cite web | title = Public Health Image Library (PHIL) | url = http://phil.cdc.gov/phil/home.asp}}</ref></SMALL></SMALL>
</gallery>
==References==
==References==
{{Reflist|2}}
{{Reflist|2}}
[[Category:Needs overvew]]
[[Category:Needs overview]]
[[Category:Parasitic diseases]]
[[Category:Parasitic diseases]]
[[Category:Infectious disease]]
 
[[Category:Disease]]
[[Category:Disease]]


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Latest revision as of 17:31, 18 September 2017

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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Overview

Diagnosis of cryptosporidiosis is made by microscopic identification of the oocysts in stool or tissue with acid-fast staining or direct immunofluorescence.

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.

Gallery

Gallery


References

  1. ↑ 1.00 1.01 1.02 1.03 1.04 1.05 1.06 1.07 1.08 1.09 1.10 1.11 "Public Health Image Library (PHIL)".


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