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

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

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

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

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

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: 48-72 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

Antigen detection Specimen: Serum/plasma, finger prick (whole blood), CSF, urine, BAL Both screening and diagnostic tools Latex agglutination Sensitivity : 93-100% 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

Advantages of CrAg-LFA Rapid turn around time (10-15 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

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

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

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

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