Cryptococcal Antigen Test: Introduction, Principle, Test Procedure, Result

Cryptococcal Antigen Test: Introduction, Principle, Test Procedure, Result Interpretation and Clinical Significance

Introduction of Cryptococcal Antigen Test

Cryptococcal antigen test is a very effective test since it can detect the infection early before it develops into life-threatening cryptococcal meningitis (Cryptococcosis). Fungal infection, Cryptococcosis is caused by the encapsulated yeast Cryptococcus neoformans (serotypes A and D), C. neoformans var. grubii (serotype A) has a worldwide distribution. Individuals with impaired cell-mediated immune (CMI) function due to acquired immunodeficiency syndrome (AIDS), lymphoproliferative disorders, steroid therapy, and organ transplantation is at increased risk of Cryptococcosis. AIDS accounts for 80-90% of cryptococcal infections where Cryptococcosis occurs in 5-10% of AIDS patients in the USA. The incidence of Cryptococcosis in other parts of the world, such as Africa, is as high as 30%. Cryptococcosis is the fourth most common opportunistic infection. In brief, the cryptococcal antigen test is also called the CrAg test. It has several advantages over traditional methods that are available in resource-limited areas for detecting Cryptococcus, that include microscopy (India Ink staining) and culture. Both of these methods have limited sensitivity, which means that the test is not always positive in people suffering from an infection. Also, identification of organisms through a culture of Cryptococcus can take weeks to obtain a final result. In contrast, antigen detection is highly sensitive and specific, and the results are available quickly by a simple attempt. CrAg test can be performed in the following ways-Lateral flow assay (LFA), Latex agglutination test (LAT), and Enzyme Immunoassay (EIA). The dipstick working on lateral flow assay is validated for use serum, plasma, CSF, or urine. This would allow for diagnosis to occur at the point-of-care (POC) and would increase access to cryptococcal diagnostics in resource-limited settings (without advanced infrastructure). Most importantly, the CrAg test can be positive in patients who do not yet have symptoms of meningitis, which makes it the ideal test to be used for screening in those who are at the highest risk of developing cryptococcal meningitis especially among people with HIV/AIDS having CD4 < 100 cells/µL.

Principle of Cryptococcal Antigen Test

Cryptococcal antigen test works on the principle of immunochromatography. The lateral flow assay (LFA) uses gold-conjugated, two monoclonal antibodies impregnated onto an immunochromatographic test strip. If CrAg is present in a sample, suspended, gold-conjugated antibodies bind to the antigen. The gold-antibody-CrAg complex moves by capillary action up the test trip interact with immobilized monoclonal antibodies against CrAg and forms a red line as shown above image.

Storage of Test Kit

The kit can be stored at room temperature for up to 2 years.

Requirements for Cryptococcal Antigen Test

Test kit-The Immy CrAg LFA kit contains 50 test strips, specimen diluent, titration diluent, and positive control.
Extra we need –

Test specimen ( serum/ plasma/CSF/urine)
Gloves
Waste disposing of bins
Hypochlorite solution

Test procedure for Cryptococcal Antigen Test

Add one drop of specimen diluent to a tube.
Now, add 40 µL (1 drop) of the patient specimen to the tube.
Insert the LFA strip into the tube
Wait for 10 minutes.
Finally, interpret the result.

Result Interpretation of Cryptococcal Antigen Test

Positive: Both red lines at the control and test regions

Negative: Only one red line at the control region

Invalid: No red line or line only at test region and this condition repeat the test with another test LFA strip

Clinical significance of Cryptococcal Antigen Test

It is a simple, rapid, highly accurate test and can be done in a simple setup even in a clinic on various samples like serum, plasma, CSF, or urine.
It can detect antigen early in serum an average of 22 days before symptoms of meningitis develop.
The occurrence of cryptococcal antigen in the serum is highly predictive of who will develop cryptococcal meningitis. It indicates that there is a window of time where cryptococcal disease can be recognized early and treated in order to prevent the early, asymptomatic infection from progressing to meningitis.
It may also apply for screening in those who are at the highest risk of developing cryptococcal meningitis especially among people with HIV/AIDS having CD4 < 100 cells/µL.

Comparison of lateral flow assay with another immunoassay as well as India ink and culture

Lateral flow assay (LFA): No need for pre-treatment of the sample

Highly accurate ( 99.5% sensitivity and 99% specificity)
No advanced setting for test needed
Rapid result (10 minutes)
The overall cost is low.

Latex agglutination test (LAT): Specimen needs pre-treatment with pronase.

Low sensitivity for CrAg of serotype C
No advanced setting for test needed
Rapid result (10-30 minutes, depending on the nature of the sample).
83-100 % sensitivity and 97-100% specificity

Enzyme Immunoassay (EIA): No need for pre-treatment of the sample

Low sensitivity for CrAg of serotypeS C and D
Testing setup with minimal or no infrastructure
Time is taken (35-45 minutes) test
Costly than the above immunoassays
99% sensitivity and 97% specificity

Microscopy (India ink staining): Less sensitive ( 80-85% in AIDS-related cryptococcal meningitis)

It needs to be trained staff to report.

Culture: Less sensitive than immunoassays

In AIDS-related CM, the sensitivity of CSF and blood cultures are approximately 90% and nearly 50-70%, respectively.
Testing setup with advanced infrastructure.
There should be a trained laboratory person.
It requires several days for the final result.

Keynotes on Cryptococcal Antigen Test

False-positive latex agglutination reactions have resulted from infection with Trichosporon beigelii and with the DF-2 bacillus, presumably because these pathogens produce antigens that are cross-reactive with Cryptococcus neoformans polysaccharide.
Worldwide, there are approximately 1 million new cases of HIV-related cryptococcal meningitis (CM) and 625,000 deaths from CM each year. Sub-Saharan Africa has the highest number of estimated cases per year, followed by East, South, and Southeast Asia.
A fingerstick Cryptococcal antigen (CrAg) lateral flow assay (LFA) represents a true point-of-care (POC) test for prevention and diagnosis of AIDS-related.
Cryptococcal antigen (CrAg) test and India ink are not useful for diagnosis of subsequent episodes of cryptococcal meningitis (CM) due to the possibility of long-term positivity and therefore a positive CSF culture for C. neoformans is the only test to confirm mycological relapse or treatment failure.
High cryptococcal burden and slow clearance of infection on treatment, together with altered mental status, are the most important drivers of acute cryptococcal-related mortality and high fungal load can be estimated with a baseline high titer of cryptococcal antigen in serum or CSF (i.e. CrAg-latex > 1:1024). In this line, baseline lateral flow assay (LFA) titers correlate with quantitative cultures and predict 2- and 10-week mortality.
Manufacturers’ data claims that LFA sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) are 99.5%, 99%, 98%, and 99.7%, respectively, using serum, plasma, CSF, or urine, when compared to culture.
Positive serum cryptococcal antigen test alone can not differentiate between early disease and meningitis.
Various studies conducted in Africa have reported the prevalence of detectable serum cryptococcal antigenemia between 2-21% in patients with CD4 < 100 cells/µL entering into HIV care.
An immunocompromised patient with fever, headache, neck stiffens, cryptococcal antigen test positive (serum) may create a situation of a clinician for lumbar puncture (LP).
In the histopathological diagnosis of cryptococcosis, the Giemsa stain and Haematoxylin and Eosin (H/E) stains are not very useful even though other stains i.e. mucicarmine and Alcian blue shows the cryptococcal capsule, Fontana-Manson detects the presence of melanin whereas Grocott’s methenamine silver stain of the fungal cell wall.

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