1. ASPERGILLOSIS A variety of diseases: pulmonary, external ears, eyes, meninges, sinuses or blood stream

2. 1.ALLERGIC 2.FUNGUS BALL 3.INVASIVE

3. Difficult to diagnose 1.Clinical symptoms are not specific 2.Radiography not specific (except fungus ball) 3.Blood cultures seldom positive 4.Serology seldom positive (early) 5.Need invasive procedures for early detection

4. A. FUMIGATUS A. NIGER A. FLAVUS

6. GEOGRAPHIC DISTRIBUTION WORLD-WIDE

7. SOIL DECAYING VEGETATION FOOD MEDICATION AIR VENTS DISINFECTANTS

8. More than 900 species Slow growing Various gross colors Spores –Size –Shape –Texture –color

10. DICHOTOMOUS BRANCHING WIDE, SEPTATE HYPHAE

12. Aspergilloma Cavity wall

14. Aspergillosis Aspergillosis is a spectrum of diseases of humans and animals caused by members of the genus Aspergillus. These include (1) mycotoxicosis due to ingestion of contaminated foods; (2) allergy to the presence of conidia or transient growth of the organism in body orifices; (3) colonization without extension in preformed cavities and debilitated tissues; (4) invasive, inflammatory, granulomatous, necrotizing disease of lungs, and other organs; and rarely (5) systemic and fatal disseminated disease. The type of disease and severity depends upon the physiologic state of the host and the species of Aspergillus involved. Distribution: World-wide. Aetiological Agents: Aspergillus fumigatus, A. flavus, A. niger, A. nidulans and A. terreus.

16. Verycommon: compost heaps, airbone dustUsuallyaffects the lungs and sinusesLess often: very agressive, spread trough outthelungs and bloodstream to brain and kidneys

17. Aspergillosis– Symptoms& Diagnosis May cause no symptoms fungus ball Rx) Reapeted coughing up of blood (severebleeding) Fever Chestpain Difficulty breathing

18. Aspergillosis-Symptoms& Diagnosis Can affect deepertissues: 1.Renalfailure 2.Liverfailure 3.Shock 4.Delirium 5.Boodclots 6.Deathcanoccurquickly Chestx-ray Computedtomography(CT) Wheneverpossible : sendmaterialto a laboratoryto confirmidentification

19. Aspergilloma found at post-mortem in the lung of a child with leukaemia. Note fungus ball occupying cavity.

20. Aspergilloma found at post-mortem in the lung of a child with leukaemia.

22. Aspergillosis in air sacs of a hen during an epidemic of aspergillosis in poultry.

23. Grocott’s methenamine silver (GMS) stained tissue section of lung showing fungal balls of hyphae of Aspergillus fumigatus.

24. Grocott’s methenamine silver (GMS) stained tissue section of lung showing dichotomously branched, septate hyphae of Aspergillus fumigatus.

25. Grocott’s methenamine silver (GMS) stained tissue sections showing Aspergillus fumigatus in lung tissue, note conidial heads forming in an alveolus.

27. Aspergillus fumigatus on Czapek dox agar showing typical blue-green surface pigmentation consisting of a dense felt of conidiophores.

28. 038 Microscopic morphology of Aspergillus fumigatus showing typical columnar, uniseriate conidial heads. Conidiophores are short, smooth-walled and have conical shaped terminal vesicles, which support a single row of phialides on the upper two thirds of the vesicle. Conidia are produced in basipetal succession forming long chains (slide 038), however, during preparation of slides the conidial chains are usually disrupted giving the more typical microscopic appearance seen in slide 039. Conidia are globose to subglobose, green and rough-walled to echinulate

29. Microscopic morphology of Aspergillus fumigatus showing typical columnar, uniseriate conidial heads. Conidiophores are short, smooth-walled and have conical shaped terminal vesicles, which support a single row of phialides on the upper two thirds of the vesicle. Conidia are produced in basipetal succession forming long chains (slide 038), however, during preparation of slides the conidial chains are usually disrupted giving the more typical microscopic appearance seen in slide 039. Conidia are globose to subglobose, green and rough- walled to echinulate.

30. Aspergillus niger on Czapek dox agar. Colonies consist of a compact white or yellow basal felt covered by a dense layer of dark-brown to black conidial heads.

31. 041 Microscopic morphology of Aspergillus niger showing large, globose, dark brown conidial heads, which become radiate, tending to split into several loose columns with age. Conidiophores are smooth-walled, hyaline or turning dark towards the vesicle. Conidial heads are biseriate with the phialides borne on brown, often septate metulae. Conidia are globose to subglobose, dark brown to black and rough-walled.

32. Aspergillus flavus on Czapek dox agar. Colonies are granular, flat, often with radial grooves, yellow at first but quickly becoming bright to dark yellow-green with age.

33. Microscopic morphology of Aspergillus flavus. Conidial heads are typically radiate, later splitting to form loose columns, biseriate but having some heads with phialides borne directly on the vesicle. Conidiophores are hyaline and coarsely roughened, often more noticeable near the vesicle. Conidia are globose to subglobose, pale green and conspicuously echinulate. Some strains produce brownish sclerotia.

34. Aspergillus nidulans on Czapek dox agar showing typical plain green colony with dark red-brown cleistothecia developing within and upon the conidial layer. Reverse may be olive to drab-grey or purple-brown.

35. Microscopic morphology of Aspergillus nidulans. Conidial heads are short columnar and biseriate. Conidiophores are usually short, brownish and smooth-walled. Conidia are globose and rough-walled.

36. Aspergillus terreus on Czapek dox agar showing typical suede-like cinnamon-buff to sand brown colonies. Reverse yellow to deep dirty brown.

37. Conidial head of Aspergillus terreus. Conidial heads are compact, columnar and biseriate. Conidiophores are hyaline and smooth-walled. Conidia are globose to ellipsoidal, hyaline to slightly yellow and smooth- walled.

38. Immunodiffusion test showing precipitins against Aspergillus.

40. Antifungal susceptibility disk test showing the in vitro activity of voriconazole against Aspergillus fumigatus with Candida krusei as a control.

41. Aspergillosis-Prognosis& Treatment Whenpresentin a sinus or single spot in the lung progresses SLOWLY Requires treatment, but no immediatedanger If infection widespread: patient isseriously ill Voriconazole Amphotericinb Itraconazole Capsofungin

42. If fungus balls grow near lungs.Blood vessels removed surgically Sinuses aspergillosis removed surgically Ear canal infection scrapingout the fungus and topics antifungal drops

43. New (and newer) antifungals for invasive aspergillosis Azoles itraconazole (i.v.) voriconazole posaconazole ravuconazole BAL 8557/4815 Echinocandins caspofungin micafungin anidulafungin aminocandin Polyenes ABLC, ABCD, AmBisome liposomal nystatin inhaled amphotericin B Note: ABLC=amphotericin B lipid complex; ABCD= amphotericin B colloidal dispersion Note: Blue text, earlier stage development

44. Invasive Aspergillosis in Transplant Recipients Type of TransplantIncidence Range, % (Mean) Mortality (%) Lung3-14% (6%)68% Liver1-8 (2)87 Heart1-15 (5)78 Kidney0-4 (1)77 Small bowel0-10 (2)66 Allogeneic stem cell5-26 (10)78-92 Autologous stem cell2-6 (5)78-92 Nonmyeloblative stem cell8-23 (11)63-67 Singh N & Paterson DL, Clin Microbiol Rev 2005;18:44-69.

45. Aspergillus spp. Isolates Submitted to San Antonio Fungus Testing Laboratory 918 Isolates; Jan. 2001-July 2004 AmB=Amphotericin B; MFC=Minimum Fungicidal Concentration; MIC=Minimum Inhibitory Concentration Sutton D et al, Advances Against Aspergillus 2004 (Abstract 16) A. nidulans 3% AmB MFC >16  A. fumigatus 24% AmB MIC>2  A. terreus 90%  A. flavus 51%  A. ustus 50%

46. Lipid Preparations of Amphotericin B: Rationale for Use Polyene: broad spectrum of activity Lipid formulations of amphotericin B –Reduced toxicities of intravenous amphotericin B deoxycholate –Improved therapeutic index: ≥5 mg/kg/d well tolerated Salvage therapy (limited efficacy  40% responded) Empiric therapy (reduced efficacy vs moulds at lower doses) Limited data for primary therapy; most studies in empirical use Target use for patients with documented need (eg Zygomycosis, intolerance or progressive infection) –Cost remains significant obstacle to use!

47. Bowman et al. Antimicrobial Agents Chemother 2002;46:3001-3012 In Vitro Effects of Echinocandins on Growth of Aspergillus In vitro activity: Not classically fungicidal or fungistatic Activity against other Aspergillus spp. (A terreus) Animal models prolonged survival Living Cells Dead Cells Control CellsAmBCaspoItra Amphotericin B 0.15  g/mL Caspofungin 0.30  g/mL Itraconazole 2.6  g/mL

48. Echinocandins in Invasive Aspergillosis Study conducted in patients with well-documented, refractory infection Efficacy –Progressive infection –Multiple prior antifungals Excellent tolerability Clinical utility for moulds –Combination therapy (not primary therapy) –Activity: Aspergillus (not Zygomycetes or other moulds) Maertens J et al. Clin Infect Dis 2004;39:1563-71 % %

49. Diagnostic Strategies in Invasive Aspergillosis Consideration of risk Role of mycological diagnosis –Predictive value of positive cultures in high risk patients Utility of radiological procedures Non-culture based diagnostics –Impact of antifungal therapies –Value of serial samples –Significance of false negative/false positive results –Role of testing in other body fluids, including CSF & BAL Role of surrogate markers in decision making & impact on mortality

50. Combination Therapy: Candins In vitro –Most interactions show synergy / additive effects (Perea, 2002) –Poor correlation between in vitro results and in vitro efficacy (Johnson, 2004) Experimental infections –Candin plus polyene (Kohno, 2000; Nakajima, 2000) –Candin plus azole (Kirkpatrick, 2002; Petraitiene, 2002) Improved sterilization of tissues Reduced tissue burden Anecdotal clinical series –Candin+polyene (Aliff, 2003; Kontoyiannis, 2003; Ratanatharathorn, 2002) –Candid plus azole (Marr, 2004)

51. Efficacy of Empirical Antifungal Therapy in Neutropenic Patients Walsh TJ et al, New Engl J Med 2002;346:225-34 21/422 (5%) 8/415 (1.9%) Voriconazole vs liposomal amphotericin B  Composite success: 26% vs 31%  High risk patients: 18% allogeneic BMT  Similar survival, fever resolution, toxicity or lack of efficacy  Fewer breakthrough infections Efficacy in high risk:  Breakthrough infections: 2/143 (2%) vs 13/143 (9%)

52. Caspofungin vs Liposomal Amphotericin B for Empirical Antifungal Therapy in Patients with Fever & Neutropenia Walsh TJ et al, New Eng J Med, 2004;351:1391-1402 *Patients may have had more than one organism Caspofungin (n=556) Liposomal Amphotericin B (n=539) Composite Success33.9%33.7% Success Baseline Infections14/27 (52%)7/27 (26%) Breakthrough Infections29 (5.2%)24 (4.5%) Etiological Agents* Aspergillus109 Candida1615 Fusarium10 Zygomycetes20 Other11

53. Invasive Aspergillosis: Polyenes, Azoles or Echinocandins? Importance of early detection –Role of radiological diagnosis –Non-culture based diagnostics Importance of serial samples Impact of prior therapy –Poorer outcomes with extensive disease Poor efficacy of amphotericin B in high risk patients –Improved responses with early effective therapy –Utility of early targeted therapy Role of new agents in invasive aspergillosis –Efficacy of voriconazole as primary therapy –Options for salvage therapy: posaconazole, echinocandins, lipid amphotericin formulations Clinical trials needed combination therapy