Cryptococcal Antigen

Cryptococcal antigen (CrAg) is the capsular polysaccharide (glucuronoxylomannan) of Cryptococcus neoformans and Cryptococcus gattii, detected in serum, plasma, whole blood (fingerstick) and CSF. Methods are lateral flow assay (LFA, the preferred test, with results in 10 minutes), latex agglutination (with titration) and enzyme immunoassay.

The LFA has sensitivity and specificity of over 95 to 99% for cryptococcal meningitis and for cryptococcal antigenaemia, and detects antigen weeks to months before symptoms appear. A semi-quantitative LFA titre predicts disease burden and outcome.

Cryptococcal meningitis causes about 190 000 deaths each year, nearly three quarters in sub-Saharan Africa, and accounts for 15 to 20% of HIV-related deaths.

Cryptococcal antigenaemia is present in 4 to 8% of people with CD4 counts under 100 cells/microlitre (up to 10 to 15% in some cohorts), and untreated asymptomatic antigenaemia progresses to meningitis in 25 to 30% within weeks, with high mortality.

Other risk groups are solid organ transplant recipients, people on high dose steroids or biologic therapies, patients with haematological malignancy, sarcoidosis, cirrhosis and idiopathic CD4 lymphopenia, and immunocompetent people (mainly with C. gattii in tropical and temperate outbreak regions).

Indications. Screening of all HIV-positive adults and adolescents with CD4 under 100 cells/microlitre (and under 200 in some programmes) before or at start of antiretroviral therapy, and in anyone who is clinically unwell. Headache, fever, confusion, neck stiffness, seizures, cranial nerve palsy or visual loss in an immunocompromised patient.

Subacute or chronic meningitis of unknown cause. Pulmonary nodules or masses in an immunosuppressed patient. Suspected disseminated fungal infection (skin lesions, bone, prostate). Monitoring after treatment (CSF titre not used to decide cure).

Specimen. Serum or plasma 0.5 to 1 mL, or fingerstick capillary blood (LFA), or CSF 0.5 to 1 mL. A lumbar puncture is done for all antigen positive patients with symptoms or in screening positive patients with neurological features, to exclude meningitis and measure opening pressure. Stable at 2 to 8 °C for days and at room temperature for hours. Urine and saliva are experimental.

Reference values. Lateral flow assay: negative (no test band). Latex agglutination: titre under 1 in 2. A positive LFA is reported with the semi-quantitative band intensity or serum titre (for example, 1 in 5 to 1 in 5120 or higher).

Interpretation.

• Positive CSF CrAg (any titre): cryptococcal meningitis, and a CSF titre of 1 in 1024 or more indicates a high fungal burden with a poor prognosis. CSF CrAg sensitivity is 93 to 100% and specificity 93 to 100%.
• Positive serum CrAg with meningitis symptoms: lumbar puncture to confirm, measure opening pressure (raised in over 50%, and over 25 cmH2O needs therapeutic drainage), and send India ink, culture and CSF CrAg.
• Positive serum CrAg without symptoms (asymptomatic antigenaemia): lumbar puncture to exclude meningitis, then pre-emptive fluconazole in those without meningitis. A serum titre of 1 in 160 or higher (LFA 1 in 160 or more) carries a high risk of meningitis and death.
• Negative CrAg in a patient with high clinical suspicion: unlikely cryptococcosis (negative predictive value over 99% in advanced HIV), though antigen can be negative in early, localised or low burden pulmonary disease, in C. gattii and in rare capsule deficient strains. Repeat and culture when suspicion remains.
• Prozone effect: very high antigen titres may produce a false negative latex or LFA result at low dilution, resolved by dilution of the sample (1 in 10 or 1 in 100).
• CSF opening pressure, white cell count (often under 20 cells/microlitre in HIV, with few inflammatory cells), protein (mildly raised), glucose (low or normal) and positive India ink or culture complete the picture.
• Serial CrAg titres decline slowly and are not used to monitor therapy. Cure is judged by clinical response and sterile CSF culture at 2 weeks.

Critical values and findings needing immediate action.

• Positive CSF CrAg or India ink: cryptococcal meningitis, start induction therapy with liposomal amphotericin B (or amphotericin B deoxycholate) with flucytosine, or a single high dose liposomal amphotericin B regimen in settings with limited access, for 1 to 2 weeks. Fluconazole consolidation for 8 weeks and maintenance follows.
• Raised CSF opening pressure (over 25 cmH2O) or symptoms of raised pressure (severe headache, vomiting, visual loss, papilloedema): daily therapeutic lumbar puncture until pressure and symptoms settle (removal of 10 to 30 mL to reduce pressure by about 50%, or to under 20 cmH2O).
• Antigen positive patient with CD4 under 100: delay antiretroviral therapy for 4 to 6 weeks after starting antifungal therapy in meningitis to reduce immune reconstitution inflammatory syndrome, but start earlier in antigenaemia without meningitis.
• Altered consciousness, seizures or focal signs: neuroimaging, urgent admission and monitoring of potassium and renal function on amphotericin.
• Transplant or biologic treated patient with positive CrAg: urgent infectious disease review and consideration of reduction of immunosuppression.

Interferences and pitfalls.

• False positive latex agglutination occurs with rheumatoid factor (treated by pronase), Trichosporon, Capnocytophaga, Stomatococcus and some disinfectants or soaps, and with contaminated reagents. The LFA has fewer false positives but occasional cross-reaction with Trichosporon and Capnocytophaga.
• False negatives from the prozone effect (dilute samples), from low level or localised infection, and from some strains with poorly encapsulated cells (rare).
• LFA reading must follow the manufacturer's time (10 minutes), and a faint band is read as positive. A faint serum band with a low titre in an asymptomatic person needs confirmation.
• Serum CrAg titre does not show treatment response, and it can remain positive for months after cure.
• A negative India ink does not exclude cryptococcal meningitis (sensitivity 70 to 90% in HIV and lower in non-HIV), and CSF CrAg and culture are more sensitive.
• Culture of CSF (Sabouraud agar, birdseed agar for C. neoformans) takes 2 to 7 days (up to 2 to 3 weeks), and confirms viable organisms and allows susceptibility testing.
• Cryptococcal antigen screening is cost effective only when CD4 counts are under 100 to 200, and screening must link to treatment and lumbar puncture capacity.
• Immune reconstitution inflammatory syndrome after antiretroviral therapy can cause new symptoms and cultures that stay negative.
• Amphotericin toxicity (nephrotoxicity, hypokalaemia, hypomagnesaemia, anaemia) requires pre-hydration and electrolyte replacement.
• Fluconazole monotherapy is inferior for meningitis and is used only for asymptomatic antigenaemia, consolidation or maintenance, or where no alternative exists.

Special groups.

• Children: less common, with presentation as meningitis, and screening in HIV-positive adolescents.
• Pregnancy: fluconazole in the first trimester is teratogenic (amphotericin B is used for treatment).
• Older adults and transplant recipients: atypical presentation, with pneumonia and subtle neurological signs.
• Immunocompetent patients: C. gattii is more likely, with large brain and lung cryptococcomas and a higher rate of raised intracranial pressure.
• Regions: sub-Saharan Africa and Southeast Asia carry the greatest burden, and point-of-care LFA screening linked to treatment programmes has reduced mortality.

Follow up tests.

• CSF opening pressure, cell count, protein and glucose, India ink, CSF fungal culture and CrAg titre
• Blood fungal culture
• CD4 count and HIV viral load
• Creatinine, potassium and magnesium (before and during amphotericin)
• Full blood count
• Chest radiograph or CT
• MRI or CT brain
• Ophthalmology (visual acuity and fundus)
• Serum CrAg titre at baseline
• Hepatitis B and C screening
• Drug susceptibility of the isolate