Upload
lucas-tobing
View
236
Download
0
Embed Size (px)
DESCRIPTION
Coccidia
Citation preview
LT Mark Simons, Ph.D.
Naval Medical Center, San Diego
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
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
Goodgame. 1996
Modified Methods to increase detection of
Coccidia/Microsporidia
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
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,
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)
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
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
Characteristics and Identification
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
MAF
MAF
Trichrome
Yeast
Crypto
DFA
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
Trichrome
MAF MAF
MAF
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
Iodine Wet Mount
MAF
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).
Joseph. 2006 DPDx
Calcofluor White Acridine Orange
Gram Stain Trichrome EM
MAF
Joseph. 2006
Trichrome
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