The LATEX-CRYPTOCOCCUS ANTIGEN TEST (CrAg) is a simple and rapid latex test (lateral flow immunoassay test also exist) for the semi-quantitative or qualitative detection of the capsular polysaccharide antigens of Cryptococcus neoformans in cerebrospinal fluid (cryptococcal meningitis or cryptomeningitis) and serum.Cryptococcal antigen latex test and lateral flow assay is more sensitive as compare to India Ink mount in the detection of Cryptococcus neoformans polysaccharide antigens in serum and CSF.Cryptococcosis in humans is an infection caused by the encapsulated yeast Cryptococcus neoformans (cryptococcal meningitis in csf).It is an opportunistic infection and is the fourth most common opportunistic, life-threatening infection among AIDS patients.It mostly affect individuals with impaired cell mediated immune (CMI) or cell mediated immunity function due to acquired immunodeficiency syndrome (AIDS) , lymphoproliferative disorders,leukemia,melanoma,neuroblastoma,mesothelioma,organ transplant,b cell lymphoproliferative disorder,malignant melanoma and steroid therapy. AIDS accounts for 80-90% of Cryptococcal infections where Cryptococcosis occurs in 5-10% of Acquired Immunodeficiency Syndrome patients in the United States.
Principle of Cryptococcus antigen test
- It is based on the agglutination of anti-Cryptococcal antibody coated latex particles with suspected specimens containing Cryptococcal capsular polysaccharide antigens
- The detection of cryptococcal antigen in serum is mostly hampered by the presence of rheumatoid factor therefore,
- A pretreatment of serum specimens with Pronase reduces nonspecific interference and enhances the detection of capsular polysaccharide antigens of Cryptococcus neoformans due to rheumatoid factor and immune complexes
Materials and reagents required for Cryptococcal antigen test
|Specimen Diluent||10 ml (Contain concentrated glycine buffered saline of pH 8.6 containing albumin and a preservative)|
|Cryptococcal Latex||1.5 ml (Contain standardized latex particles sensitized with rabbit anti-cryptococcal globulin in glycine buffered saline containing a preservative) DO NOT FREEZE|
|Cryptococcal Antigen Positive Control||1 ml (Purified capsular polysaccharide antigens containing a preservative)|
|Negative Control||1 ml (Normal goat serum containing a preservative)|
|Pronase||1.75 ml (Lyophilized Pronase containing a preservative)|
|Pronase Control||2 ml (Goat anti-rabbit globulin containing a preservative)|
|Pronase Inhibitor||6 ml (Contains an inhibitor for the Pronase)|
|Disposable Ring Slides||xxxxxx|
Possible precaution when performing Cryptococcus antigen test
- Do not use reagents containing foreign matter, particulates or aggregates, which indicate contamination or improper storage or handling.
- Specimens must not contain bacteria, visible lipids, or other obvious signs of contamination.
- NEVER heat inactivate Pronase Control as this could cause aberrant control reactions.
- Do not store specimens in a frost-free type freezer. Repeated freezing and thawing of the specimens can affect test results.
- Do not store rehydrated Pronase in a frost-free type freezer.
- Care should be taken not to introduce syneresis fluid, which is present in various types of agar, into any specimens prior to testing as this may cause spurious results.
- When handling patient specimens, adequate measures should be taken to prevent exposure to etiologic agents potentially present in the specimen.
- All reagents are preserved with sodium azide [0.095% (w/w)], which is a skin irritant. Avoid skin contact with the kit components.
- Do not mix reagents with acid as this may result in the formation of hydrazoic acid, an extremely toxic gas.
- Additionally, disposal of reagents containing sodium azide into lead or copper plumbing can result in the formation of explosive metal azides.
- It is therefore recommended that excess reagents simply be discarded in an appropriate waste receptacle.
Preparation of cryptococcal reagents
The preparation of cryptococcus reagents is done by reconstituting or diluting the following reagents with the indicated volume of distilled or deionized water.
- Pronase 1.75 ml : The reconstituted Pronase should be aliquoted into test tubes in 0.05 ml (50 µl) amounts, sealed, and frozen immediately at –20°C or colder. Also do not use siliconized tubes.
- Specimen Diluent Dilute 1:10
- Latex solutions must appear as homogeneous suspensions.
- When using the negative control for the first time, heat inactivate at temperature 56°C for 30 minutes.
- Also the ring slides should be discarded after each use.
Preparation of specimen for cryptococcus antigen test
Suspected specimens required for this diagnostic are Serum and Cerebrospinal fluid(CSF)
How to prepare serum for Cryptococcus antigen test
- Start by collecting the whole blood aseptically following accepted procedures.It should be collected in a plain tube without anticoagulant as this will invalidate the test
- Allow the blood to clot for 10 minutes or more at room temperature in the tube.
- Now centrifuge the specimen at 1500 rpm for 15 minutes
- Carefully aspirate the serum into a sterile container and seal it.
- The specimen may be processed immediately or preserve by refrigeration, freezing at -20°C or by adding thimerosal to provide a final concentration of 0.01%.
- Add 300 µl of serum to 50 µl aliquot of Pronase and seal the tube with parafilm.
- Incubate serum/Pronase solution at 56°C for 30 minutes.
- Add 1 drop of Pronase Inhibitor then mix to terminate enzymatic digestion.
- From here,the specimen is ready for testing
How to prepare CSF for Cryptococcus antigen test
- Collect the specimen aseptically following accepted procedures.
- Then you can centrifuge it at 1500 rpm for 15 minutes to ensure the separation of all white cells and particulate matter.
- Then,carefully aspirate the CSF into a sterile container and seal it.
- This specimen may be processed immediately or preserve by refrigeration,freezing at –20°C or by adding thimerosal to provide a final concentration of 0.01%. 5.
- Incubate csf fluid specimen at 100°C for 5 minutes.
- The specimen is ready for testing
Cryptococcus antigen test procedure (Screening method)
- Start by adding 25 µl of Cryptococcus Antigen Positive Control, Negative Control and each heat-treated CSF and/or Pronase-treated serum specimen onto separate rings of the ring slide. Take note to use a new pipette tip for each specimen and reagent.
- Now add 25 µl of Cryptococcal Latex to each ring.
- Using separate applicator sticks, thoroughly mix the contents of each ring.
- Then rotate by hand or place the ring slide on a rotator set to 100 rpm (+/- 25) for 5 minutes at room temperature.
- Read the reactions immediately
Cryptococcus antigen test procedure (Titration method)
Titration method is initiated when screening method is reactive (1 + or greater reaction).This is done by
- Adding 100 µl of Specimen Diluent to each of 10 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.
- Add 100 µl of patient specimen to tube number 1 and mix well.
- Then transfer 100 µl from tube number 1 to tube number 2 and mix well.
- Continue this dilution procedure through tube until tube number 10 (Serial dilution)
- Add 25 µl of Cryptococcus Antigen Positive Control , Negative Control and each specimen dilution onto separate rings of the ring slide.
- Add 25 µl of Cryptococcal Latex to each ring.
- Using separate applicator sticks, thoroughly mix the contents of each ring.
- Rotate by hand or place the ring slide on a rotator set to 100 rpm (+/- 25) for 5 minutes at room temperature.
- Read the reactions immediately
Reading Cryptococcus antigen test result
The reactions is read immediately over a dark background and rate on a scale from negative to 4+. For comparison, the Cryptococcus Antigen Positive Control should give a 2+ or greater reaction while the Negative Control should be less than 1+
The graduations of the test reaction strengths are as follows:
|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 a clear background|
|Four Plus (4+)||large clumps against a very clear background|
Interpretation of Cryptococcus antigen result (Serum and csf interpretation)
|Control Reactions||The Cryptococcal Antigen Positive Control must be 2+ or greater, and the Negative Control must be less than 1+ with the Cryptococcal Latex. If either control is incorrect, one or both of the reagents is unsatisfactory (or the tests were performed improperly) and any patient tests with the reagents are invalid. A positive reaction with the Negative Control may indicate possible contamination or freezing of the Cryptococcal Latex, which could produce false positive results in patient specimens. The Pronase Control detects the presence of rabbit globulin on the latex particles. Failure of the Pronase Control to give a positive reaction indicates that one of the reagents is unsatisfactory.|
|Negative Test||If the screening test performed on the undiluted patient specimen was negative or a 1 + reaction, then the test should be reported as negative. However, 1 + reactions may be suggestive of Cryptococcosis . If the clinical symptoms of the patient are suggestive of Cryptococcosis, subsequent specimens and culture are strongly recommended. Weakly reactive specimens (e.g. 1+) should be checked for prozone effect of high titers by testing using the Titration Procedure . If prozoning is suspected, repeat the test with both a 1:10 and a 1:100 dilution of the specimen.|
|Positive Test||If a 2+ or greater reaction is observed in the Screening Procedure, then the specimen is titrated using the Titration Procedure. The positive titer is reported as the highest dilution showing a 2+ or greater reaction.|
Possible limitation of the test method
- Note that a negative test does not exclude the possibility of cryptococcal infection, particularly when a single patient specimen has been tested and the patient has symptoms consistent with Cryptococcosis.False-negative reactions may be caused by low titers, early infection, presence of immune complexes, prozone effect of high titers or poorly encapsulated strains with low production of polysaccharide.
- False-positive reactions can occur due to the presence of rheumatoid factor,agar syneresis fluid,Capnocytophaga canimorsus, Trichosporon beigelii, hydroxyethyl starch, sera with >200 mg Fe3+/dL (10), improper cleaning of the ring slide and non-specific reactivity in HIV-infected patients.
- Also Pronase treatment has been shown to reduce false positives,increase titers and increase sensitivity in serum specimens.
- D. Palmer, L. Kaufman, W. Kaplan, and J. Cavallaro (eds.) 1977. Slide Latex Agglutination test for Cryptococcal Antigen, p. 94- 106. Serodiagnosis of Mycotic Diseases. CC Thomas, Springfield, IL.
- Bauman, S. K. Unpublished Data. 2002.
- Bennett, J. E. and J. W. Bailey. 1971. Control for rheumatoid factor in the latex test for cryptococcosis. Am.J.Clin.Pathol. 56:360-365.
- Bennett, J. E., H. F. Hasenclever, and B. S. Tynes. 1964. Detection of Cryptococcal polysaccharide in serum and spinal fluid: Value in diagnosis and prognosis. Trans.Assoc.Am.Physicians 77:145-150.
- Blevins, L. B., J. Fenn, H. Segal, P. Newcomb-Gayman, and K. C. Carroll. 1995. False-positive cryptococcal antigen latex agglutination caused by disinfectants and soaps. J.Clin.Microbiol. 33:1674-1675.
- Bloomfield, N., M. A. Gordon, and F. Elmenforf. 1963. Detection of Cryptococcus neoformans Antigen in Body Fluids by Latex Particle Agglutination. Proc.Soc.Exp.Biol.Med. 114:64-67.
- Boom, W. H., D. J. Piper, K. L. Ruoff, and M. J. Ferraro. 1985. New cause for false-positive results with the cryptococcal antigen test by latex agglutination. J.Clin.Microbiol. 22:856-857.
- Clumeck, N., J. Sonnet, H. Taelman, F. Mascart-Lemone, M. De Bruyere, P. Vandeperre, J. Dasnoy, L. Marcelis, M. Lamy, and C. Jonas. 1984. Acquired immunodeficiency syndrome in African patients. N.Engl.J.Med. 310:492-497
- Diamond, R. D. and J. E. Bennett. 1974. Prognostic factors in cryptococcal meningitis. A study in 111 cases. Ann.Intern.Med. 80:176-181.
- Eberhard, T. H. 1993. False-positive reactions in cryptococcal antigen determination. Am.J.Clin.Pathol. 100:364.
- Engler, H. D. and Y. R. Shea. 1994. Effect of potential interference factors on performance of enzyme immunoassay and latex agglutination assay for cryptococcal antigen. J.Clin.Microbiol. 32:2307-2308.
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