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Cryptococcal Antigen

Latex Agglutination
test
Introduction
The latex agglutination for Cryptococcus Neoformans antigen is a simple and
rapid procedure for the diagnosis of cryptococcal meningitis. Since the test is
sensitive, care must be taken to prevent contamination of the sample, resulting in
false-positive results. This test detect the capsular antigen of Cryptococcus
Neoformans in cerebrospial fluid (CSF) and serum.
Principle

Cryptococcal Antigen Latex Agglutination test is


based upon the principle that anti-Cryptococcal
antibody-related latex particles will agglutinate
with specimens containing Cryptococcal
capsular polysaccharide antigens.
Procedure
• Specimen Preparation:
SERUM
1.Collect whole blood aseptically following accepted
procedures.
2. Allow the blood to clot for 10 minutes or more in the room
temperature.
3.Centrifuge for 15 minutes at 1000 RPF
4.Carefully aspirate the serum into a sterile tube seal.
5.Add 300 ul of serum to 50ul of aliqout of Pronase and seal
the tube with parafilm
6.Incubate the sample at 50o C for 30 minutes.
7.Add 1 drop of Pronase Inhibitor and mix to terminate the
Procedure
• Specimen Preparation:
CSF
1. Collect CSF aseptically following accepted procedures.
2. Centrifuge at 1000 RPM for 15 minutes to ensure the removal of all white
cells and particulate matter.
3. Carefully aspirate the serum into a sterile tube seal.
4. Specimen may be processed immediately, refrigerated, preserved by freezing
at -20o C or by adding thimerosal to provide a final concentration of 0.01%
5. Incubate the sample at 100o C for 5 minutes.
Procedure
• Screening Procedure:
1. Add 25 ul of Cryptococcus Antigen positive and negative
control and heat-treated CSF/Pronase-treated serum specimen
onto separate rings of the ring slide. Use a new pipette tip for
each reagent and specimen.
2. Add 25 ul of Cryptococcal Latex to each ring.
3. Using separate aplicator sticks, mix thoroughly the content of
each ring.
4. Place the ring slide on a rotator set to 100 RPM (+/- 25) for 5
minutes at room temperature.
5. Read the reactions immediately.
Procedure
• Titration Procedure:
Patients showing a 1+ or greater reactions should be titrated.

1. Add 100 ul of specimen diluent to each test tubes labeled 1-10 and place
in a rack (1:2 through 1:1024). Additional dilutions may be necessary if
the specimen is positive at 1:1024.
2. Add 100 ul of patient’s specimen to tube #1 and mix well.
3. Transfer 100 ul from tube #1 to and mix well.
4. Transfer 100 ul from tube #1 to tube # 2 and mix well. Continue this
dilution up to tube #10.
5. Add 25 ul of Cryptococcus Positive , negative control and each specimen
dilution onto separate rings of the ring slide.
6. Add 25 ul of Cryptococcus Latex to each ring.
7. Place the ring slide on a rotator set to 100 RPM (+/- 25) for 5 minutes at
room temperature.
8. Read the reactions immediately.
Results
• Reporting:
 negative (-) : a homogeneous suspension of particles with no visible clumping.

 One plus (1+) : fine granulation against a milky background.

 Two plus (2+) : small but definite clumps against a slightly cloudy background.

 Three plus (3+) : large and small clumps against clear background.

 Four plus (4+) : large clumps against a very clear background.


Precautions
• All reagents are intended for diagnostic use only.
• Specimens must not contain bacteria, visible lipids, or other
obvious signs of contamination.
• NEVER heat Pronase Control as this could cause aberrant
control reactions.
• Care should be taken not to introduce syneresis fluid into
any specimens prior to testing as this may cause spuriuos
results.
• All reagents are preserved with sodium azide (which is a
skin irritant). Avoid contact the kit components. Do not mix
reagents with acid as this may resluts in the formation of
hydrazoic acid, an extremely toxic gas.
Disease
 Cryptococcosis
– A systemic infection caused by the
fungus Cryptococcus Neoformans . This
organism typically gains entrance to the
body causing a lung infection and from
there, it can rapidly spread to the
central nervous system.
Cryptococcosis

Cryptococcus fungi

Cryptococcus found
on the lung of patient
with AIDS.
False Positive Cryptococcal Antigen
Agglutination caused by Disinfectants
and soaps
• Summary:
Five disinfectants or soaps were tested to
determine if any could be responsible for false-
positive results obtain with the Latex Antigen
detection system kit (Immuno-Mycologics, Inc).
Three disinfectants or soaps produced false-
positive agglutination after repeated washing or
ring slides during testing of a known negative
CSF specimen
Cryptococcal Antigen Test Revisited:
Significance for Cryptococcal Therapy
monitoring in a Tertiary Chinese hospital
• Summary:
For a total of 29 non-human immunodeficiency virus 1
cryptococcal meningitis cases, titer changes in the latex
agglutination test before and after therapy were reviewed
along with the clinical manifestations, laboratory findings,
and therapy regimens. The cryptococcal antigen titer
decreased for every cased after therapy and was correlated
to fungal clearance as defined by the fungus smear and
after/or culture. However, cryptococcal antigen can remain
at low titers for long periods of time after therapy, even when
fungus smears and/or cultures become negative.
False positive Reactions in the Latex
Agglutination Test for Cryptococcus
neoformans Antigen
• Summary:
The Latex Agglutination Test for Cryptococcus neoformans Antigen
is a simple and rapid procedure for the diagnosis of cryptococcal
meningitis. Since the test is sensitive, care must taken to
prevent the contamination which may lead to false-positive
results. It was discovered in the laboratory that immersion
substances of a platinum inoculating wire loop into a sample of
CSF prior to testing introduced interfering substances leading to
nonspecific agglutination. After further studies, it was
determined that trace amounts of surface condensation
(syneresis fluid) from agar, either added to the CSF or adhering
to the loop were the probable source of contamination. It is
suggested that the Latex Agglutination test for C. neoformans
antigen be performed prior to culture or on a separate sample.