DPDX

BLOOD SAMPLES
Safety
Follow universal precautions for the prevention of bloodborne pathogens when working with human
serum and other body fluids. These include:
Wear personal protective equipment such as safety glasses, gloves, laboratory coats.
If you have cuts or abrasions on the skin of your hands, cover them with adhesive dressing.
Use needles and lancets only once, and dispose of them in a "sharps" container for
decontamination.
Remove gloves and wash your hands after completing any task involving the handling of
biological material.
For more information on safety, visit the website of the Centers for Disease Control and
Prevention to view biosafety guidelines or the website of the Occupational Safety and Health
Administration (OSHA) .
Specimen Collection
Timing:
Whenever possible, specimens should be collected before treatment is initiated. When malaria and
babesiosis are suspected, blood smears should be obtained and examined without delay. Since the
parasitemia may fluctuate, multiple smears might be needed. These can be taken at 8 to 12 hour
intervals for 2 to 3 days.
Microfilariae exhibit a marked periodicity depending on the species involved, therefore the time of
specimen collection is critical. If a filarial infection is suspected, the optimal collection time for
demonstrating microfilariae is:
Loa loamidday (10 AM to 2 PM)

Brugia or Wuchereriaat night, after 8 PM
Mansonellaany time
Onchocercaany time
Type of Sample:
Venous blood samples provide sufficient material for performing a variety of diagnostic tests, including
concentration procedures (filariasis, trypanosomiasis). However, in some parasitic diseases (e.g., for
diagnosis of malaria in particular), anticoagulants in the venous blood specimen can interfere with
parasite morphology and staining characteristics; this problem can be further compounded by
excessive delays prior to making the smears. In such cases, capillary blood samples are preferable. If
PCR is required, please refer to the molecular diagnosis section for appropriate blood collection
procedures.
Capillary blood obtained by fingerstick:
1.
2.
3.
Label pre-cleaned slides (preferably frosted-end) with the patient's name (or other identifier)
and date and time of collection.
Clean the site well with alcohol; allow to dry.
Prick the side of the pulp of the 3rd or 4th finger (alternate sites include ear lobe, or in infants
large toe or heel).
4.
Wipe away the first drop of blood with clean gauze.
5.
Prepare at least 2 thick smears and 2 thin smears.
Venous blood obtained by venipuncture:

1.
2.
3.
4.
Label collection tubes and pre-cleaned slides (preferably frosted-end) with the patient's name
(or other identifier) and date and time of collection.
Clean the site well with alcohol; allow to dry.
Collect the venous blood in a vacuum tube containing anticoagulant (preferably EDTA);
alternatively, collect the blood in a syringe and transfer it to a tube with anticoagulant; mix well.
Prepare at least 2 thick smears and 2 thin smears as soon as possible after collection.
Specimen Processing
Preparing Blood Smears
If you are using venous blood, blood smears should be prepared as soon as possible after collection
(delay can result in changes in parasite morphology and staining characteristics).
Thick smears
Thick smears consist of a thick layer of dehemoglobinized (lysed) red blood cells (RBCs). The blood
elements (including parasites, if any) are more concentrated (app. 30) than in an equal area of a
thin smear. Thus, thick smears allow a more efficient detection of parasites (increased sensitivity).
However, they do not permit an optimal review of parasite morphology. For example, they are often
not adequate for species identification of malaria parasites: if the thick smear is positive for malaria
parasites, the thin smear should be used for species identification.
Prepare at least 2 smears per patient!
1.
Place a small drop of blood in the center of the pre-cleaned, labeled slide.
2.
Using the corner of another slide or an applicator stick, spread the drop in a circular pattern
until it is the size of a dime (1.5 cm2).
3.
A thick smear of proper density is one which, if placed (wet) over newsprint, allows you to
barely read the words.
4.
Lay the slides flat and allow the smears to dry thoroughly (protect from dust and insects!).
Insufficiently dried smears (and/or smears that are too thick) can detach from the slides during
staining. The risk is increased in smears made with anticoagulated blood. At room temperature,
drying can take several hours; 30 minutes is the minimum; in the latter case, handle the smear
very delicately during staining. You can accelerate the drying by using a fan or hair dryer (use cool
setting). Protect thick smears from hot environments to prevent heat-fixing the smear.
5.
Do not fix thick smears with methanol or heat. If there will be a delay in staining smears, dip
the thick smear briefly in water to hemolyse the RBCs.
Scratch Method for Thick smears

The scratch method is an alternate method for making thick films that allows for improved adherence
and faster turnaround times. The process is similar to making a normal thick film, but instead of using
a stick to spread the blood, the edge of a glass microscope slide is used, while applying firm pressure
to create small scratches in the underlying slide. The scratches allow for improved adherence of the
blood film to the slide without affecting the smear morphology. The smear can then be stained as soon
as it is dry, generally within 20-30 minutes of smear preparation.
Reference: Norgan AP, Arguello HE, Sloan LM, Fernholz EC, Pritt BS. Malaria Journal 2013; 12: 231.
Thin smears
Thin smears consist of blood spread in a layer such that the thickness decreases progressively toward
the feathered edge. In the feathered edge, the cells should be in a monolayer, not touching one
another.
Prepare at least 2 smears per patient!
A thin smear being prepared.
1.
2.
3.
Place a small drop of blood on the pre-cleaned, labeled slide, near its frosted end.
Bring another slide at a 30-45 angle up to the drop, allowing the drop to spread along the
contact line of the 2 slides.
Quickly push the upper (spreader) slide toward the unfrosted end of the lower slide.
4.
Make sure that the smears have a good feathered edge. This is achieved by using the correct
amount of blood and spreading technique.
5.
Allow the thin smears to dry. (They dry much faster than the thick smears, and are less
subject to detachment because they will be fixed.)
6.
Fix the smears by dipping them in absolute methanol.
Note: Under field conditions, where slides are scarce, national malaria programs (and CDC staff)
prepare both a thick and a thin smear on the same slide. This works adequately if one makes sure
that of the two smears, only the thin smear is fixed.
Special Procedures for Detecting Microfilariae

Blood microfilariae:
A.
Capillary (fingerstick) blood

Since microfilariae concentrate in the peripheral capillaries, thick and thin smears prepared from
fingerstick blood are recommended.
B.
Anticoagulated (EDTA) venous blood (1 ml) should be concentrated by one of the following
methods:
1.
Centrifugation (Knott's technique)

a.
b.
c.
d.
2.
Prepare 2% formaldehyde (2 ml of 37% formaldehyde + 98 ml H2O).

Mix 9 ml of this 2% formaldehyde with 1 ml of patient's venous blood.
Centrifuge at 500 g for 10 minutes; discard supernatant. Sediment is composed of
WBCs and microfilariae (if present).
Examine as temporary wet mounts.
Prepare thick and thin smears; allow to dry; dip in absolute methanol before
Giemsa staining to enhance staining of microfilariae.
Filtration
a.
Place Millipore or Nucleopore membrane filter (5 m pore) in filter holder

with syringe attachment.
b.
Mix 1 ml of venous blood (in EDTA) with 10 ml of 10% Teepol 610 (Shell
Co.); allow to stand for several minutes to allow lysis; transfer to a 10 ml Luer-Loc
syringe; attach the filter apparatus.
c.
Force the solution through the 5 m pore filter, followed by several syringes of
water to wash out the remaining blood, then 1 or 2 syringes full of air to clear excess fluid.
d.
Prepare a temporary wet mount by removing the filter and placing it on a glass
slide, adding a drop of stain or dye and a coverslip.
e.
For permanent preparations, pass 2 to 3 ml of methanol through the filter

while it is still in the holder; remove filter and dry it on a glass slide; then stain it with
Giemsa stain, horizontally (so that the filter does not wash off the slide); coverslip filter
before examining.
Shipping blood smears for microscopic examination

1.
Place labeled dried blood smears (stained and unstained) in a slide box, with grooves to
separate the slides.
2.
Pack the slide box inside another box, cushioned so that the slides are protected from
breakage.
3.
Include information:
a.
Submitter's name, address and phone number
b.
Physician's name, address and phone number
c.
Patient's name, travel history (places and dates) and treatment information
d.
Specimen collection date
e.
What organism is suspected
4.
Ship via mail or package carrier.
Shipping whole blood for isolation of parasites (culture or animal inoculation),

molecular diagnosis or drug level
1.
Place labeled tube of anticoagulated (EDTA) blood in enough absorbent material to contain any
leakage, and place in a sealed plastic bag or 50 ml screw cap centrifuge tube.
2.
Pack this bag or container in a box, cushioned so that the blood tube is protected from
breakage.
3.
4.
Include information:
a.
Submitter's name, address and phone number
b.
Physician's name, address and phone number
c.
Patient's name, travel history (places and dates) and treatment information
d.
Specimen collection date
e.
What tests are requested and what organisms are suspected
Ship via courier or overnight delivery to permit optimum recovery of parasites; refrigeration
during shipment may be necessary and should be discussed beforehand with the receiving
laboratory.
Staining
Staining Blood Smears
Stain only one set of smears, and leave the duplicates unstained. The latter will prove useful if a
problem occurs during the staining and/or if you wish later to send the smears to a reference
laboratory.
Wright (Wright-Giemsa) stain

Used in hematology, this stain is not optimal for blood parasites. It can be used if rapid results are
needed, but should be followed up when possible with a confirmatory Giemsa stain, so that Schffner's
dots can be demonstrated.
Giemsa stain
Recommended for detection and identification of blood parasites.
1.
Stock 100 Giemsa Buffer - 0.67 M
Na2HPO4
NaH2PO4H2O
Deionized water
2.
Autoclave or filter-sterilize (0.2 m pore). Sterile buffer is stable at room temperature for one
year.
3.
Working Giemsa Buffer - 0.0067M, pH 7.2
Stock Giemsa Buffer

Deionized water
4.
Check pH before use. Should be 7.2. Stable at room temperature for one month.
5.
. Triton X-100 5%
Deionized water (warmed to 56C)

Triton X-100
6.
Prewarm the deionized water and slowly add the Triton X-100, swirling to mix.
7.
Stock Giemsa stain (Giemsa stain is available commercially, but the following formulation
gives more constant results and does not expire)
Glass beads, 3.0 mm

Absolute methanol, acetone-free
Giemsa stain powder (certified)
Glycerol
a.
Put into a 500 ml brown bottle the glass beads and the other ingredients, in the order
listed. Screw cap tightly. Use glassware that is clean and dry.
8.
b.
Place the bottles at an angle on a shaker; shake moderately for 30 to 60 minutes

daily, for at least 14 days.
c.
Kept tightly stoppered and free of moisture, stock Giemsa stain is stable at room
temperature indefinitely (stock stain improves with age).
d.
Just before use, shake the bottle. Filter a small amount of this stock stain through
Whatman #1 filter paper into a test tube. Pipet from this tube to prepare the working Giemsa
stain.
Working Giemsa stain (2.5%): make fresh for each batch of smears
Working Giemsa buffer

Giemsa Stain Stock
5% Triton X-100
Staining
1.
Prepare fresh working Giemsa stain in a staining jar, according to the directions above. (The
40 ml fills adequately a standing Coplin jar; for other size jars, adapt volume but do not change
proportions).
2.
Pour 40 ml of working Giemsa buffer into a second staining jar. Add 2 drops of Triton X-100.
Adapt volume to jar size.
3.
4.
5.
Place slides into the working Giemsa stain (2.5%) for 45-60 minutes.
Remove thin smear slides and rinse by dipping 3-4 times in the Giemsa buffer. Thick smears
should be left in buffer for 5 minutes.
Dry the slides upright in a rack.
Note: As alternates to this 45-60 minutes in 2.5% Giemsa stain, the smears could be stained for
shorter times in more concentrated stains. One alternate is 10 minutes in 10% Giemsa; the shorter
stains yield faster results, but use more stain and might be of less predictable quality.
Staining Procedure: Quality Control

To ensure that proper staining results have been achieved, a positive smear (malaria) should be
included with each new batch of working Giemsa stain. Since good quality control smears are not
available commercially, they may be prepared from a patient's blood and stored for future use in the
following manner:
1.
Choose a patient blood specimen, anticoagulated with EDTA, that has enough parasites so that
at least one is found in every 2 to 3 fields.
2.
Make as many thin smears as possible, preferably within one hour after the blood was drawn
from the patient.
3.
Allow the smears to dry quickly, using a fan or blower at room temperature.
4.
Fix the smears in absolute (100%) methanol; allow them to dry.
5.
Place them, touching front to back, in a box without separating grooves.
6.
Label the outside of the box with the species, date and "Giemsa control slides."
7.
Store at -70C (or colder) if the purpose is to make quality control slides.
8.
Just before use, remove the smear from the box and allow the condensation to evaporate;
label the slide "+ malaria" and the present date. The smear is now ready for staining since it was
previously fixed.
Microscopic Examination
Examining thick smears
Since the erythrocytes (RBCs) have been lysed and the parasites are more concentrated, the thick
smear is useful for screening for parasites and for detecting mixed infections.
1.
First screen the entire smear at a low magnification (10 or 20 objective lens), to detect
large parasites such as microfilaria.
2.
Then examine the smear using the 100 oil immersion objective lens. Select an area that is
well-stained, free of stain precipitate, and well-populated with white blood cells (WBCs) (10-20
WBCs/field).
3.
If you see parasites, make a tentative species determination on the thick smear and then
examine the thin smear to determine the species present. Most often, the thin smear is the
appropriate sample for species identification.
4.
Determination of "No Parasites Found" (NPF): For malaria diagnosis, WHO recommends that at
least 100 fields, each containing approximately 20 WBCs, be screened before calling a thick smear
negative. Assuming an average WBC count of 8,000 per microliter of blood, this gives a threshold
of sensitivity of 4 parasites per microliter of blood. In nonimmune patients, symptomatic malaria
can occur at lower parasite densities, and screening more fields (e.g., 200, 300, or even the whole
smear) might be warranted, depending on the clinical context and the availability of laboratory
personnel and time. NCCLS standards recommend examination of at least 300 fields using the
100 oil immersion objective.
Examining thin smears

Thin smears are useful for species identification of parasites already detected on thick smears,
screening for parasites if adequate thick smears are not available, and a rapid screen while the thick
smear is still drying.
1.
Screen at low magnification (10 or 20 objective lens) if this has not been done on the thick
smears.
2.
Carefully examine the smear using the 100 oil immersion objective lens. NCCLS standards
recommend examination of at least 300 fields using the 100 oil immersion objective.
Quantifying parasites
In some cases (especially malaria) quantification of parasites yields clinically useful information. If this
information is needed by the physician, malaria parasites can be quantified against blood elements
such as RBCs or WBCs.
To quantify malaria parasites against RBCs, count the parasitized RBCs among 500-2,000 RBCs on the
thin smear and express the results as % parasitemia.
% parasitemia = (parasitized RBCs/total RBCs) 100
If the parasitemia is high (e.g., > 10%) examine 500 RBCs; if it is low (e.g., <1%) examine 2,000
RBCs (or more); count asexual blood stage parasites and gametocytes separately. Only the former are
clinically important and gametocytes of P. falciparum can persist after elimination of asexual stages by
drug treatment.
To quantify malaria parasites against WBCs: on the thick smear, tally the parasites against WBCs, until
you have counted 500 parasites or 1,000 WBCs, whichever comes first; express the results as
parasites per microliter of blood, using the WBC count if known, or otherwise assuming 8,000 WBCs
per microliter blood.
Parasites/microliter blood=(parasites/WBCs) WBC count per microliter<or 8,000>
Results in % parasitized RBCs and parasites per microliter blood can be interconverted if the WBC and
RBC counts are known, or otherwise (less desirably) by assuming 8,000 WBCs and 4,000,000 RBCs
per microliter blood.
Detection of blood parasites using fluorescent dyes

Fluorescent dyes that stain nucleic acids have been used in the detection of blood parasites. In
the Kawamoto technique, blood smears on a slide are stained with acridine orange and examined with
either a fluorescence microscope or a light microscope adapted with an interference filter system. This
results in a differential staining of nuclear DNA in green and of cytoplasmic RNA in red, which allows
recognition of the parasites. The method has been applied to malaria parasites (and to a lesser extent,
African trypanosomes).
In the Quantitative Buffy Coat (QBC; Becton Dickinson) method, blood samples are collected in a
special tube containing acridine orange, an anticoagulant, and a float, and then are centrifuged in a
microhematocrit centrifuge. After centrifugation, the tubes are examined using a fluorescence
microscope with a stage adapter, or a light microscope with a customized fluorescence attachment.
Malaria parasites concentrate below the granulocyte layer in the tube. The QBC method is reported to
have a good sensitivity for detection of malaria parasites, and has also been applied (albeit to a lesser
extent) to other parasites such as trypanosomes, microfilaria and Babesia spp.
Molecular Diagnosis
Specimen Collection
Microscopic examination of stained blood smears is considered the gold standard for diagnosis
ofmalaria and babesiosis. When species determination cannot be made by microscopic examination,
analysis by polymerase chain reaction (PCR) is helpful. Collect a 1-5 ml blood sample in Vacutainer
EDTA tubes prior to anti-parasitic therapy and ship at 4C to a reference laboratory. Alternatively,
blood can be collected on filter papers (e.g., products available through
Whatman http://www.whatman.com ). Punching the spots may increase the risk of crosscontamination among specimens. Spot the paper directly from whole blood or finger stick. Follow
allshipment guidelines and requirements. Blood smears should always accompany the EDTA blood
sample. The blood smears will be examined first; PCR will be performed only if species determination
cannot be made from the blood smears.
The following procedure describes how a specimen will be accepted for PCR analysis at CDC. Prior
arrangements should be made to determine the appropriateness of PCR as an adjunct for the
diagnosis of malaria and babesiosis. At this time, PCR analysis takes approximately one week for
completion.
DNA has to be extracted from the blood specimens for PCR detection. Click to view the DNA
extraction protocols recommended for molecular diagnosis of malaria and babesiosis.
Species-specific PCR for Diagnosis of Malaria

Plasmodium sp. genomic DNA is extracted from 200 l whole blood using the QIAamp Blood Kit (Cat.
No. 29106; Qiagen Inc., Chatsworth, CA) or a similar product that can yield the comparable
concentration of genomic DNA from the same volume of blood. Detection and speciation
of Plasmodium is done with a two step nested PCR using the primers of Snounou et al 1993. In the
first step (PCR1), 1 l of extracted DNA is amplified using genus specific primers; in the second step
(PCR2), 1 l of PCR1 amplification product is further amplified using primers specific for
each Plasmodium species. Ten microliters of each PCR2 amplified DNA product is electrophoretically
resolved on a 2% agarose gel, stained for 15 min with ethidium bromide and visualized by UV
illumination for analysis of results.
Species-specific PCR for Diagnosis of Babesia

Babesia sp. genomic DNA is extracted in the same way as Plasmodium sp. DNA (see above). Detection
of Babesia microti is done with a two step nested PCR using the primers of Persing et al. In the first
step (PCR1), 1 l of extracted DNA was amplified using B. microti specific primers, Bab1 and Bab4; in
the second step (PCR2), 1 l of PCR1 amplification product was further amplified using internal
primers, Bab2 and Bab3. Ten microliters of PCR2 amplified DNA product was electrophoretically
resolved on a 2% agarose gel, stained for 15 minutes with ethidium bromide and visualized by UV
illumination for analysis of results.
Extraction of DNA from Blood Specimens

Whole blood
This method is used for whole blood collected in Vacutainer EDTA tubes. Use preferably the QIAamp
Blood Kit (Cat. No. 51106; Qiagen Inc., Valencia, CA, http://www.qiagen.com ). Similar products
that can yield the comparable concentration of genomic DNA from the same volume of blood can be
used if QIAamp is not available.
Note: This protocol is a summary of the procedure included in the QIAamp Blood kit manual provided
by the manufacturer. Please refer to the manual for detailed product information and protocols.
Before you start, be sure to do the following:
a.
Equilibrate samples to room temperature.
b.
Heat one water bath or heat block to 56C.
c.
Ensure that buffer AW1, Buffer AW2, and QIAGEN protease have been prepared according to
the instructions.
d.
All centrifugation steps should be carried out at room temperature.
e.
Use carrier DNA if the sample contains less than 10,000 genome equivalents.
f.
200 l of the whole blood yields 3-12 g of DNA. Preparation of buffy coat is recommended if a
higher yield is required.
Procedure
1.
Pipet 200 l whole blood, 20 l QIAGEN Protease, and 200 l Buffer AL into a 1.5 ml low
binding microcentrifuge tube (e.g., Cat. No. T6050G, Marsh Biomedical Products, Rochester, NY).
Mix by vortexing.
2.
Incubate at 56C for 10 minutes.
3.
Spin down briefly to remove drops from the inside of the tube.
4.
Add 200 l of 96-100% ethanol and mix by vortexing.
5.
Carefully apply the mixture from step above to a QIAamp spin column. Centrifuge 1 minute at
full speed (15,000 g). Discard the tube containing the filtrate. The DNA will be bound to the
filters in the spin columns. Place column in a clean 2 ml collection tube.
6.
Open the QIAamp spin column and add 500 l Buffer AW1 without hitting the rim. Close the
cap and centrifuge 1 minute at full speed. Place QIAamp spin column in a clean 2 ml collection
tube.
7.
Carefully open the QIAamp spin column and add 500 l Buffer AW2. Close the cap and
centrifuge the QIAamp column for 3 minutes at maximum speed.
8a. Discard the 2 ml collection tube containing the filtrate, place the QIAamp spin column in a new
collection tube and spin for 1 minute to remove residual buffer AW2.
8.
Place the spin column in a clean 1.5 ml low binding microcentrifuge tube. Add 200 l Buffer AE
or distilled water to the spin column. Incubate at room temperature for 5 minutes to elute the
DNA. Centrifuge 1 minute at full speed. Use 1l of extracted DNA for a 50l PCR.
9.
Store at 4C.
Extraction from filter containing blood

Use preferably the Schleicher and Schuell IsoCode Stix (Cat. No. 10495015; Schleicher & Schuell,
Keene, NH, now Whatman ). Spot papers directly from whole blood or finger stick. Do not use blood
collected in EDTA to prepare the IsoCode Stix. Refer to the IsoCode Stix brochure provided by the
manufacturer for detailed product information.
1.
2.
Label one 1.5 ml microcentrifuge for each IsoCode Stix to be extracted. Do not add the
IsoCode Stix to the tubes yet! Label one additional tube for water to be used in step 7.
Add 500 l of deionized sterile water to each tube that will receive the IsoCode Stix. Add
enough deionized sterile water in the additional tube to provide 50 l per sample to be extracted.
Place the tubes under ultraviolet light (UV) for 20 minutes. Do not expose the IsoCode Stix
to UV light!
3.
At the end of this incubation, hold the stick with triangle of dried blood over the respective
tube containing water that was exposed to UV. While closing the cover, pull the scored end of the
stick so that the triangle detaches and falls directly into the tube. If the water turns even faintly
pink, the IsoCode Stix was not dry and should be discarded.
4.
Vortex the tubes containing the triangles at full power 3 for at least 5 seconds to wash.
5.
After vortexing, centrifuge the tubes for few seconds and remove the water with a sterile
pipette.
6.
Centrifuge the tubes again for 5 seconds, and pipette off the residual water.
7.
Add 50 l of deionized sterile water that was exposed to UV (from the additional tube prepared
in steps 1 and 2), to each tube, using a new tip for each addition. Completely immerse the triangle
by a brief centrifugation.
8.
Place each tube in a 95C heat block for 30 minutes. Caution should be taken to prevent the
lids from opening during incubation. Lid locks can be used for this purpose.
9.
After incubation, gently tap tube several times (around 20 times) as most of the fluid is on the
tube sides and lid. Spin down tubes for a few seconds to prevent leaking when opening.
10.
Remove the supernatant to a clean siliconized 1.5 ml microcentrifuge tube. For PCR
amplification, use 5 l of the extracted DNA per 50 l volume PCR mix. Alternatively, 2 l of the
extracted DNA per 20 l of PCR mix can be used.
11.
Store extracted DNA at 4C.
Detection of Parasite Antigens

Rapid diagnostic tests for malaria have been developed that employ immunochromatographic methods
based on the detection of malarial antigens present in peripheral blood. Most tests use monoclonal
antibodies and detect particular malarial antigens in blood specimens. Tests have been developed that
detect antigens including the histidine-rich protein II (HRP-II), aldolase, and parasite lactate
dehydrogenase (pLDH). These tests generate results within 15 minutes and do not require skilled
microscopists.
Commercially available kits for HRP-II detect P. falciparum HRP-II only and therefore diagnose onlyP.
falciparum malaria. The HRP-II antigen is synthesized and released by trophozoite and immature
gametocyte stages and persists in peripheral blood. Therefore, HRP-II tests can remain positive for up
to 2 weeks following chemotherapy and parasite clearance, as confirmed by microscopy. These tests
have low sensitivities for detecting infections with low level parasitemias (<100 parasites/l) and
mature gametocytes. In contrast, trained microscopists can diagnose infections with parasitemias as
low as 5-10 parasites/l. The reported specificities of these tests are high (>90%). Early tests
reported false positives due to cross-reactions with rheumatoid factor, but these issues have
reportedly been addressed and corrected.
Parasite lactate dehydrogenase (pLDH) is produced by asexual and sexual stages (gametocytes) of
malaria parasites. Test kits that are currently available detect pLDH from all four species
ofPlasmodium. They can distinguish P. falciparum from the non-falciparum species, but cannot
distinguish between P. malariae, P. ovale, and P. vivax. Tests that detect pLDH do not generate
persistent positive results following chemotherapy, like the HRP-II test.
Isolation of Organisms
The diagnosis of Leishmania spp. is made by microscopic identification of the nonmotile, intracellular
form (amastigote) in stained sections from lesions, and by culture of the motile, extracellular form
(promastigote) on suitable media. Slides should be fixed and stained before they are sent unless
reagents are not available. Serologic tests are also available to detect for anti-leishmanial antibodies;
however, these tests are often not sensitive, particularly for diagnosing cutaneous leishmaniasis, and
do not provide definitive diagnosis.
Special Tests: MQ Testing

Mefloquine is recommended by CDC as a prophylactic against malaria. When individuals who are
presumably on mefloquine prophylaxis exhibit signs of malaria, blood samples are collected and
analyzed for the presence of the drug. The drug is extracted from the blood and the concentration is
determined by high-performance liquid chromatographic methods. Determining the level of mefloquine
in the blood helps assess if the individual was adherent with his/her medication. This procedure is also
useful to determine treatment failure due to a mefloquine resistant form of malaria.
STOOL SPECIMEN
Safety
Laboratorians working with stool specimens face potential risks including ingestion of eggs or cysts,
skin penetration by infective larvae, and infection by nonparasitic agents found in stool and biologic
fluids. These risks can be minimized by adopting universal precautions as well as standard
microbiological laboratory practices (Biosafety Level 2). These include:
Wear protective safety glasses, gloves and laboratory coat when processing specimens.
Use biological safety cabinets as needed.
Do not eat, drink, smoke, apply cosmetics or manipulate contact lenses in work area.
Decontaminate work surface at least once a day and after any spill of potentially infectious
material.
If you use any sharp instruments, dispose of them in a "sharps" container for
decontamination.
Remove gloves and wash your hands after completing any task involving the handling of fecal
material.
Note: These precautions should be taken even with stool specimens that have been fixed in
preservatives because they may still be infectious. For example, fixation in formalin takes days or
weeks to kill some parasite cysts or oocysts that are protected by a thick shell. Eggs of Ascaris
lumbricoides may continue to develop and are infectious even when preserved in formalin.
Prevention to view biosafety guidelines or the website of the Occupational Safety and Health
Administration (OSHA) .
Specimen Collection
1.
Collect the stool in a dry, clean, leakproof container. Make sure no urine, water, soil or other
material gets in the container.
2.
The image on the right demonstrates the distribution of protozoa in relation to stool
consistency and should be taken into consideration when specimens are received.
3.
Fresh stool should be examined, processed, or preserved immediately. An exception is

specimens kept under refrigeration when preservatives are not available; these specimens are
suitable for antigen testing only.
4.
Preserve the specimen as soon as possible. If using a commercial collection kit, follow the
kit's instructions. If kits are not available, the specimen should be divided and stored in two
different preservatives, 10% formalin and PVA (polyvinyl-alcohol), using suitable containers. Add
one volume of the stool specimen to three volumes of the preservative.
5.
Insure that the specimen is mixed well with the preservative. Formed stool needs to be well
broken up.
6.
Insure that the specimen containers are sealed well. Reinforce with parafilm or other suitable
material. Insert the container in a plastic bag.
7.
Certain drugs and compounds will render the stool specimens unsatisfactory for examination.
The specimens should be collected before these substances are administered, or collection must
be delayed until after the effects have passed. Such substances include: antacids, kaolin, mineral
oil and other oily materials, non-absorbable antidiarrheal preparations, barium or bismuth (7-10
days needed for clearance of effects), antimicrobial agents (2-3 weeks), and gallbladder dyes (3
weeks).
8.
Specimen collection may need to be repeated if the first examination is negative. If possible,
three specimens passed at intervals of 2-3 days should be examined.
Preservation of specimens is necessary when stool specimens cannot be examined within the
prescribed time interval. Various preservatives are available (see table), with the two most commonly
used being 10% aqueous formalin and PVA (polyvinyl-alcohol). If molecular detection (PCR) is
required, refer to the molecular diagnosis section to obtain specific information on how to collect,
preserve, and ship the specimens.
Distribution of protozoa in relation to stool consistency
Preservative
10% Formalin
Advantages
All purpose fixative
Easy to prepare
Long shelf life
Good preservation of morphology of

helminth eggs, larvae, protozoan cysts, and
coccidia
Disadvantages
Not suitable for some

permanent smears stained
trichrome
Inadequate preservation
morphology of protozoan
trophozoites
Can interfere with PCR,

especially after extended
Preservative
MIF
merthiolate-iodineformaldehyde)
Advantages
Suitable for concentration procedures and

UV fluorescence microscopy
Suitable for acid-fast, safranin, and

chromotrope stains
Compatible with immunoassay kits and UV

fluorescence microscopy
Components both fix and stain organisms
Easy to prepare
Long shelf life
Useful for field surveys
Suitable for concentration procedures
LV-PVA
(low viscosity polyvinylalcohol)
SAF
(sodium acetate-acetic acidformalin)

protozoan trophozoites and cysts
Easy preparation of permanent smears
stained with such as trichrome (solution both
preserves organisms and makes them
adhere to slides)
Preserved samples remain stable for
several months
Suitable for both concentration procedures

and preparation of permanent stained
smears
Easy to prepare
Long shelf life
Suitable for acid-fast, safranin, and
Disadvantages
fixation time
Not suitable for some

permanent smears stained
trichrome
morphology of protozoan
trophozoites
Iodine interferes with oth

stains and fluorescence
Iodine may cause distort

protozoa
morphology of helminth e
and larvae, coccidia, and
microsporidia
Contains mercuric chlorid
Difficult and expensive to

dispose of
Difficult to prepare in the

laboratory
Not suitable for concentr

procedures
Cannot be used with

immunoassay kits
Not suitable for acid-fast

safranin and chromotrope
Requires additive (e.g.,

albumin-glycerin) for adhe
of specimens to slides
Permanent stains not as

as with PVA or Schaudinn'
fixative
Preservative
Advantages
Disadvantages
chromotrope stains
Compatible with immunoassay kits
Schaudinns Fixative
Modified PVA
copper or zinc
One-Vial Fixatives
(such as Ecofix, Parasafe,
Unifix, Proto-fix, STF, and
others that may be available)

protozoan trophozoites and cysts
Permanent smears can be made and

stained with trichrome
Zinc is preferred over copper
No mercuric chloride
Concentrate and permanent smear can be

made out of one vial
Immunoassays can be done on most
No mercuric chloride
Contains mercuric chlorid
morphology of helminth e
and larvae, coccidia, and
microsporidia
Poor adhesion of liquid o

mucoid specimens to slide
Staining not consistent
Organism morphology m
poor
Copper-morphology of cy
and trophozoites is poor
Zinc-better morphology b
not comparable to LV-PVA
Certain one-vial fixatives

use certain stains
Color difference of stain
Staining not always cons
Sometimes more expens

than formalin and LV-PVA
Easy preparation of permanent stained

smears
Less suitable for concent

procedures
Because 10% formalin and PVA have complementary advantages (see table), it is recommended that
the specimen be divided and preserved in both types of preservatives (add one volume of stool to
three volumes of the preservative). Commercial two-vials kits are available for this purpose.
Preserved specimens can be stored for several months.
Specimen Processing
Stool specimens can be examined fresh or preserved.
Examination of fresh specimens permits the observation of motile trophozoites, but this must be
carried out without delay. Liquid (diarrheic) specimens (which are more likely to contain trophozoites)
should be examined within 30 minutes of passage (not within 30 minutes of arrival in the laboratory!),
and soft specimens (which may contain both trophozoites and cysts) should be examined within one
hour of passage. If delays cannot be avoided, the specimen should be preserved to avoid
disintegration of the trophozoites. Formed specimens (less likely to contain trophozoites) can be kept
for up to one day, with overnight refrigeration if needed, prior to examination.
The flow chart on the right shows how specimens preserved in formalin and PVA are processed and
tested at CDC.
Specimens preserved in formalin can be tested directly (wet mount, immunoassay, chromotrope
stain, UV fluorescence) or can be concentrated prior to further testing.
Concentration procedure separate parasites from fecal debris and increase the chances of detecting
parasitic organisms when these are in small numbers. They are divided into flotation techniques and
sedimentation techniques.
This flow chart shows how specimens preserved in formalin and PVA are processed and tested at CDC.
Flotation techniques (most frequently used: zinc sulfate or Sheather's sugar) use solutions which have
higher specific gravity than the organisms to be floated so that the organisms rise to the top and the
debris sinks to the bottom. The main advantage of this technique is to produce a cleaner material than
the sedimentation technique. The disadvantages of most flotation techniques are that the walls of
eggs and cysts will often collapse, thus hindering identification. Also, some parasite eggs do not float.
Sedimentation techniques use solutions of lower specific gravity than the parasitic organisms, thus
concentrating the latter in the sediment. Sedimentation techniques are recommended for general
diagnostic laboratories because they are easier to perform and less prone to technical errors. The
sedimentation technique used at CDC is the formalin-ethyl acetate technique, a diphasic
sedimentation technique that avoids the problems of flammability of ether, and which can be used with
specimens preserved in formalin, MIF or SAF.
Formalin-Ethyl Acetate Sedimentation Concentration

1.
Mix the specimen well.
2.
Strain 5ml of the fecal suspension (more or less depending on its consistency) through wetted
cheesecloth-type gauze placed over a disposable paper funnel into a 15 ml conical centrifuge tube.
(Conical paper cups with the tips cut off are sufficient).
3.
Add 0.85% saline or 10% formalin through the debris on the gauze to bring the volume in the
centrifuge tube to 15 ml. Distilled water may be used; however, Blastocystis hominis may be
deformed or destroyed.
Centrifuge at 500 g for 10 minutes.
4.
5.
Decant supernatant. Add 10 ml of 10% formalin to the sediment and mix thoroughly with
wooden applicator sticks.
6.
Add 4 ml of ethyl acetate, stopper the tube, and shake vigorously in an inverted position for
30 seconds. Carefully remove the stopper.
7.
8.

Free the plug of debris from the top of the tube by ringing the sides with an applicator stick.
Decant the top layers of supernatant.
9.
10.
Use a cotton-tipped applicator to remove debris from sides of the centrifuge tube.
Add several drops of 10% formalin to resuspend the concentrated specimen. Proceed with
applicable testing.
*Commercial fecal concentration tubes are available that decrease processing time and supplies
needed for concentrating specimens (e.g., Fecal Parasite Concentrator, Evergreen Scientific).
Specimens preserved in PVA are mostly used for permanent staining with trichrome. Prior to
staining, they are processed as follows:
1.
Insure that the specimen is well mixed.
2.
Prepare a smear using 2 to 3 drops of the specimen depending on density.
3.
Heat fix on slide warmer set at 60C for 5 minutes or air dry completely at room temperature.
Slides may be trichrome stained or kept for several months in a protective slide tray or box for future
staining.
Shipment
Submit the specimen to the appropriate city, county or state health department laboratory
(seehttp://www.aphl.org ) for processing and examination. That facility will refer specimens to
CDC if necessary. Click on the following link to see proper labeling and packaging of specimens:
(http://www.cdc.gov/od/ohs/biosfty/bmbl4/b4acf1.htm).
Note: When you ship a specimen to CDC, make sure your package will arrive on a weekday and
willnot arrive at CDC on the weekend or a federal holiday.
Shipment of Unpreserved Specimens

On some occasions, unpreserved stool is requested in order to isolate a known or suspected pathogen
(i.e., culture for microsporidia, PCR testing). In these cases, the specimen must be placed in a clean
container as quickly as possible and kept under refrigeration until necessary arrangements are made
for pick-up and delivery by an overnight courier. The sender must ensure that the specimen remains
cold during transport by using available packing materials such as cold-packs. CDC follows regulations
describing the requirements for proper packaging and shipment of biomedical materials (42 CFR Part
72 - Interstate Shipment of Etiologic Agents) which can be located
at http://www.cdc.gov/od/ohs/biosfty/shipregs.htm.
Shipment of Preserved Specimens

The rules for the shipment of preserved specimens are the same as those for unpreserved specimens
except that they do not need refrigeration.
Packaging
Stool specimens must be packaged so as to avoid leakage. The package should be able to withstand
rough handling and passage through routinely used sorters, conveyer belts, etc. There are two sets of
instructions for packaging, depending on the volume:
Volume not exceeding 50 ml.
1.
The material to be shipped shall be placed in a securely closed, watertight tube, vial, or similar
container that is referred to as the primary container.
2.
The primary container is then placed in a durable, watertight container referred to as the
secondary container.
3.
Several primary containers can be placed in one secondary container as long as the combined
volume does not exceed 50 ml.
4.
Cold packs along with absorbent material must be placed around the primary containers.
There must be enough material to absorb all of the contents of the primary container in case of
leakage. The absorbent material should not be particulate such as sawdust or vermiculite.
5.
Each set of primary and secondary containers is then placed in an outer shipping container
constructed of corrugated fiberboard, cardboard, wood, or other like material.
6.
The shipping container must have a "Etiologic Agents - Biomedical Material" label affixed in a
prominent location.
Volume greater than 50 ml.

Packaging of these larger volumes of material must comply with all of the requirements of the above
but in addition:
1.
Shock absorbent material, in volume at least equal to that of the material used between the
primary and secondary containers, should be placed on all sides around the secondary container
before placing in the outer shipping container.
2.
Single primary containers shall not contain more than 1000 ml; however, two or more primary
containers, whose combined volume does not exceed 1000 ml can be placed in a single secondary
container.
3.
The maximum amount of specimen within one shipping container may not exceed 4000 ml.
Staining Procedures
Modified Acid-Fast Staining Procedure
This technique is useful for the identification of oocysts of the coccidian species
(Cryptosporidium,Cystoisospora, and Cyclospora), which may be difficult to detect with routine stains
such as trichrome. Unlike the Ziehl-Neelsen Modified Acid-Fast Stain, this stain does not require the
heating of reagents for staining.
Specimen:
Concentrated sediment of fresh or formalin-preserved stool may be used. Other types of clinical
specimens such as duodenal fluid, bile, pulmonary samples (induced sputum, bronchial wash,
biopsies) may also be stained.
Reagents:
There are four steps to this procedure requiring the following solutions:
1.
Absolute Methanol
2.
Acid Alcohol: 10 ml Sulfuric Acid + 90 ml Absolute ethanol. Store at room temperature.
3.
Kinyoun's Carbol fuchsin: may be purchased commercially.
4.
3% Malachite green: dissolve 3 g of malachite green in 100 ml of distilled water. Store at room
temperature.
Procedure:
1.
Prepare a smear with 1 to 2 drops of specimen on the slide and dry on a slide warmer at 60C
until dry. Do not make the smears too thick!
2.
Fix with absolute methanol for 30 seconds.
3.
Stain with Kinyoun's carbol fuchsin for one minute. Rinse briefly with distilled water and drain.
4.
Destain with acid alcohol for 2 minutes. Rinse with distilled water and drain.
5.
Counterstain with Malachite green for 2 minutes. Rinse briefly with distilled water and drain.
6.
Dry on a slide warmer at 60C for about 5 minutes. Mount with a coverslip using desired
mounting media.
7.
Examine 200 to 300 fields using 40 or higher objectives. To confirm internal morphology, use
100 oil immersion objective.
Quality Control:
A control slide of Cryptosporidium spp. from a 10% formalin preserved specimen should be included
with each staining run. Cryptosporidium spp. stains a pinkish-red color. The background should stain
uniformly green.
Chromotrope Staining Procedure

This staining method was developed at CDC using various components of the trichrome staining
method to differentiate microsporidia spores from background fecal elements.
Specimen:
Prepare a thin smear using approximately 10 l of 10% formalin fixed stool suspension
(unconcentrated) on a glass slide. Formalin concentrates may also be used but the number of
organisms will be essentially the same as before concentration. Heat fix on a slide warmer at 60C
until completely dry (5-10 minutes).
Reagents:
There are six steps to this procedure requiring the following solutions:
1.
2.
Absolute methanol
Chromotrope Stain:
Chromotrope 2R
Fast green
Phosphotungstic acid
Glacial acetic acid
3.
Mix ingredients and allow to stand for 30 minutes. Then add 100 ml of distilled water. Prepare
fresh for use every month.
4.
Acid alcohol:
90% ethanol
Glacial acetic acid
5.
6.
7.
95% ethanol
100% ethanol
Xylene or xylene substitute
Procedure:
1.
Fix smear in absolute methanol for 5 minutes.
2.
Place in chromotrope stain for 90 minutes.
3.
Destain in acid alcohol for only 1 to 3 seconds.
4.
Rinse in 95% ethanol by dipping.
5.
Place in two changes of 100% ethanol for 3 minutes each.
6.
Place in two changes of xylene or xylene substitute for 10 minutes each.
7.
Drain slide and mount with coverslip using mounting media (e.g., permount). Examine smear
after drying using at least 100 objective oil immersion or higher. Examine at least 200 to 300 oil
immersion fields.
Quality Control:
A control slide of microsporidia from a 10% formalin preserved specimen should be included with each
staining run. Spore walls of microsporidia stain a pinkish-red color and measure about 1 m. Change
all solutions subsequent to chromotrope stain after every 10 slides to obtain proper rinsing and
dehydration. A microscope with good optics is recommended for accuracy. Use at least 100 objective
oil immersion magnification to detect organisms; higher magnifications are better. Because of the
difficulty of identification of these small spores, it is recommended that a second reader confirm a
positive diagnosis.
Quick-Hot Gram-Chromotrope Staining Procedure

This is an alternative stain to the chromotrope procedure that is a fast, reliable, and simple method of
staining smears to demonstrate microsporidian spores in fecal and other clinical specimens.
Specimen:
Prepare a thin smear of the material to be stained (such as feces, urine, sputum, saliva, and cell
culture supernatant) and air dry.
For formalin-fixed, paraffin-embedded tissue sections, deparaffinize as usual, hydrate in a series of
alcohols, and bring the slides to water before performing the Gram's stain.
Reagents:
1.
2.
Gram Stain Kit

Chromotrope Stain:
Chromotrope 2R
Fast green
Phosphotungstic acid
Glacial acetic acid
3.
Mix dry ingredients then add acetic acid. Let stand for 30 minutes and then add 100 ml
distilled water. Prepare fresh for use every month.
4.
Acid alcohol:
90% ethanol
Glacial acetic acid
5.
6.
95% ethanol
100% ethanol
Note: In addition to the listed reagents, a method for heating reagents and maintaining a specific
temperature is required. Since only the chromotrope stain needs to be warmed, a hot plate will
suffice.
Procedure:
1.
Heat-fix smear (3 times for 1 second each over a low flame or 5 minutes on a slide warmer set
at 60C). Cool to room temperature.
2.
Perform Gram's stain omitting the safranin step as follows:

a.
b.
Flood slides into gentian violet solution and let stand for 30 seconds. For tissue
sections, extend time to 1 minute.
Rinse off excess stain gently with water.
c.
Flood slides into Gram's iodine solution and allow to remain on the slide for 30
seconds. For tissue sections, extend time to 1 minute.
d.
Remove Gram's iodine solution by gently rinsing with decolorizer solution. Hold the
slide at an angle and add the decolorizer solution dropwise until it flows off the slide colorless.
Take extra care during this step to achieve correct staining of spores.
e.
3.
Wash the slide gently with cold water to remove excess decolorizer solution.
Perform chromotrope stain as follows:
a.
Place the slide in warmed (50 to 55C) chromotrope stain for at least 1 minute. For
tissue sections, extend time 30 seconds.
b.
Rinse in 90% acid-alcohol for 1 to 3 seconds. Take extra care during this step to
achieve correct staining of spores.
c.
d.
Rinse in 95% ethanol for 30 seconds.

Rinse twice, 30 seconds each time, in 100% ethanol (two separate containers are
required for this step). Let dry then mount with Cytoseal60 (Stephens Scientific) or other
suitable sealer, following instructions. For tissue sections, we recommend that the slides be
washed briefly in a solution of 50% ethyl alcohol/50% xylene for 15 seconds, before
mounting.
Quality Control:
A control slide of microsporidia spores from a 10% formalin-preserved specimen should be included
for each staining run.
In fecal samples, microsporidia spores should appear as dark staining violet ovoid structures against a
pale green background. Yeast cells, if present, should stain either dark violet or pinkish-red and are
easily differentiated from microsporidia spores.
In cytologic preparations, microsporidia spores should stain deep violet to pink violet and may contain
gram-positive granules. Occasionally, spores will demonstrate a prominent equatorial belt-like stripe.
Reference:
Moura H, Schwartz DA, Bornay-Llinares F, et al 1997. A new and Improved "Quick-Hot GramChromotrope" Technique That Differentially Stains Microsporidian Spores in Clinical Samples, Including
Paraffin-Embedded Tissue Sections. Arch Pathol Lab Med.121:888-893.
Modified Safranin Technique (Hot Method) Staining Procedure

For Cyclospora, Cryptosporidia, and Cystoisospora species:
Oocysts of Cyclospora in clinical specimens are routinely demonstrated using modified acid-fast stain
(cold). However, with that technique, the oocysts stain variably from nonstaining to full staining
leading to possible misidentification. The modified safranin technique produces a more uniform
staining of these oocysts. The stain needs to be heated to boiling using either a hot plate or
microwave.
Specimen:
Concentrated sediment of fresh or formalin-preserved stool may be used. Other types of clinical
specimens such as duodenal fluid may also be stained.
Reagents:
There are three steps to this procedure, requiring the following solutions:
1.
2.
3.
Acid Alcohol (3%HCL/Methanol): slowly add 3 ml of hydrochloric acid to 97 ml of absolute

methanol. Store at room temperature in a tightly closed container.
Safranin stain (Available in Gram stain kits)
3% Malachite Green: dissolve 3 g of malachite green in 100 ml of distilled water. Store at
room temperature.
Procedure:
1.
Prepare a thin smear on the slide and dry on slide warmer.
2.
Fix in acid alcohol for 5 minutes.
3.
Rinse with distilled water.
4.
Place in boiling safranin for 1 minute.
5.
Rinse with distilled water.
6.
Counterstain with malachite green for 1 minute.
7.
Rinse briefly with distilled water.
8.
Dry the slide and mount with a coverslip using desired mounting media.
Quality Control:
A control slide of Cyclospora spp. from a 10% formalin preserved specimen should be included with
each staining run. Cyclospora spp. oocysts will stain reddish-orange. The background should stain
uniformly green.
Trichrome Staining Procedure

It is generally recognized that stained fecal films are the single most productive means of stool
examination for intestinal protozoa. The permanent stained smear facilitates detection and
identification of cysts and trophozoites and affords a permanent record of the protozoa encountered.
Small protozoa, missed by wet mount examinations (of either unconcentrated or concentrated
samples) are often seen on the stained smear. The Wheatley Trichrome technique for fecal specimens
is a modification of Gomori's original staining procedure for tissue. It is a rapid, simple procedure,
which produces uniformly well-stained smears of the intestinal protozoa, human cells, yeast, and
artifact material.
Specimen:
The specimens usually consist of fresh stool or stool fixed in polyvinyl alcohol (PVA) smeared on
microscope slides and allowed to air dry or dry on a slide warmer at 60C. Stool preserved in sodium
acetate-acetic acid-formalin (SAF) or some of the one-vial fixatives can also be used.
Reagents:
There are seven steps to this procedure, requiring the following solutions:
1.
70% Ethanol plus iodine: prepare a stock solution by adding iodine crystals to 70% alcohol
until you obtain a dark solution. To use, dilute the stock with 70% alcohol until a dark reddish
brown color or strong tea color is obtained.
2.
70% Ethanol (twice)
3.
Trichrome Stain: may be purchased commercially
4.
90% Acid Ethanol
90% ethanol
Acetic acid (glacial)
5.
95% ethanol
6.
100% ethanol (twice)
7.
Xylene or xylene substitute (twice)
Procedure:
1.
For PVA smears, place the slide in 70% ethanol plus iodine for 10 minutes. For other fixatives,
follow the manufacturer's instructions. Omit the iodine step for preservatives that do not contain
mercuric chloride.
2.
Place slide in 70% Ethanol for 5 minutes.
3.
Place in second 70% Ethanol for 3 minutes
4.
Place in Trichrome stain for 10 minutes.
5.
Destain in 90% ethanol plus acetic acid for 1 to 3 seconds.
6.
Rinse several times in 100% ethanol.
7.
Place in two changes of 100% ethanol for 3 minutes each.
8.
Place in two changes of xylene or xylene substitute for 10 minutes.
9.
Mount with coverslip using mounting medium (e.g., permount).
10.
Examine the smear microscopically utilizing the 100 objective. Examine at least 200 to 300
oil immersion fields.
Quality Control:
A control slide of a known protozoan such as Giardia spp. from a PVA preserved specimen should be
included with each staining run. When the smear is thoroughly fixed and the stain is performed
correctly, the cytoplasm of protozoan trophozoites will have a blue green color sometimes with a tinge
of purple. Cysts tend to be slightly more purple. Nuclei and inclusions (chromatoid bodies, red blood
cells, bacteria) and Charcot-Leyden crystals have a red color sometimes tinged with purple. Glycogen
is dissolved by the solvents and appears as a clear area in the organism. The background material
usually stains green providing a nice color contrast with the protozoa.
Calcofluor White Staining Procedure

This chemofluorescent technique is useful for the detection of
microsporidia, Acanthamoeba spp., Pneumocystis jiroveci, and Dirofilaria spp. Chemofluorescent
agents, such as Calcofluor, Fungi-Fluor or Uvitex 2B, also known as optical brightening agents, are
rapid and inexpensive screening agents. These reagents are sensitive but nonspecific since many
objects and organisms other than parasites may fluoresce. This test should be used as a quick
screening tool and not for species identification.
Specimen:
Prepare a thin smear using approximately 10 l of fresh or preserved specimens on a glass slide.
Specimens may include stool, urine, culture or other types of samples. Heat fix on a slide warmer at
60C until completely dry (5-10 minutes).
Reagents
There are two steps to this procedure requiring the following solutions:
1.
Absolute methanol
2.
0.01% Calcofluor white reagent: Prepare a 0.01% solution in 0.1M Tris-buffered saline, pH 7.2
Procedure
1.
Prepare a thin smear of fecal, culture, or other sample material.
2.
Fix the smear in methanol for 30 seconds.
3.
Stain with 0.01% calcofluor white reagent for 1 minute.
4.
Rinse with distilled water and let the smear dry.
5.
Mount with a #1 thickness cover slip.
6.
Examine with an UV fluorescence microscope equipped with a blue violet filter cube with a
wavelength of 400 nm or less.
7.
To screen for microsporidia, examine the smear with a 50 or 100 oil immersion objective.
The microsporidia spores will fluoresce a brilliant blue-white color on a black background.
Quality Control
A control slide of microsporidia preserved in 10% formalin prepared from culture or from a stool
specimen should be included with each staining run.
Microscopic Examination
Calibration of Microscopes Using an Ocular Micrometer:
A correctly calibrated microscope is crucial because size is an important characteristic for identification
of parasites. This section assumes that an ocular micrometer disk has been installed in one of the
oculars and that a stage micrometer is available for calibrating the ocular micrometer. This calibration
should be done for each of the microscope's objectives.
Place the stage micrometer on the microscope stage and focus on the micrometer scale, until you can
distinguish between the large (0.1 mm) and the small (0.01 mm) divisions of the scale. Adjust the
stage micrometer so that the "0" line on the ocular micrometer is superimposed with the "0" line on
the stage micrometer. Without changing the stage adjustment, find a point as distant as possible from
the two superimposed "0" lines where two other lines are also exactly superimposed. Determine the
number of ocular micrometer spaces, as well as the number of millimeters on the stage micrometer,
between the two points of superimposition.
For example: Suppose 48 ocular micrometer spaces (units) equal 0.6 mm. Calculate the number of
mm/ocular micrometer space.
0.6 mm x 48 ocular micrometer spaces = 0.0125 mm/ocular micrometer space
Since most measurements of microorganisms are given in m rather than mm, the value calculated
above must be converted to m by multiplying it by 1000 m/mm.
For example:
0.125 mm ocular space 1000 m/mm = 12.5 m/ocular micrometer space
Thus in this case, 1 ocular micrometer space (unit) is the equivalent of 12.5 m.
Follow the above steps for each objective. Calibration readings should be posted on each microscope
and the microscope should be recalibrated after every cleaning or changing of objectives or oculars.
Wet Mount Preparation:
Figure A
Figure B
Figure C
Before preparing a wet mount slide, the microscope should be calibrated. The objectives and oculars
used for the calibration procedure should be used for all measurements on the microscope. The
calibration factors should always be posted on the side of the microscope.
Protozoan trophozoites, cysts, oocysts, and helminth eggs and larvae may be seen and identified using
a wet mount identification technique. To prepare a wet mount, obtain a microscope slide and the stool
specimen. Take a small amount of the specimen and place it on a microscope slide. If the stool
specimen is still somewhat solid, add a drop or two of saline to the specimen and mix. Ideally, two
smears can be prepared on one slide, of which one can be stained with iodine. Thickness of the wet
mount should be as Figure A on the right illustrates.
If desired the coverslip(s) can be sealed. A preparation of petroleum jelly and paraffin in a 1:1 ratio
can be applied with a cotton tip swab as illustrated in Figure B on the right. It must be heated to
approximately 70C to both mix and use. Sealing the coverslip keeps organisms from moving when
using oil immersion objectives and prevents the preparation from drying out. To seal, secure the four
corners by placing a drop of hot sealant to anchor the coverslip. Spread a thin layer around the edges.
Other suitable sealing preparations can be used if desired.
Systematically scan the entire coverslip area using the 10 objective as illustrated in Figure C on the
right. If something suspicious is seen, a higher magnification may be necessary.
CAUTION: Bringing high power objectives too near the edge of the slide will result in the sealant
smearing the objective and interfering with the optors.
Stained Slide Preparation:

Permanent stained slides are used for identification of protozoan trophozoites and cysts and for
confirmation of species. It also permits consultation reference and diagnosis when needed as well as
providing a permanent record of organism(s) observed. The microscope should be calibrated before
examination begins. Positive microscope slides as well as reference material (plates, photographs,
digital images) should be available by the workstation to compare morphological details and
organisms. Refer to the staining section of stools for additional information regarding which stains to
use.
Normally 3 1 slides are used to prepare permanent stained slides. If the specimen is unpreserved,
prepare a thin even smear of the material by streaking the material back and forth on the slide with
an applicator stick. If necessary dilute feces with saline. For PVA fixed specimens, apply two or three
drops of the specimen to the slide and with a rolling motion or an up and down dabbing motion spread
the specimen evenly to cover an area roughly the size of a 22 by 22 mm coverslip. For other fixatives,
check manufacturers instructions.
After the staining process is complete, systematically examine the smear microscopically utilizing the
100 oil objective. Examine at least 200 to 300 oil immersion fields. Report protozoa seen as either
trophozoites and/or cysts as applicable.
UV Fluorescence Microscopy Procedure:

The demonstration of Cyclospora oocysts in wet preparations is greatly enhanced by using UV
fluorescence microscopy. Despite the age of the specimen or sample, Cyclospora oocysts exhibit
intense blue color when observed under a fluorescence microscope (UV excitation filter set at 330-365
nm). If this filter set is not available, a less intense green fluorescence can be obtained with blue
excitation (450-490 nm). Under bright-field (differential interference contrast or DIC)
microscopy, Cyclospora oocysts appear as refractile spheres (8-10 m) with a distinct oocyst wall. The
utilization of both bright-field (DIC) and fluorescence microscopy provides an efficient and reliable
approach to diagnosis. However, it does not provide a permanent stained slide that can be archived.
Detection of Parasite Antigens

The diagnosis of human intestinal protozoa depends on microscopic detection of the various parasite
stages in feces, duodenal fluid, or small intestine biopsy specimens. Since fecal examination is very
labor-intensive and requires a skilled microscopist, antigen detection tests have been developed as
alternatives using direct fluorescent antibody (DFA), enzyme immunoassay (EIA), and rapid, dipsticklike tests. Antigen detection methods can be performed quickly and do not require an experienced and
skilled morphologist. Much work has been accomplished on the development of antigen detection
tests, resulting in commercially available reagents for the intestinal
parasites Cryptosporidium spp., Entamoeba histolytica, Giardia duodenalis, andTrichomonas vaginalis.
In addition, antigen detection tests using blood or serum are available
forPlasmodium and Wuchereria bancrofti.
Specimens for antigen detection

Fresh or preserved stool samples are the appropriate specimen for antigen detection testing with most
kits, but refer to the recommended collection procedures included with each specific kit.
Amebiasis
EIA kits are commercially available for detection of fecal antigens for the diagnosis of intestinal
amebiasis. Organisms of both the pathogenic E. histolytica and the nonpathogenic Entamoeba
dispar strains are morphologically identical. These assays use monoclonal antibodies that detect the
galactose-inhibitable adherence protein in the pathogenic E. histolytica. The primary drawback of
these assays is the requirement for fresh, unpreserved stool specimens. Several EIA kits for antigen
detection of the E. histolytica/E. dispar group are available in the U.S., but only the TechLab kit is
specific for E. histolytica.
Cryptosporidiosis
Immunodetection of antigens on the surface of organisms in stool specimens, using monoclonal
antibody-based DFA assays, is the current test of choice for diagnosis of cryptosporidiosis and provides
increased sensitivity over modified acid-fast staining techniques. There are commercial products (DFA,
IFA, EIA, and rapid tests) available in the United States for the diagnosis of cryptosporidiosis. Several
kits are combined tests for Cryptosporidium, Giardia, and E. histolytica. Factors such as ease of use,
technical skill and time, single versus batch testing, and test cost must be considered when
determining the test of choice for individual laboratories. The most sensitive (99%) and specific
(100%) method is reported to be the DFA test, which identifies oocysts in concentrated or
unconcentrated fecal samples by using a fluorescein isothiocyanate (FITC)-labeled monoclonal
antibody. A combined DFA test for the simultaneous detection ofCryptosporidium oocysts
and Giardia cysts is available.
Some commercial EIA tests are available in the microplate format for the detection
of Cryptosporidium antigens in fresh or frozen stool samples and also in stool specimens preserved in
formalin, or sodium acetate-acetic acid-formalin (SAF) fixed stool specimens. Concentrated or
polyvinyl alcohol-treated (PVA) samples are unsuitable for testing with available antigen detection EIA
kits. The kits are reportedly superior to microscopy, especially acid-fast staining, and show good
correlation with the DFA test. Kit sensitivities and specificities reportedly range from 93 to 100% when
used in a clinical setting. Laboratories which use these EIA kits need to be aware of potential problems
with false-positive results and take steps to monitor kit performance.
Rapid immunochromatographic assays are available for the combined antigen detection of
either Cryptosporidium and Giardia or Cryptosporidium, Giardia, and E. histolytica. These offer the
advantage of short test time and multiple results in one reaction device. Initial evaluations indicate
comparable sensitivity and specificity to previously available tests.
The Meridian Merifluor DFA Kit for Cryptosporidium/Giardia, modified acid-fast stain
for Cryptosporidium spp., or Wheatley's trichrome stain for Giardia spp. are used at CDC for routine
identification of these parasites. These techniques can be used to confirm suspicious or discrepant
diagnostic results.
Giardiasis
Detection of antigens on the surface of organisms in stool specimens is the current test of choice for
diagnosis of giardiasis and provides increased sensitivity over more common microscopy techniques.
Commercial products (DFA, EIAs, and rapid tests) are available in the United States for the
immunodiagnosis of giardiasis. DFA assays may be purchased that employ FITC-labeled monoclonal
antibody for detection of Giardia cysts alone or in a combined kit for the simultaneous detection
of Giardia cysts and Cryptosporidium oocysts. The sensitivity and specificity of these kits were both
100% compared to those of microscopy. They may be used for quantitation of cysts and oocysts, and
thus may be useful for epidemiologic and control studies.
Some commercial EIA tests are available in the microplate format for the detection of Giardia antigen
in fresh or frozen stool samples and also in stool specimens preserved in formalin, MIF, or SAF
fixatives. Concentrated or PVA samples are not suitable for testing with EIA kits. EIA kit sensitivity
rates were recently reported as ranging from 94-100% while specificity rates were all 100%.
Rapid immunochromatographic assays are available for the combined antigen detection of
either Cryptosporidium and Giardia or Cryptosporidium, Giardia, and E. histolytica. These offer the
advantage of short test time and multiple results in one reaction device. Initial evaluations indicate
comparable sensitivity and specificity to previously available tests.
The Meridian Merifluor DFA Kit for Cryptosporidium/Giardia, modified acid-fast stain
for Cryptosporidium spp., or Wheatley's trichrome stain for Giardia spp. are used at CDC for routine
identification of these parasites. These techniques can be used to confirm suspicious or discrepant
diagnostic results.
Trichomoniasis
Trichomoniasis, an infection caused by Trichomonas vaginalis, is a common sexually transmitted
disease. Diagnosis is made by detection of trophozoites in vaginal secretions or urethral specimens by
wet mount microscopic examination, DFA staining of specimens, or culture. Sensitivity of the assays
were reported as 60% for wet mounts and 86% for DFA when compared to cultures. A kit which
employs FITC- or enzyme-labeled monoclonal antibodies for use in a DFA or EIA procedure is available
for detection of whole parasites in fluids. A latex agglutination test for antigen detection in vaginal
swab specimens is available; the manufacturer's evaluation indicated good sensitivity and specificity.
Organism
Cryptosporidium spp.
Kit name
Manufacturer distributora
Ty
T
Crypto CELISA
Cellabs
EIA
PARA-TECT Cryptosporidium
Antigen 96
Medical Chemical
Corporation
EIA
ProSpecT Rapid
Remel
EIA
ProSpecT
Remel
EIA
Cryptosporidium
TechLab
EIA
Cryptosporidium
Wampole
EIA
Organism
Cryptosporidium spp./Giardia duodenalis
Cryptosporidium spp./Giardia
duodenalis/Entamoeba histolytica/dispar
Entamoeba histolytica
Entamoeba histolytica/E. dispar

Giardia duodenalis
Kit name
Ty
T
Crypto CEL
Cellabs
IFA
XPect Crypto
Remel
Rap
PARA-TECT
Cryptosporidium/Giardia DFA 75
Medical Chemical
Corporation
DFA
Merifluor
Meridian
DFA
ProSpecT
Remel
EIA
Crypto/Giardia CEL
Cellabs
IFA
ColorPAC*
Becton Dickinson
Rap
ImmunoCard STAT!*
Meridian
Rap
XPect
Remel
Rap
Triage
BioSite
Rap
Entamoeba CELISA
Cellabs
EIA
E. histolytica
Wampole
EIA
E. histolytica II
TechLab
EIA
ProSpecT
Remel
EIA
Giardia CELISA
Cellabs
EIA
PARA-TECT Giardia Antigen 96
Medical Chemical
Corporation
EIA
ProSpecT
Remel
EIA
Giardia II
TechLab
EIA
Giardia
Wampole
EIA
GiardiaEIA
Antibodies, Inc.
EIA
Giardia CEL
Cellabs
IFA
ProSpecT
Remel
Rap
Organism
Wuchereria bancrofti
Kit name
Ty
T
Simple-Read Giardia
Medical Chemical
Corporation
Rap
Filariasis CELISA
Cellabs
EIA
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 a preservative that
is compatible with molecular detection. Fixatives/preservatives with acceptable performance for PCR
include TotalFix, Unifix, modified PVA (Zn- or Cu-based), and Ecofix. Stool specimens in these
preservatives can be stored and shipped at room temperature. Alternatively, stool specimens can be
collected in a clean vial and kept unpreserved; however, these specimens must be stored cold or
frozen and shipped either refrigerated (4C) or frozen (shipped with dry ice). Trichrome stained
smears (for 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. Click here
for more information about shipping stool specimens to CDC.
Fixatives/preservatives that are not recommended for molecular detection include formalin, SAF, LVPVA, and Protofix.
For specific applications or when commercial fixatives are not an option, the stool can be mixed in
potassium dichromate 2.5% (1:1 dilution) or in absolute ethanol (1:1 dilution) and shipped
refrigerated.
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 Cyclospora cayetanensis, Entamoeba histolytica, and E. dispar is
performed at CDC by both conventional PCR and real-time PCR. Conventional PCR is available
for 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 (404) 718-4120.
Real-time PCR Illustrations

SYBR Green
The principle of SYBR Green detection in real-time PCR is outlined in the figure above. 1 The fluorescent dye SYBR
Green is added to the PCR mixture (1). SYBR Green is a DNA binding dye that fluoresces strongly when bound to
double-stranded DNA. At the start of the reaction, very little double stranded DNA is present, and so the
fluorescent signal detected by the thermocycler is low (3). As the reaction proceeds and PCR product accumulates,
the amount of double-stranded DNA increases and with it the fluorescence signal (4-5). The signal is only
detectable during annealing and extension, since the denaturation step contains predominantly single-stranded
DNA (6).
TaqMan
The principle of TaqMan real-time PCR is depicted in the schematic above. 1The TaqMan probe is designed to be
5
complementary to a specific sequence spanned by the PCR primers. The TaqMan probe has a reporter dye at its 5
end and a quencher dye at its 53 end. As long as the probe is intact and the reporter and the quencher dyes are in
close proximity, no fluorescence signal is emitted due to the quenching effect (black arrow in 1, 2, and 3) (1). After
the annealing of the TaqMan probe (2) and the primers (3), the primers are extended by the DNA polymerase. As
the polymerase reaches the TaqMan probe, it uses its exonuclease activity to remove the probe one nucleotide at
the time (4). This releases the reporter from the proximity of the quencher and allows for the release of a
fluorescence signal from the reporter (5).
References
SERUM/PLASMA SPECIMENS
Safety
Universal precautions for the prevention of bloodborne pathogens must be followed when working with
human serum and other body fluids. These include:
Wear personal protective equipment such as safety glasses, gloves, laboratory coats.
Use needles and lancets only once, and dispose of them in a sharps container for
decontamination.
Remove gloves and wash your hands after completing any task involving the handling of
biological material.
Preventionto view biosafety guidelines or the website of the Occupational Safety and Health
Administration (OSHA)
Specimen Requirements
Serum/plasma is required for all parasitic disease immunodiagnostic tests. A single sample is usually
sufficient; acute and convalescent specimens are not necessary. CSF and eye fluids (vitreous or
aqueous) are acceptable for selected diseases (see below) and MUST be accompanied by a serum
specimen.
Serum for all tests: 0.5 ml serum/plasma separated from RBC's before shipping.
CSF: 0.5 ml. Acceptable only for cysticercosis and baylisascariasis testing.
Eye fluids: 0.1 ml neat fluid (no washings). Acceptable only for toxocariasis.
Specimen Submission
To submit a specimen for antibody testing for parasitic diseases:
1.
For routine requests, submit the specimen to the appropriate city, county or state health
department laboratory for processing and examination (see http://www.aphl.org ). The
specimens will be referred to CDC if necessary.
2.
For an emergency, call the Immunodiagnostic Laboratory of the Division of Parasitic Diseases
at (770) 488-4431 to make arrangements.
Shipping Requirements
Clinical specimens such as serum, CSF, and eye fluids must be triple packed in a primary receptacle,
water tight secondary packaging, and a durable outer package for shipment as required by
international regulations for biological agents of human disease. Figure 1shows correct labeling and
packaging of specimens (http://www.cdc.gov/od/ohs/biosfty/bmbl4/b4acf1.htm):
1.
2.
Label the specimen tube with the patient ID and seal with waterproof tape. Wrap the tube in
absorbent packing material.
Place the specimen tube in secondary packaging.
3.
Place the secondary package in the outer packaging (i.e., the shipping container). Label the
shipping container "Clinical Specimen" on the outside of the package.
4.
Include the following information: submitter's name, address, phone number, fax number, and
e-mail address; patient's name, age, brief clinical history, and travel history; specimen collection
date; and test(s) requested.
5.
Ship at room temperature by overnight/2 day carrier.
Additional information about shipping regulations may be found at:

1.
2.
3.
4.
Guidelines for the Safe Transport of Infectious Substances and Diagnostic Specimens, (World
Health Organization )
The IATA Dangerous Goods Regulations, (International Air Transport
Association) http://www.iata.org/ps/publications/dgr/Pages/index.aspx
Title 49 Code of Federal Regulations, Parts 100-185. Hazardous Materials regulations
(Department of Transportation)http://www.phmsa.dot.gov/hazmat/regs
Title 42 Code of Federal Regulations, Part 72. Interstate shipment of etiologic agents
(Department of Health and Human Services)http://www.cdc.gov/od/ohs/biosfty/shipregs.htm
Antibody Detection Tests Offered at CDC
Disease
Organism
Test
Acceptable
Specimens
Amebiasis
Entamoeba histolytica
Enzyme immunoassay
(EIA)
Serum
Babesiosis
Babesia microti
Babesia sp. WA1
Immunofluorescence
(IFA)
Serum
Baylisascariasis
Baylisascaris procyonis
Immunoblot
Serum or CSF
Chagas disease
Trypanosoma cruzi
IFA
Serum
Cysticercosis
Larval Taenia solium
Immunoblot (Blot)
Serum, CSF
Echinococcosis
Echinococcus granulosus
EIA, Blot
Serum
Filariasis
Wuchereria
bancrofti and Brugia malayi
EIA
Serum
Disease
Organism
Test
Acceptable
Specimens
Leishmania braziliensis
Leishmaniasis
IFA
Serum
Malaria
Plasmodium falciparum
P. malariae
P. ovale
P. vivax
IFA
Serum
Paragonimiasis
Paragonimus westermani
Blot
Serum
Schistosomiasis
Schistosoma sp.
S. mansoni
S. haematobium
S. japonicum
L. donovani
L. tropica
FAST-ELISA
Serum
Blot
Strongyloidiasis
Strongyloides stercoralis
EIA
Serum
Toxocariasis
Toxocara canis
EIA
Serum, vitreous
fluid
EIA
Serum
Trichinellosis(Trichinosis) Trichinella spiralis
OTHER SPECIMENS
Shipment
Note: When you ship a specimen to CDC, make sure your package will arrive on a weekday and
willnot arrive at CDC on the weekend or a federal holiday.
1.
In emergencies, call the Epidemiology Branch of the Division of Parasitic Diseases at (770)
488-7760 to make special arrangements.
2.
For routine requests, submit the specimen to the appropriate city, county or state health
department laboratory (see http://www.aphl.org ) for processing and examination. That facility
will refer specimens to CDC if necessary.
Figure 1 shows correct labeling and packaging of specimens
(http://www.cdc.gov/od/ohs/biosfty/bmbl4/b4acf1.htm).
3.
4.
If shipping a specimen, please refer to shipping regulations and guidelines at the following
addresses:
a.
b.
c.
d.
e.
f.
Guidelines for the Safe Transport of Infectious Substances and Diagnostic Specimens,
(World Health Organization )
The IATA Dangerous Goods Regulations, (International Air Transport
Association)http://www.iata.org/ps/publications/dgr/Pages/index.aspx
Title 49 Code of Federal Regulations, Parts 100-185. Hazardous Materials regulations
(Department of Transportation) http://www.phmsa.dot.gov/hazmat/regs
Title 42 Code of Federal Regulations, Part 72. Interstate shipment of etiologic agents
(Department of Health and Human Services)http://www.cdc.gov/od/ohs/biosfty/shipregs.htm
Title 42 Code of Federal Regulations, Part 72.6. Additional requirements for facilities
transferring or receiving select agents (Department of Health and Human
Services)http://www.selectagents.gov/
Biosafety in Microbiological and Biological and Biomedical Laboratories
(CDC/NIH)http://www.cdc.gov/od/ohs/biosfty/bmbl/bmbl-1.htm
Tissue Specimens
Tissue samples, including biopsies, surgical or necropsy specimens, collected for possible detection of
parasites, should be submitted to the hospital pathologist. Specimens may be forwarded to the
Division of Parasitic Diseases at CDC if further identification is necessary. Specimens may be submitted
in several forms, including sectioned and stained slides, tissue blocks, or preserved tissues from cases
that have a high suspicion of containing parasite material. In most instances, routinely stained
hematoxylin and eosin sections are suitable for examination for parasitic agents, and many times, an
accurate identification of the parasite can be established in tissue sections. Occasionally, additional
sections are required to further the diagnosis. Ship samples according to shipping guidelines and
requirements (see shipping guidelines). Care should be taken to pack glass slides securely, as they
can be damaged in shipment if not packed in a crush proof container.
Tissue Specimens for Free-living Amebae (FLAs)

Tissue specimens, including biopsy, surgical or necropsy specimens, may be collected for the detection
of free-living amebae (Naegleria, Balamuthia, and Acanthamoeba). The desired specimens include:
tissue slides stained with hematoxylin and eosin (H&E).
unstained slides (for indirect Immunofluorescence, or IIF).
unfixed brain tissue or CSF for PCR.
unfixed corneal scrapings (for Acanthamoeba).
paraffin-embedded tissue block.
Ship samples according to shipping guidelines and requirements (see shipping guidelines). Unfixed
specimens for culture should be sent at ambient temperature by overnight priority mail. For PCR,
sterile unfixed specimens or specimens in 70-90% ethanol should be sent by overnight priority mail on
ice packs. Care should be taken to pack glass slides securely, as they can be damaged in shipment if
not packed in a crush-proof container. Specimen submission forms for free-living amebae testing can
be requested by emailing dpdx@cdc.gov.
Isolation of Leishmania Organisms

The diagnosis of leishmaniasis is made by microscopic identification of the nonmotile, intracellular
form (amastigote) in stained sections from lesions, and by culture of the motile, extracellular form
(promastigote) on suitable media. Slides should be fixed with methanol and Giemsa stained before
they are sent unless reagents are not available. Serologic tests are also available to detect for antileishmanial antibodies; however, these tests are often not sensitive, particularly for diagnosing
cutaneous leishmaniasis, and do not provide a definitive diagnosis. Examination of scrapings,
impression smears, and biopsy smears can be performed at CDC, with prior notification of the
appropriate state health laboratory.
Leishmanial culture is also available on needle aspirates or punch biopsy lesions. CDC can provide
culture medium (typically Novy-MacNeal-Nicolle (NNN) medium). Keep this refrigerated until it is used
(stable for 2-4 weeks) and bring it to room temperature right before inoculation. Once inoculated,
keep the culture at room temperature and mail as soon as possible by overnight mail. If culture
medium is needed, call 404-718-4195 or 404-718-4110.
The needle aspirate should be obtained by first drawing approximately 0.1 ml of preservative-free
sterile 0.9% NaCl into a 1.0 to 3.0ml syringe. Insert the needle (23-27 gauge) through intact skin into
the dermis of the active border. Move the needle back and forth repeatedly under the skin
simultaneously rotating the syringe and applying gentle suction until pink-tinged tissue juice is noted
in the hub of the syringe. After the aspirate is obtained, discharge into the leishmanial culture
medium. For biopsy specimens, obtain 1 or 2 full thickness punch specimens at the active border of
the lesion. Use 1 specimen for culture and the other for impression smears. For culture, place the
punch biopsy into the culture medium. The culture tubes should be kept at room temperature prior to
and during shipment. The cultures will be kept 4 weeks at CDC and if the culture becomes positive and
grows, isoenzyme studies will be performed for species identification.
Sputum Specimens
Microscopic examination of sputum is used in identifying Paragonimus westermani eggs,Strongyloides
stercoralis larvae, Ascaris lumbricoides larvae, hookworm larvae, and rarelyEntamoeba histolytica.
Sputum should be obtained from the lower respiratory passages rather than a sample consisting
mainly of saliva. Sputum specimens should be collected first thing in the morning. A sputum sample
can be examined in several ways:
The unfixed specimen may be centrifuged and then the sediment examined as a direct wet
mount.
If the sputum is too viscous, an equal volume of 3% sodium hydroxide may be added, then
centrifuge, and examine the sediment.
The specimen may be preserved in 10% formalin and a formalin-ethyl acetate concentration
procedure may be completed and the sediment examined using either a wet mount or a stained
preparation.
The specimen may also be preserved in PVA if protozoa are suspected and stained with
trichrome stain.
Aspirates
Duodenal aspirates may be useful in demonstrating Giardia duodenalis or Strongyloides
stercoralis larvae. Material collected following intubation through the nose and stomach into the upper
small intestine may be submitted to the laboratory. Centrifuge the specimen at 500 g for 2 to 3
minutes and examine the wet mount. An unfixed specimen can be examined immediately or if the
specimen cannot be examined within 1 to 2 hours after collection, it should be preserved in 10%
formalin.
Sigmoidoscopy material and abscesses of the liver and lung may demonstrate amebic
trophozoites. Material from the mucosal surface or from visible lesions should be aspirated. This
material can be examined immediately in a 0.85% saline wet mount preparation (or part of this
material could be placed in formalin) or can be fixed in PVA. Once fixed in PVA, the material can be
stained using trichrome stain and examined for trophozoites of Entamoeba histolytica. A real-time PCR
test is available for confirmation of amebiasis that detects E. histolytica at the species level. If
molecular diagnosis is necessary, specimens should be unpreserved and kept refrigerated or frozen.
Lymph node material, bone marrow, and spleen may be examined for the presence of motile
trophozoites of Trypanosoma brucei gambiense or Trypanosoma brucei rhodesiense. ForLeishmania
donovani infections, material obtained by needle aspiration from bone marrow or spleen can be used
to demonstrate amastigote stages. Smears can then be prepared by fixing in methanol and staining
with Giemsa stain.
Skin ulcers may demonstrate the amastigote stages in cutaneous and mucocutaneous leishmaniasis.
Permanent stained smears made with these specimens by fixing in methanol and staining with Giemsa
stain.
Sputum, induced sputum and bronchoalveolar lavage (BAL) for Pneumocystis jirovecii
Sputum, induced sputum, and bronchoalveolar lavage (BAL) material are commonly used for
diagnosing Pneumocystis jirovecii (previously classified as Pneumocystis carinii) infections. To examine
these specimens for cysts of P. jiroveci, prepare smears of the sediment and examine after staining
with the recommended procedure (Giemsa or methenamine silver stain).
Staining Procedures
Stain only one set of smears and leave the duplicates unstained. The latter will prove useful if a
problem occurs during the staining and/or if you wish later to send the smears to a reference
laboratory.
Preparation of the specimen

Refer to safety section prior to handling the specimens (link)
1.
Treat specimens containing mucus (sputum mainly) with a mucolytic agent (e.g., Sputalysin)
by mixing the agent in a 1:1 ratio with the specimen.
2.
3.
Discard supernatant.
4.
Treat sediment containing blood with red cell lytic agent (e.g., saponin*). If this is the case,
centrifuge once more following conditions described above and discard the supernatant.
5.
Resuspend the remaining sediment.
6.
Place drops of the sediment on the center of a microscopic slide.*
7.
Spread the drop on the slide with a pipette to evenly distribute on the slide.
8.
Air dry and stain following the required procedure.
*For specimens containing blood, separate an aliquot that has not been subjected to lyses and prepare
smears from that as well.
Giemsa Stain
1.
Stock 100 Giemsa Buffer - 0.67 M
Na2HPO4
NaH2PO4H2O
Deionized water
2.
Autoclave or filter-sterilize (0.2 m pore). Sterile buffer is stable at room temperature for one
year.
3.
Working Giemsa Buffer - 0.0067M, pH 7.2
Stock Giemsa Buffer

Deionized water
4.
Check pH before use. Should be 7.2. Stable at room temperature for one month.
5.
Triton X-100 5%
Deionized water (warmed to 56C)

Triton X-100
6.
Prewarm the deionized water and slowly add the Triton X-100, swirling to mix.
7.
Stock Giemsa stain

Giemsa stain is available commercially, but the following formulation gives more constant results
and does not expire
Glass beads, 3.0 mm
Absolute methanol, acetone-free

Giemsa stain powder (certified)
Glycerol
8.
a.
Put into a 500 ml brown bottle the glass beads and the other ingredients, in the order
listed. Screw cap tightly. Use glassware that is clean and dry.
b.
Place the bottles at an angle on a shaker; shake moderately for 30 to 60 minutes

daily, for at least 14 days.
c.
Kept tightly stoppered and free of moisture, stock Giemsa stain is stable at room
temperature indefinitely (stock stain improves with age).
d.
Just before use, shake the bottle. Filter a small amount of this stock stain through
Whatman #1 filter paper into a test tube. Pipet from this tube to prepare the working Giemsa
stain.
Working Giemsa stain (2.5%): make fresh for each batch of smears
Working Giemsa buffer

Giemsa Stain Stock
5% Triton X-100
Staining
1.
Prepare fresh working Giemsa stain in a staining jar, according to the directions above. (The
40 ml fills adequately a standing Coplin jar; for other size jars, adapt volume but do not change
proportions).
2.
Pour 40 ml of working Giemsa buffer into a second staining jar. Add 2 drops of Triton X-100.
Adapt volume to jar size.
3.
4.
5.
Place slides into the working Giemsa stain (2.5%) for 45-60 minutes.
Remove thin smear slides and rinse by dipping 3-4 times in the Giemsa buffer. Thick smears
should be left in buffer for 5 minutes.
Dry the slides upright in a rack.
Note: As alternates to this 45-60 minutes in 2.5% Giemsa stain, the smears could be stained for
shorter times in more concentrated stains; one alternate is 10 minutes in 10% Giemsa; the shorter
stains yield faster results, but use more stain and might be of less predictable quality.
Staining Procedure: Quality Control

To ensure that proper staining results have been achieved, a positive smear (malaria) should be
included with each new batch of working Giemsa stain.
Methenamine silver stain (Gomori's silver methenamine)

Store reagents at room temperature (25C) except the silver nitrate, which must be stored at 4C.
1.
CrO3 10% solution (stable for 1 year)
CrO3
Distilled H2O
2.
Methenamine 3% solution (stable for 6 months)
(CH2)6N4 (Hexamethylenetetramine)
Distilled H2O
3.
Silver Nitrate 5% solution (stable for 1 month).
AgNO3
Distilled deionized H2O
4.
Sodium borate (Borax) 5 % solution (stable for 1 year)
Na2B4O7 . 10 H2O
5.
Sodium bisulfate 1% solution (stable for 1 year)
Na2HSO3
6.
Gold Chloride 1 % solution (stable for 1 year)
Gold chloride
7.
Sodium Thiosulfate 5% solution (stable for 1 year)
Na2S2O3 . 5 H2O
8.
Stock Light green (stable for 1 year)
Light green, S. F. (yellow) (CI no. 42095)

CH3COOH
9.
Working Light Green (stable for 1 month)
Stock Light Green

10.
Alcohols
Ethanol 100% and 95%

Methanol 100%
11.
Xylene or Xylene substitute
PROCEDURE:
1.
2.
Add chromic acid 10% solution to cover the smears. Incubate for 10 min at room temperature.
Prepare working methanine solution by mixing 20 ml of 3% methanine, 1 ml of 5% silver
nitrate, 1.5 ml of 5% sodium borate, and 17 ml of distilled deionized H2O.
3.
Wash slide with distilled H2O.
4.
Cover slides with 1% sodium bisulfate. Incubate for 1 min at room temperature.
5.
Wash slides with distilled H2O.
6.
Place slides in a Coplin jar containing the working methanine solution. Cover with a cap and
place in a microwave oven.
7.
Microwave it at 50% power for 35 seconds. Gently rotate the jar and microwave again for 35
seconds. Leave the slides in the hot methanine solution for 1-3 minutes. Microwave on 600 Watts
for 35 seconds. If a microwave oven is not available, use a water bath heated at 80C. Prewarm
the solution by putting the Coplin jar in the bath in the bath for 6 minutes, prior to adding the
slides. Add the slides and incubate in the water bath for additional 5 minutes.
8.
Wash the slides with distilled and then with deinonized H2O.
9.
Dip the slides several times in the in the 1% gold chloride solution.
10.
11.
12.
Place the slides on a rack, cover with 5% sodium thiosulfate solution and incubate for 1
minute at room temperature.
Wash the slides with distilled H2O.
Cover the slides with the working light green solution (counterstain) and incubate for 1 minute
at room temperature.
13.
Wash the slides with distilled H2O. Drain the excess of water and air dry.
14.
Examine the slides.
P. jiroveci was previously classified as a protozoa, but currently it is considered a fungus.
Vaginal Swabs for Detection and Susceptibility of Trichomonas

Demonstration of Trichomonas vaginalis trophozoites is usually done by preparing wet mounts
made from vaginal swabs or scrapings. If the specimen cannot be examined immediately, it should be
preserved in PVA and stained smears examined later.
Susceptibility Testing
T. vaginalis is usually highly susceptible to metronidazole. However, cases of resistant T. vaginalishave
been reported and may be increasing. There are no standardized guidelines for the identification and
treatment of these organisms. In 1978, an in vitro drug susceptibility test was developed by J.G.
Meingassner. It can be used to determine the extent of resistance of a trichomonad isolate to the 5
nitroimidazoles. This test is performed at the Division of Parasitic Diseases on isolates that are
received from across the country. Test results are forwarded to the CDC/NCHSTP/DSTDP and are then
communicated to the submitting physician.
Commercially available tests for detection of Trichomonas
a.
b.
Chemicon, 28835 Single Oak Dr., Temecula, CA 92590; PanBio InDx, 1756 Sulfur Spring Rd.,
Baltimore, MD 21227
DFA = direct fluorescent antibody; LA = latex agglutination
Cellulose Tape or Swube Tube Procedure for Demonstration of Pinworm Eggs

The most reliable and widely used technique for demonstrating pinworm eggs (Enterobius
vermicularis) is the cellulose tape or swube tube procedure. The adhesive part of the swube tube or
tape is applied to the perianal area first thing in the morning. Specimens should be collected on three
consecutive mornings prior to bathing. If an infection is present, eggs and sometimes adult worms
of Enterobius vermicularis will be present on the tape and can be seen under the microscope.
Urine Specimens
The definitive diagnosis of urinary schistosomiasis (Schistosoma haematobium) is established by
demonstration of S. haematobium eggs in urine. An increased number of eggs is shed in the urine
around midday, so an optimum urine specimen for diagnosis should be collected at noon. The
specimen should be immediately centrifuged at 400 g and the sediment examined by wet mount.
Trichomonas vaginalis motile trophozoites may also be found in the urine, especially in infected
male patients. To look for the presence of trophozoites, the urine specimen should be centrifuged at
400 g, the sediment mixed with a drop or two of saline, and examined by wet mount. Temporary
stains, such as methylene blue or malachite green, are also helpful.

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BLOOD SAMPLES

Loa loamidday (10 AM to 2 PM)

Wipe away the first drop of blood with clean gauze.

Prepare at least 2 thick smears and 2 thin smears.

Venous blood obtained by venipuncture:

Scratch Method for Thick smears

Fix the smears by dipping them in absolute methanol.

Special Procedures for Detecting Microfilariae

Capillary (fingerstick) blood

Centrifugation (Knott's technique)

Prepare 2% formaldehyde (2 ml of 37% formaldehyde + 98 ml H2O).

Place Millipore or Nucleopore membrane filter (5 m pore) in filter holder

For permanent preparations, pass 2 to 3 ml of methanol through the filter

Shipping blood smears for microscopic examination

Submitter's name, address and phone number

Physician's name, address and phone number

Specimen collection date

What organism is suspected

Ship via mail or package carrier.

Shipping whole blood for isolation of parasites (culture or animal inoculation),

Submitter's name, address and phone number

Physician's name, address and phone number

Specimen collection date

What tests are requested and what organisms are suspected

Wright (Wright-Giemsa) stain

Stock 100 Giemsa Buffer - 0.67 M

Working Giemsa Buffer - 0.0067M, pH 7.2

Stock Giemsa Buffer

Deionized water (warmed to 56C)

Glass beads, 3.0 mm

Place the bottles at an angle on a shaker; shake moderately for 30 to 60 minutes

Working Giemsa buffer

Staining Procedure: Quality Control

Fix the smears in absolute (100%) methanol; allow them to dry.

Place them, touching front to back, in a box without separating grooves.

Examining thin smears

Detection of blood parasites using fluorescent dyes

Species-specific PCR for Diagnosis of Malaria

Species-specific PCR for Diagnosis of Babesia

Extraction of DNA from Blood Specimens

Equilibrate samples to room temperature.

Heat one water bath or heat block to 56C.

All centrifugation steps should be carried out at room temperature.

Incubate at 56C for 10 minutes.

Add 200 l of 96-100% ethanol and mix by vortexing.

Extraction from filter containing blood

Store extracted DNA at 4C.

Detection of Parasite Antigens

Special Tests: MQ Testing

Use biological safety cabinets as needed.

Fresh stool should be examined, processed, or preserved immediately. An exception is

Distribution of protozoa in relation to stool consistency

All purpose fixative

Long shelf life

Good preservation of morphology of

Not suitable for some

Can interfere with PCR,

Suitable for concentration procedures and

Suitable for acid-fast, safranin, and

Compatible with immunoassay kits and UV

Components both fix and stain organisms

Long shelf life

Useful for field surveys