Parasito Intro
Transcript of Parasito Intro
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Diagnosis of Protozoan
infections
Laboratory methods
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Observation of several
parasite life cycle stages
Trophozoites trope, nourishment + zoon, animal
Cystsresting or dormant stage
Oocyststhick-walled spore phase of certainparasites
Sporesstructure that is adapted for dispersaland surviving for extended periods of time in
unfavorable conditions
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Processing blood specimens
Thick and thin blood smears-Fresh whole blood
- blood + EDTA
Low parasitemiaIdentification of characteristic features
-Stained with:Giemsa-MeOHWrightWright-GiemsaCDC recommends:3% Giemsa sol.(pH7.2) 30-45 min.
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microscopic examination
1. screen the entire smear at 10X or 20X todetect large parasites such as microfilaria
2. examine the smear using the 100X
3. Select a well-stained area with 10-20WBCs/field
4. if you see parasites, make a tentativespecies determination on the thick smear
5. examine the thin smear to determine thespecies present
6. NPF : "No Parasites Found" (at least 300
fields for malaria diagnosis)
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Quantifying parasites
1. Carefully examine the smear at 100X
and identify species
2. count the parasitized RBCs among
500-2,000 RBCs
3. express the results as % parasitemia=
#parasitized RBCs/total RBCsat 100X
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Stool specimensO&P examination
can be examined fresh or preserved
Wet Mount preparation of fresh stools:
for observation of motile trophozoites
should be examined after 30 min of passage
Take a small amount of the specimen (not fixed) andplace it on a microscope slide. If the stool specimen isstill somewhat solid, add a drop or two of saline to the
specimen and mix Systematically scan the entire
coverslip area using the 10X objective
a higher magnification may be necessary
if something is identified
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(Polyvinyl alcohol)
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Concentration procedure
Flotation technique or formalin-ethyl acetatetechnique organisms rise to the top and the debris sinks to
the bottom
ADVANTAGE: produce a cleaner material thanthe sedimentation technique
DISADVANTAGE: walls of eggs and cysts willoften collapse, and some parasite eggs do not
float Sedimentation techniques
using Zinc sulfate or Sheather's sugar
organisms are concentrated in the sediment
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Staining ProcedureTrichrome staining Most commonly used. is a three-color staining
protocol used in histology. Uses iron hematoxylinand three differentsolutionsChromotrope modifiedtrichrome staining
USE: differentiates microsporidia spores
Modified Acid-Fast (does not require heating)
USE: the identification of oocysts of the coccidian species difficult todetect with routine stains such as trichrome
Quick-Hot Gram-Chromotrope alternative stain to the chromotropeprocedure
USE: demonstrates microsporidian spores in fecal and other clinicalspecimens.
Modified Safranin Technique (Hot Method)USE: produces uniformly well-stained smears of the intestinal
protozoa, human cells, yeast, and artifact material.Calcofluor White chemofluorescent technique used only as a
screening tool (sensitive but nonspecific)
USE: detection of microsporidia,Acanthamoeba spp., Pneumocystisjiroveci, and Dirofilaria spp.
http://www.dpd.cdc.gov/dpdx/HTML/DiagnosticProcedures.htm
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Calibration of microscopes usingan ocular micrometer
Important for size determination oftrophozoites, cysts, oocysts, and helmintheggs and larvae of Protozoan
ocular micrometer disk is installed in one ofthe oculars and a stage micrometer is usedfor calibrating the ocular micrometer
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GU specimens
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Aspirates Duodenal aspirates useful for Giardia lamblia or Strongyloides
stercoralis larvae.
examine unfixed specimens sediment in wet mount within 1 to 2 hours
after collection or preserved in 10% formalin.
Sigmoidoscopy material and abscesses of the liver and lung
for amebic trophozoites.
examined immediately in saline wet mount preparation or fix in PVA.
fixed material can be stained using trichrome stain and examined for
trophozoites ofEntamoeba histolytica.
Lymph node material, bone marrow, and spleen for motile
trophozoites ofTrypanosoma bruceigambiense or Trypanosoma
brucei rhodesiense. For Leishmania donovaniinfections, to see amastigote stages
Fixed smears can be stained with Giemsa stain.
Skin ulcers may demonstrate the amastigote stages in cutaneous
and mucocutaneous leishmaniasis.
Fixed smears can be stained with Giemsa stain.
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CSF
CSF sediment stained with:
Giemsa for trypanosomes or trypomastigotes,
amebas and microsporidia Trichrome, chromotrope, or calcofluor for
amebas
Acid-fast stain for calcofluor, or modified
trichome for microsporidia Culture of amebas such as NaegleriaandAcanthamoebassp.on agar with E.coli
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Immunodiagnostics
Organism Type of TestbCryptosporidium spp. EIACryptosporidium spp.Giardia lamblia EIA
IFACryptosporidium spp.
Giardia lamblia
Entamoeba histolytica/disparDFA
Entamoeba histolytica DFAEIAIFA
Entamoeba histolytica
E. dispar RapidGiardia lamblia RapidWuchereria bancrofti EIA
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Molecular methods
PCR SpecimenCollection
stool specimen must becollected in absence of
preservatives kept andshipped eitherrefrigerated (4C) orfrozen (shipped with dryice)
can also be mixed inpotassium dichromate2.5% (1:1 dilution) or inabsolute ethanol (1:1dilution) and shippedrefrigerated.
Agarose gel (2%) analysis of a PCR diagnostictest for detection ofCryptosporidium parvumDNA. PCR was performed using primersCPBDIAGF and CPBDIAGR.1 Lane S: Molecularbase pair standard (100-bp ladder). Blackarrows show the size of standard bands. Lane1:C. parvum positive fecal specimen. The red
arrow shows the diagnostic band of 435 bp forzoonotic Cryptosporidium parvum.
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