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Molecular Diagnosis
Microscopic examination is still considered the “gold standard” for the diagnosis of parasitic diseases. If an unequivocal identification of the parasite can not be made, the stool specimen can be analyzed using molecular techniques such as polymerase chain reaction (PCR). PCR amplified fragments can be analyzed by using restriction fragment length polymorphisms (RFLP) or DNA sequencing if further characterization is needed.
Specimen
Collection
If PCR is being requested on a stool specimen, the specimen must be
collected in absence of preservatives kept and shipped either refrigerated
(4°C) or frozen (shipped with dry ice). Alternatively stool specimens can
also be mixed in potassium dichromate 2.5% (1:1 dilution) or in
absolute ethanol (1:1 dilution) and shipped refrigerated. Trichrome
stained smears (for G. lamblia or E. histolytica/E. dispar)
or acid-fast smears (for C. parvum or C. cayetanensis)
should accompany the stool specimen when requesting PCR for any of these
protozoa. All stained smears will be read first and if an identification
of the parasite can be made, PCR will not be performed. PCR results take
approximately 24 hours for completion. Click here for more information
about shipping
stool specimens to CDC.
DNA
Extraction
It is
necessary to extract DNA from the stool specimens for PCR detection.
Click to view the DNA extraction protocols recommended for
molecular diagnosis of intestinal parasites.
PCR
Analysis
Molecular detection of
Cryptosporidium sp.,
Cyclospora
cayetanensis,
Entamoeba
histolytica, and
E. dispar
is performed at CDC by both conventional PCR and real-time PCR. Conventional PCR is available for
Giardia lamblia
and
microsporidia. Click on the links above to learn more about the
specific tests and to view analysis of PCR results for the respective parasites listed.
Conventional PCR:
DNA preparations
extracted from fecal samples are tested by PCR with diagnostic primers.
Amplified DNA fragments are electrophoretically resolved on an agarose gel for analysis of results.
Real-Time PCR:
In real-time PCR, the DNA amplification is
monitored by measuring the fluorescence signal generated
in the reaction vessel. The fluorescence signal is measured every cycle and is
proportional to the amount of accumulated PCR product.
The real-time PCR assays for parasite detection at CDC use either the DNA-binding dye SYBR Green or sequence-specific TaqMan probes as fluorescence detection mechanisms. Probe-based assays have the advantages of high specificity and the possibility to detect multiple targets in the same vessel by combining probes labeled with dyes with different fluorescent spectra. Assays using SYBR Green can be easier and less expensive, but caution should be exercised since all double-stranded DNA is detected, including primer-dimers and other PCR artifacts. The specificity of these assays can be improved by including a dissociation (melting) curve analysis. This helps to distinguish the correct product from possible artifacts, thus avoiding false-positive results. See illustrations for details about the fluorescence detection mechanisms using SYBR Green and TaqMan probes.
For additional information on molecular diagnosis using stool specimens, call the Division of Parasitic Diseases at (770) 488-4474.
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