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

2. 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

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

4. 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

5. 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

6. Patchy changes seen on chest radiograph of patient with invasive aspergillosis

7. 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

8. Aspergillus hyphae in a sputum sample Microscopy of a mucoraceous mould

9. Absidia corymbifera

10. 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

11. 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

12. 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

13. Scedosporium apiospermum Scedosporium prolificans

14. 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

15. Fusarium solani

16. Cryptococcosis in the UK 1980 - 1999 Year No. cases AIDS-defining 24 18 24 illness Knight et al. 1993 J Infect 27:185-191

17. 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

18. Indian ink stain of Cryptococcus neoformans

19. Pneumocystis carinii var. hominis = Pneumocystis jiroveci

20. 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

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

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

23. 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 25 - 30ºC 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

24. 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

25. Histoplasmosis

26. Direct microscopy Small, oval, budding yeast cells seen in Giemsa stained smears Cells often seen within macrophages Culture Mycelial colonies after incubation at 25 - 30ºC 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

27. 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