cocci dial
DESCRIPTION
CoccidiaTRANSCRIPT
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LT Mark Simons, Ph.D.
Naval Medical Center, San Diego
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Stool preserved in Zn/PVA and 10% formalin
Zn/PVA prep slide smear, Trichrome stain Look at 40-100x for protozoans
Organisms commonly ID: Giardia lamblia, Entamoeba spp.,
Endolimax nana, Iodamoeba bustschlii, Dientamoeba fragilis,
Balantidium coli, Trichomonas spp.
10% formalin concentrate, Iodine wet mount Look at 10-40x for helminth ova, larvae, and some protozoa
Organisms commonly ID: Hookworm, Taenia spp., Ascaris
lumbricodes, Enterobius vermicularis (pinworm), Trichuris
trichura (whipworm), Capillaria philippiensis,
Diphylobothrium latum, Hymenolepsis spp., Fasciola spp.,
Schistosoma spp., Paragonimus spp., Clonorchis sinensis
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Disease:
Usually asymptomatic or self-limiting diarrhea in
immunocompetent persons: travellers diarrhea
Severe watery diarrhea to prolonged chronic
diarrhea in immunocompromised persons
(children, elderly, HIV)
Small spherical protozoa (1-12mm size)
Small size and poor staining by traditional
Trichrome, requires special staining procedures
Often misidentified as yeasts or debris
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Goodgame. 1996
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Modified Methods to increase detection of
Coccidia/Microsporidia
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Concentrate specimen prior to making slide
(10 minutes at 500 X g)
Make dime-sized spot on slide, air dry
Fix with 100% MetOH, 1 minute air dry Stain with Kinyouns carbol fuchsin, 5 minutes
Rinse with water
Decolorize with 1% sulfuric acid or 0.5% AcOH, 2 minutes
Rinse with water
Counterstain with methylene blue, 1-2 minutes
Rinse with water air dry Look for pink/red coccidia vs. blue background
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Concentrate specimen prior to making slide
(10 minutes at 500 X g)
Make dime-sized spot on slide, air dry
Fix with 100% MetOH, 5-10 minutes air dry Stain with Trichrome (10x concentration), 90 minutes
Rinse in AcOH,
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Concentrate specimen prior to making slide
(10 minutes at 500 X g)
Make dime-sized spot on slide, air dry
Fix with 100% MetOH, 5-10 minutes air dry Stain with Kinyouns carbol fuchsin, 10 minutes
Rinse with water
Decolorize with 0.5% AcOH rinse with water Stain with Trichrome (10x concentration), 30 minutes
**staining at 37oC increases staining of spores
Rinse AcOH < 10s Dip 95% EtOH 95% EtOH for 30s air dry
Look for pink coccidia w/ pink stripe (polar tubule)
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Only available for Cryptosporidium/Giardia
Requires fluorescent microscope
Very sensitive and easy to use
More expensive than traditional methods
Being developed for other coccidian protozoans
Giardia
Cryptosporidium
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Many different kits avaiable for Giardia,
Cryptosporidium, Entamoeba
Very good sensitivity and specificity for the
target organism
No Ag detection for other intestinal coccidia
More expensive than traditional stains
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Characteristics and Identification
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C. parvum and C. hominis
Fecal-oral transmission, low
inoculum to cause disease
Associated with water (pools),
day care, and HAI
Watery diarrhea
1-2 weeks in healthy persons
Chronic, life-threatening in
AIDS
Autoinfective cycle allows high
organism load w/o repeated
ingestion/exposure
TMP-SMX Tx of choice
Size 4-6 mm, seen using MAF
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MAF
MAF
Trichrome
Yeast
Crypto
DFA
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Worldwide distribution
Causes flu-like symptoms,
nausea/vomiting, explosive
diarrhea, and weight loss Associated with water and fresh
produce (raspberries, basil, lettuce)
Usually self-limiting in 3-4 days for
immunocompetent, may relapse for
4-7 weeks
Severe and persistent disease (12
weeks) in AIDS
TMP-SMX Tx of choice
Size 8-12mm, seen by MAF or
modified Trichrome
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Trichrome
MAF MAF
MAF
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Food, water contamination with
fecal sources
Malabsorptive diarrhea, watery
and profuse
May last for weeks to months to
even years depending on immune
status
More severe in infants/kids
Tx w/ TMP-SMX
Large size (20-30 mm), best seen
using wet mounts w/ iodine
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Iodine Wet Mount
MAF
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Closely related to fungi, may be
reclassified
Very small (1-2mm) and faintly
staining = difficult detection
7 genera infective to humans:
Encephalitozoon, Enterocytozoon,
Nosema most common
Associated with chronic diarrhea,
increased recognition with AIDS
resulting in mortality Associated with Cryptosporidium in
AIDS (30%)
Tx with various antiparasitics (i.e.
albendazole, metronidazole, etc).
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Joseph. 2006 DPDx
Calcofluor White Acridine Orange
Gram Stain Trichrome EM
MAF
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Joseph. 2006
Trichrome
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Garcia, LS. 2007. Diagnostic Medical Parasitology, 5th
ed. ASM Press; Washington, DC.
Garcia, LS. 2002. Laboratory Identification of the
Microspordia. J Clin Micro 40:1892.
Goodgame, RW. 1996. Understanding Intestinal Spore-
forming Protozoa: Cryptosporidia, Microsporidia,
Isospora, and Cyclospora. Ann Intern Med 124:429.
Joseph J, et al. 2006. Histopathological evaluation of
ocular microspordiosis by different stains. BMC Clin
Path 6:1.
Centers for Disease Control. DPDx online: Laboratory
Identification of Parasites of Public Health Concern.
www.dpd.cdc.gov/dpdx