Cyclospora
How to identify C. cayetanensis under the microscope
I
n wet mounts containing
little debris, Cyclospora
cayetanensis oocysts are seen
as non-refractile spheres;
however, it can be very
difficult to identify these
structures in a wet mount
unless the oocyst load is
heavy.
By Lynne Garcia
Lynne Garcia is currently the
Director of LSG & Associates
(consultation in Diagnostic
Medical Parasitology/Health
Care Management).
Lynne has presented at over
350 national and international
events.
Her works include over 150
publications, including:
Diagnostic Medical
Parasitology, 5th ed., 2007,
ASM Press.
Practical Guide to Diagnostic
Parasitology, 1999, ASM Press.
Editor-in-Chief, Clinical
Microbiology Procedures
Handbook
Editor-in-Chief of Clinical
Laboratory Management in the
st
21 Century, 2004, ASM Press
Reviewer: 9 journals
Fellow of the American
Academy of Microbiology (AAM)
The oocysts are acid-fast
variable with one of the
modified acid-fast stains;
those that are unstained appear
as glassy, wrinkled spheres.
Modified acid-fast stains stain
the oocysts light pink to deep
red/purple, and some will
contain granules or have a
bubbly appearance. However,
the oocysts are not yet
sporulated when passed, so
they will not contain any
sporozoites like those often
seen in Cryptosporidium
oocysts.
Remember that the acid
decolorizer should be 1-3%,
with many laboratories using
1% only. This will provide
good staining of Cyclospora,
Cryptosporidium, and
Cystoisospora. Often, if the
acid decolorizer is too strong,
the color will be removed
from the Cyclospora oocysts.
It is very important to be
aware of these organisms
when one of the modified
acid-fast stains is examined,
because Cryptosporidium spp.
and other similar but larger
structures (approximately
twice the size of
Cryptosporidium oocysts)
may be seen in the stained
smear.
Figure 1: The large pink/red
and clear structures are
Cyclospora (8-10 µm), middle
size pink/red structure is
Cryptosporidium (4-6 µm), and
the small pink object is an
artifact (1.5-2 µm).
It is important for laboratories
to measure all modified
acid-fast oocysts, particularly
if they appear to be somewhat
larger than those of
Cryptosporidium.
Cryptosporidium will measure
approximately 4-6 microns,
while those of Cyclospora will
measure 8-10 microns. In
many of these smears, there
may also be artifacts that stain
the same pink to red color, but
measure only 1.5-2 microns
(see Fig: 1).
The oocysts will autofluoresce
green (450 to 490 DM
excitation filter) or blue (365
DM excitation filter) under
UV epifluorescence.
However, if you currently
perform the combo
Giardia/Cryptosporidium FA
tests, you will be used to
seeing fluorescence at 3-4+.
Cyclospora will usually
autofluoresce at a lower
intensity (usually around 13+).
It is strongly recommended
that during concentration
(formalin-ethyl acetate) of
stool specimens, the
centrifugation be carried out
for 10 min at 500  g. The
sediment can then be stained
using one of the modified
acid-fast stains or the hot
safranin stain.
Normally, these diagnostic
procedures for Cyclospora are
performed on request only;
however, in a potential
outbreak situation,
laboratories may decide to test
for the organism, particularly
Figure 2: This photomicrograph of a fresh stool
sample, which had been prepared using a 10%
formalin solution, and stained with safranin,
revealed the presence of three uniformly
stained Cyclospora cayetanensis oocysts in the
field of view. Photo from CDC/ DPDx - Melanie
Moser.
if the number of positives has
been documented to be higher
than normal.
In some specialized settings,
molecular testing may be
used; however, these are not
available in most routine
clinical laboratories.
Multiplex PCR has also been
documented to detect
Cyclospora, Cystoisospora,
Encephalitozoon intestinalis,
and Enterocytozoon bieneusi
in stool specimens. (Taniuchi,
M, et al, 2011, Diagn
Microbiol Infect Dis 71:366390).
Lynne S. Garcia, M.S.,
MT(ASCP), CLS(NCA),
BLM(AAB), F(AAM)
Figure 3: This photomicrograph of a fresh stool
sample, which had been prepared using a 10%
formalin solution, and stained with modified
acid-fast stain, revealed the presence of four
Cyclospora cayetanensis oocysts in the field of
view. Compared to wet mount preparations,
the oocysts are less perfectly round and have a
wrinkled appearance due to this method of
fixation. Most importantly, the staining is
variable among the four oocysts. Photo from
CDC/ DPDx - Melanie Moser.
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