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