ORGANISM IDENTIFICATION & DIAGNOSTIC

Protozoa

What is the most effective technique for the identification of the intestinal protozoa? 

Although some protozoan cysts can be seen and identified on the wet preparation smear (direct mount, concentration sediment wet mount), the permanent stained smear is the procedure that is recommended as the most relevant and accurate for the identification of the intestinal protozoa.  These preparations are examined using the oil immersion objective (100X) for a total magnification of X1000.  At least 300 oil immersion fields should be examined prior to reporting the permanent stained smear result.

Are trophozoites ever seen in the wet mounts of stool? 

Usually the trophozoites are not seen in the concentration sediment wet mount preparations unless they are prepared from SAF fixed material.  Motile trophozoites can occasionally be seen in the direct wet smear, but the number of times this occurs is rare.

What are some of the tips to consider when reporting Entamoeba hartmanni?

The measurements of 10 microns or less for the Entamoeba hartmanni cyst refer to wet preparation measurements, so the measurement should be decreased ~1 micron on the permanent stained smear.  When you see a cyst on the permanent stained smear, there is often a halo representing shrinkage.  The cyst needs to be measured to include that halo.  On the bench, the measurements for this cyst generally run from around 9.5 down to about 8 microns and they are morphologically definitely E. hartmanni.  The E. hartmanni cyst generally contains more chromatoidal bars than are seen in E. histolytica/E. dispar.  Also the E. histolytica/E. dispar cysts tend to measure routinely on the bench (trichrome slides) from about 10.5 up to about 13 or so.  Also, on the bench, the E. hartmanni nuclei, particularly in the troph, will tend to look like a "bulls eye" - very sharp line of nuclear chromatin with the karyosome right in the middle.

Do nonpathogenic protozoa ever cause symptoms?

Endolimax nana, Iodamoeba bütschlii, Chilomastix mesnili, and Pentatrichomonas hominis (as examples) have been categorized as nonpathogens.  Although rare, patients have been documented who have been symptomatic with a nonpathogen. However, it is sometimes difficult to determine from the case history the extent of the workup, including coccidia and the microsporidia.  Before I would assign symptoms to nonpathogenic protozoa, I would certainly look for other proven pathogens.

What color is the autofluorescence seen with Cyclospora cayetanensis?

The color depends on the particular FA filters used.  If the filters are used for Calcofluor white, the oocysts will appear as pale blue rings; if the filters are used for FITC, the oocysts will appear to be more yellow-green.  Fluorescence intensity will vary from about 1+ to 2+; it is rare to see stronger autofluorescence with these oocysts.

Helminths

Why can’t all helminth eggs be recovered using the flotation concentration rather than the sedimentation concentration?

Some helminth eggs are quite heavy and will not float, even using zinc sulfate with a specific gravity of 1.20.  Other helminth eggs are operculated; when the egg is placed in a high specific gravity solution, the operculum “pop” open and the egg fills with fluid and sinks to the bottom of the tube.

Why do helminth larvae need to be identified to the species level; shouldn’t all larvae recovered in stool be that of Strongyloides stercoralis?

Although helminth larvae in stool are normally Strongyloides stercoralis, there is always the possibility that in fresh stool hookworm eggs have continued to mature and may hatch before the stool is processed and/or placed in fixatives.  It is important to make sure that the larvae seen are, in fact, the rhabditiform (non-infectious) larvae of S. stercoralis rather than larvae of hookworm.  The agar plate culture is the most sensitive method for the recovery of S. stercoralis larvae.  Also, remember that migrating larvae could also be recovered from respiratory specimens (sputum, BAL, etc.).

Are there any specific recommendations for the detection of schistosome eggs?

When trying to diagnose schistosomiasis, regardless of the species suspected you should be examining both stool (several different stool specimens) and urine (spot urines plus 24 hour specimen) (collected with no preservatives).  Occasionally adult worms get into blood vessels where they aren't normally found (Example:  S. mansoni eggs found in urine only).  When you perform a sedimentation concentration, use saline to prevent premature egg hatching.  Once you are ready to try a hatching procedure, then you can put the sediment into spring water (dechlorinated water) to stimulate hatching.  When examining wet mounts under the microscope, you need to look for the movement of cilia on the larvae within the egg shell (thus the need to collect the specimens with no preservatives).  You want to be able to tell the physician whether the eggs, if present, are viable or all you see are dead egg shells.  If you suspect schistosomiasis, I would recommend looking at a number of wet mounts, particularly if you aren't going to perform a hatching test.

What is the most sensitive test for the diagnosis of strongyloidiasis?

Agar plate cultures are recommended for the recovery of S. stercoralis larvae and tend to be more sensitive than some of the other diagnostic methods.  Stool is placed onto agar plates and the plates are sealed to prevent accidental infections and held for two days at room temperature.  As the larvae crawl over the agar, they carry bacteria with them, thus creating visible tracks over the agar.  The plates are examined under the microscope for confirmation of larvae, the surface of the agar is then washed with 10% formalin, and final confirmation of larval identification is made via wet examination of the sediment from the formalin washings.

In a study looking at the prevalence of S. stercoralis in three areas of Brazil, the diagnostic efficacy of the agar plate culture method (included in this chapter) was as high as 93.9% compared to only 28.5% and 26.5% by the Harada-Mori filter paper culture and fecal concentration methods, when fecal specimens were processed using all three methods.  Among the 49 positive samples, about 60% were confirmed as positive only using the agar plate method.  These results indicate that the agar plate approach is probably a much more sensitive diagnostic method and is recommended for the diagnosis of strongyloidiasis.

It is important to remember that more than half of S. stercoralis-infected individuals tend to have low-level infections.  The agar plate method continues to be documented as a more sensitive method that the usual direct smear or fecal concentration methods.  Daily search for furrows on agar plates for up to 6 consecutive days results in increased sensitivity for diagnosis of both S. stercoralis and hookworm infections.  Also, a careful search for S. stercoralis should be made in all patients with comparable clinical findings before deciding on a diagnosis of idiopathic eosinophilic colitis, because consequent steroid treatment may have a fatal outcome by inducing widespread dissemination of the parasite. 

Where can one get serologies for a Baylisascaris procyonis infection?
Dr. Kevin Kazacos
1243 Veterinary Pathology Bldg
Purdue University
West Lafayette IN 47907-1243
Phone: 765 494-7556
Fax: 765 494-9830
Email: kkazacos@vet.purdue.edu