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, <10 seconds
Dip 95% EtOH
95% EtOH for 5 minutes, repeat
100% EtOH for 10 minutes
Xylene or substitute (Histosol), 10 minutes air dry
Look for pink/red/purple coccidia w/ internal stripe vs.
blue (Ryan) or green (Weber) 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 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

Trichrome
Crypto

Yeast

MAF

MAF

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

MAF

Iodine Wet Mount

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).

Calcofluor White
EM

MAF

Gram Stain

DPDx

Acridine Orange
Trichrome

Joseph. 2006

Trichrome

Joseph. 2006

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 Sporeforming 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