1. Diagnosis of Cryptococcal disease
Professor Malcolm D. Richardson
Mycology Reference Centre, Manchester
Manchester University NHS Foundation Trust

2. Intended Learning Outcomes
To be aware of the different diagnostic modalities available
To understand the advantages and disadvantages of each diagnostic modality
To be aware of the histological differential diagnoses for Cryptococcus

3. How is cryptococcosis diagnosed?
Microscopy
Radiology
Antigen detection tests
Culture
Molecular identification
Histopathology
Compared to other fungal infections, diagnosis of Cryptococcosis is less difficult: Isolation of crypto in culture from body fluids, tissues or detection of capsular antigens in blood, CSF or urine
Biochemistry

4. Full workup of a suspected case of cryptococcosis
Cerebrospinal fluid: India ink smear, fungal culture, and cryptococcal antigen (CrAg) testing
Cutaneous lesions: Biopsy with fungal stains and cultures
Blood: Fungal culture, cryptococcal serology, and CrAg testing
Urine/seminal fluid: Fungal culture, CrAg testing
Sputum/BAL: Fungal cultures

5. Microscopy
Sample: CSF, host fluids or secretions
India ink or nigrosin used as background stain.
Encapsulated elongated yeast-like cells (blastoconidia).
Most rapid method of identification.
India ink preparation showing narrow-based budding yeast cell & capsular halo
India ink is usually positive in 70-80% of HIV patients with cryptococcal meningitis compared to 30-50% positivity in HIV negative individuals.
Persistent India ink positivity in a patient on treatment may signify treatment failure.
False +ve: intact lymphocytes
India ink is less sensitive than cryptococcal antigen

6. Culture Sample: CSF, blood, sputum, skin biopsy
Isolated in 75-90% of CSF samples of meningitis cases and ~35-70% blood cultures
Grows best at 30-35◦C on standard microbiological agar.
Encapsulates better on Chocolate agar
Aerobically incubated
Observations: hours and longer (up to 4 weeks) for patients on antifungal therapy
White-cream, mucoid (milky) colonies of Cryptococcus
Turn orange-tan or brown on prolonged incubation
Special agar plates:
Bird-seed agar and
Canavanine-glycine-bromothymol blue (CGBTB) agar can be used to differentiate C. gattii from C. neoformans
CGBTB agar
Bird seed agar

7. Antigen detection
Specimen: Serum/plasma, finger prick (whole blood), CSF, urine, BAL
Both screening and diagnostic tools
Latex agglutination
Sensitivity : %
Specificity: 93-98%
Lateral flow assay
Sensitivity & specificity >99%
Serum, plasma, whole blood, CSF
Sensitivity 85%
Urine sample
False positive:
Rheumatoid factor,
Trichosporon spp,
Stomatococcus mucilaginosus,
K. pneumoniae infections and
contamination with syneresis fluid
False negative:
Prozone effect due to high fungal load
Low concentration of CrAg
Poorly encapsulated /acapsular strains
Latex particle agglutination test

8. Advantages of CrAg-LFA
Rapid turn around time ( minutes)
Minimal requirements for laboratory infrastructures
Can be done at bedside
No specimen pre-treatment required
Stability at room temperature
Wider capture of both C. neoformans and C. gattii antigens
Semi-quantitative

9. Histopathology Yeasts that reproduce by narrow-based budding Samples
Tissues: Lung, skin, bone marrow, brain etc.
Centrifuged CSF/body fluid sediments
More sensitive than India ink
Stains
Polysaccharide capsule
Periodic Acid Schiff (PAS), mucicarmine
Melanin
Fontana-Masson
Cell wall
Calcofluor
Grocott’s (Gomori) methenamine silver stain (GMS)
Yeasts that reproduce by narrow-based budding
Grocott’s Methenamine silver stain
Argentaffin reaction seen in the cell wall ofCryptococcus neoformans (Fontana-Masson).It is of note that C. neoformans is stained with Fontana-Masson's argentaffin reaction. The other brown fungi are also positive. The size of the yeasts becomes much larger, when they grow outside the cell.
Fontana-Masson

10. Histological differential diagnoses for Cryptococcus
Histoplasma spp.
3-5µm var. capsulatum
5-15µm var. duboisii
Narrow-necked budding
Candida glabrata
3-15µm in size
No pseudo hyphae

11. Recommendation
Early HIV diagnosis and initiation of antiretroviral therapy for primary prevention of cryptococcal meningitis
Rapid diagnostic testing for cryptococcal meningitis in meningitis suspects with highly-sensitive point-of-care CrAg lateral flow assay
All HIV+ patients with CD4<100 should be tested for CrAg
Diagnosis and management of common co-morbidities, e.g., tuberculosis and bacterial sepsis

12. END