Cryptococcal Antigen Latex Agglutination test JOSEPH.ppt

Cryptococcal Antigen Latex Agglutination test

IntroductionThe 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 enzymatic

digestion.

• 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

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 found on the lung of patient with

AIDS.

Cryptococcus fungi

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.