Respiratory mycoses (reduced version) Dr Elizabeth M. Johnson, Director HPA Mycology Reference Laboratory, Bristol

Kingdom Fungi Eucaryotic organisms Eucaryotic organisms No chlorophyll No chlorophyll Absorb nourishment from living or dead organic material Absorb nourishment from living or dead organic material Filamentous form: septate or aseptate hyphae Filamentous form: septate or aseptate hyphae Yeasts: unicellular fungi Yeasts: unicellular fungi Sexual and asexual forms Sexual and asexual forms

Inhalation of fungal spores Spores 8 µm SINUSITIS PULMONARY ASPERGILLOSIS DISEASE SPECTRUM ALLERGY ASPERGILLOMA DISEMINATEDASPERGILLOSIS 2-4 µm

Schizophyllum commune Basidiomycete often found on rotting wood Basidiomycete often found on rotting wood Increasingly recognised as a cause of sinusitis Increasingly recognised as a cause of sinusitis Produces raised fruiting bodies on prolonged incubation Produces raised fruiting bodies on prolonged incubation Look for clamp connections and spicules Look for clamp connections and spicules

Presence of risk factors Clinical signs Laboratory Microscopy & Culture of sputum Microscopy & Culture of sputum and BAL Serology - detection of antibodies and fungal antigens Detection of fungal DNA using PCR and BAL Serology - detection of antibodies and fungal antigens Detection of fungal DNA using PCR Diagnosis of respiratory mycoses

Patchy changes seen on chest radiograph of patient with invasive aspergillosis

Aspergillosis: radiological findings in the lungs Halo sign on CT scan - early finding (1-10 days) Crescent-shaped lesions on chest radiograph - late sign following neutrophil recovery - late sign following neutrophil recovery Halo sign Halo sign Air crescent Air crescent

Aspergillus hyphae in a sputum sample Microscopy of a mucoraceous mould

Absidia corymbifera

Fungal serology - Antigens Latex agglutination ELISA Tests for antigenaemia Asp Cryp Asp Cryp TARGET Cell wall components :Cell wall components : mannans mannans galactomannan galactomannan 1,3 beta-D-glucans 1,3 beta-D-glucans Cytoplasmic antigensCytoplasmic antigens MetabolitesMetabolites Aspergillus ELISA Rat EBA-2 monoclonal antibody directed against galactomannan sensitivity - 1 ng/ml G - test Amebocyte limulus lysate test Horse shoe crab coagulation cascade sensitivity - 1 pg/ml

Pitfalls of PCR Choice of sample Blood, Serum or Plasma BAL Tissue Method of DNA extraction Mechanical Chemical Enzymes Contamination: Environmental Amplicon Absence of standardization of reagents and targets

Infection with Scedosporium spp. Pseudallesheriosis - sexual form (teleomorph) Pseudallescheria boydii Pseudallesheriosis - sexual form (teleomorph) Pseudallescheria boydii Clinically indistinguishable from invasive aspergillosis Clinically indistinguishable from invasive aspergillosis Specific features: Infection often follows near-drowning accidents Neutropenia is a risk factor Refractory to treatment

Scedosporium apiospermum Scedosporium prolificans

Infection with Fusarium spp. Clinically very similar to invasive aspergillosis Specific features: Cutaneous lesions (70%) Blood culture often positive (50-70%) Often water borne Sinus infection may lead to disseminated disease

Fusarium solani

Cryptococcosis in the UK Year No. cases AIDS-defining illness Knight et al J Infect 27:

Cryptococcus neoformans CAN infect healthy, non-immunocompromised hosts CAN infect healthy, non-immunocompromised hosts Today mainly associated with HIV-infected patients Today mainly associated with HIV-infected patients AIDS-defining illness AIDS-defining illness HAART has had an impact HAART has had an impact Mode of infection is inhalation but haematogenous Mode of infection is inhalation but haematogenous dissemination leads to meningitis dissemination leads to meningitis Diagnosis by direct microscopy or LPA antigen test Diagnosis by direct microscopy or LPA antigen test

Indian ink stain of Cryptococcus neoformans

Pneumocystis carinii var. hominis = Pneumocystis jiroveci

Coccidioides immitis Cause of coccidioidomycosis Cause of coccidioidomycosis Endemic areas southwestern USA, Endemic areas southwestern USA, Central and South America Central and South America Infection by inhalation of arthroconidia Infection by inhalation of arthroconidia Mild transient pulmonary infection Mild transient pulmonary infection 1% develop disseminated disease 1% develop disseminated disease

Coccidioides immitis distribution Endemic areas: southwestern USA Central and South America From: Medical Mycology Kwon-Chung and Bennett 1992 Lea & Febiger

Coccidioides immitis life cycle From: Medical Mycology Kwon-Chung and Bennett 1992 Lea & Febiger

Direct microscopy Endosporulating spherules seen on direct microscopy of pus, sputum or joint fluid Difficult to obtain in culture Culture Mycelial colonies after incubation at ºC for 2-7 days Growth on actidione Typical arthroconidia Exoantigen test to confirm Gene-probe also available Laboratory diagnosis of coccidioidomycosis Serology Immunodiffusion and complement fixation

Histoplasmosis Guano-enriched soil source Guano-enriched soil source Tissue form: budding yeasts Tissue form: budding yeasts Reticuloendothelial system Reticuloendothelial system Chronic cases - calcifications Chronic cases - calcifications Widespread geographically Widespread geographically - especially Eastern USA, & Africa - especially Eastern USA, & Africa Histoplasma var. capsulatum and var. duboisii Histoplasma var. capsulatum and var. duboisii

Histoplasmosis

Direct microscopy Small, oval, budding yeast cells seen in Giemsa stained smears Cells often seen within macrophages Culture Mycelial colonies after incubation at ºC for 2-7 days Conversion to yeast on blood agar or BHI agar at 37ºC, 2-6 weeks Laboratory diagnosis of histoplasmosis Serology Immunodiffusion and complement fixation

Sensitive and specific Sensitive and specific High positive predictive value High positive predictive value High negative predictive value High negative predictive value Simple, rapid and inexpensive Simple, rapid and inexpensive The ideal diagnostic test