parasito intro

7/31/2019 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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