1. Invasive aspergillosis in patients taking steroids
Alessandro C. Pasqualotto Santa Casa de Porto Alegre

2. Potential conflicts of interest
Research Grants
Myconostica, Pfizer, Merck, Sigma-Tau, CAPES, CNPq, Fungal Research Trust
Travel Grants
Pfizer, United Medical, Schering (now Merck), Bagó, Merck
Speaker honoraria
Pfizer, United Medical, Merck, Schering (now Merck), Biometrix

3. Aspergillus love steroids
A fact:
Aspergillus love steroids

4. Steroids and Aspergillus
Lymphocytes
Lymphopenia, decreased lymphokine production (e.g, TNF, -INF),Th1/Th2 dysregulation
Neutrophils
Defective chemotaxis, phagocytosis, degranulation, NO production, adherence
Lionakis M, Kontoyiannis DP. Lancet 2003; 362:

5. Steroids and Aspergillus
Monocytes / macrophages
Monocytopenia
Inhibition of pro-inflammatory cytokine production
Decreased chemotaxis
Impaired phagocytosis
Impaired antigen-presenting capacity by DC
DC = dendritic cells
Lionakis M, Kontoyiannis DP. Lancet 2003; 362:

6. Steroids enhance Aspergillus growth 30-40% increase in growth rate
Ng TTC, et al. Microbiology 1994; 140:

7. Neutrophil-mediated damage of A
Neutrophil-mediated damage of A. fumigatus hyphae is reduced after exposure to dexamethasone
Lewis RE, Kontoyiannis DP. Med Mycology 2008; S1-11

8. What about clinical data?

9. IA in allogeneic HSCT Marr K, et al. Blood 2002; 100: 4358-66
This has been recently confirmed in another investigation by Kieren Marr and colleagues. Here we should mention that IA occurs late in many of these patients
Marr K, et al. Blood 2002; 100:

10. IA in SOT recipients Renal transplantation
Risk correlates with steroid dosage
Prednisone >1.25 mg/kg/d
Gustafson and colleagues reported the risk of IA significantly increases in renal transplant patients with prednisone doses greater than 1 mg/kg/day.
Gustafson TL, et al. J Infect Dis 1983; 148: 230-8

11. IA in SOT recipients Renal transplantation
Risk correlates with steroid dosage
Prednisone >1.25 mg/kg/d
Liver, heart and lung tx recipients
Peri-operative steroid administration and boluses given to prevent rejection
Similarly, peri-operative glucocorticoid administration and boluses administered to prevent acute rejection were found to predispose patients to aspergillosis in liver, heart and lung transplant recipients.
Patterson JE. Transpl Infect Dis 1999; 1:

12. IA after neurosurgery n=25 Steroids: 52.0%
Many of these patients were also on steroids
Pasqualotto AC, Denning DW. Clin Microbiol Infect 2006; 12:

13. IA in patients with solid tumours
Series with 13 patients
Only 1 was neutropenic
46% received steroids within 30 days
Median total cumulative dose 695 mg
Ohmagari N, et al. Cancer 2004; 10:

14. Aspergillus causing VAP
Meersseman W, et al. Am J Respir Crit Care Med 2008; 177: 27-34

15. IA, COPD and steroids 57 cases over a decade in Madrid
98% taking steroids
Cumulative dosage >700 mg in 73.6%
GOLD staging
III (63.2%); IV (33.8%)
Overall mortality was 72%
Guinea J, et al. ICAAC 2008 (Abstract M-2161)

16. IA and inhaled steroids
Case reports only (rare)
Fluticasone
COPD / asthma
Ha relatos ate de ai associada com steroids inalatorios; neste caso, em pts usando steroids potentes e com dca pulmonar. Assim, nao apenas a droga e’ importante como fator de risco, mas tb a doenca do paciente
Peter E, et al. Clin Infect Dis 2002; 35: 54-56
Leav BA, et al. N Engl J Med 2000; 343: 586

17. Emerging groups Chronic GVHD SOT Multiple myeloma
Solid tumours / lymphoma
SLE / Wegener disease
AIDS
Concluir aqui, entao, que varios grupos sao considerados como de risco elevado para aspergilose invsiva, em funcao de steroids. Apos, que o eortc/msg reconhece isto.
Similar findings have been noted in expanding patient groups receiving high-dose glucocorticoid therapy
Nedel WL, Kontoyiannis DP, Pasqualotto AC. Rev Iberoamer Micol 2009; 26:

18. IFD definitions - Host factors
Neutropenia
Neutropenia
>3 weeks steroids
>3 weeks steroids
<36oC or >38oC and:
- Prior mycosis
- AIDS
- Immunosuppressive drugs
- >10 days neutropenia
Allogeneic HSCT
Treatment with other recognized T-cell immune suppressants
O eortc/msg reconhece a importancia de steroids, e coloca o risco como > 3 semanas
> 4 days unexplained fever despite antibiotics
Inherited severe immunodeficiency
GVHD
Donnelly JP

19. A ‘threshold dose’? Not properly defined
Overall risk for infection increases if:
Prednisone >20 mg/daily
Cumulative dose >700 mg
Largely variable
Mas existe uma dose limiar?
Stuck AE, et al. Rev Infect Dis 1989; 11:
Lionakis MS, Kontoyiannis DP. Lancet 2003; 362:

20. Identical to what is observed for neutropenic patients?
Clinical features
Identical to what is observed for neutropenic patients?

21. Clinical features Diagnosis is often delayed Low index of suspicion
Lewis RE, Kontoyiannis DP. Med Mycol 2008: S1-11

22. Clinical features Diagnosis is often delayed Low index of suspicion
Non-specific signs and symptoms
Suppression of fever / cough / chest pain
Co-infections are frequent
Lewis RE, Kontoyiannis DP. Med Mycol 2008: S1-11

23. Differences in pathogenesis?

24. Pathogenesis of IA
Dagenais TRT, Keller NP. Clin Microbiol Rev 2009;

25. Steroids vs. neutropenia
Chemotherapy
BAL fluid
Rapid PMN influx
No PMN influx
Balloy V, et al. Infect Immun 2005; 73:
Chamilos, et al. Haematologica 2006; 91: 986

26. Steroids vs. neutropenia
Chemotherapy
BAL fluid
Rapid PMN influx
No PMN influx
Pathology
Neutrophil infiltration
No angioinvasion
Pyogranulomatous reaction
No neutrophil infiltration
Angioinvasion
Coagulative necrosis
Haemorrhagic infarction
Balloy V, et al. Infect Immun 2005; 73:
Chamilos, et al. Haematologica 2006; 91: 986

27. Steroids vs. neutropenia
Chemotherapy
BAL fluid
Rapid PMN influx
No PMN influx
Pathology
Neutrophil infiltration
No angioinvasion
Pyogranulomatous reaction
No neutrophil infiltration
Angioinvasion
Coagulative necrosis
Haemorrhagic infarction
Fungal development
Small numbers
of conidia
Large numbers
of hyphae
Balloy V, et al. Infect Immun 2005; 73:
Chamilos, et al. Haematologica 2006; 91: 986

28. Steroids vs. neutropenia
Chemotherapy
BAL fluid
Rapid PMN influx
No PMN influx
Pathology
Neutrophil infiltration
No angioinvasion
Pyogranulomatous reaction
No neutrophil infiltration
Angioinvasion
Coagulative necrosis
Haemorrhagic infarction
Fungal development
Small numbers
of conidia
Large numbers
of hyphae GM
Very low
High
Balloy V, et al. Infect Immun 2005; 73:
Chamilos, et al. Haematologica 2006; 91: 986

29. Steroids Neutropenia H&E GMS x100 x100
Chamilos G, et al. Haematologica 2006; 91: 986-9

30. Does that have any impact
on the performance
of diagnostic tests?

31. Typical CT findings in IA
Day 0:
Halo
Day 4:
 nodule,
halo
Day 7:
Air crescent
Caillot, et al. J Clin Oncol 1997; 15:

32. The ‘Halo sign’ Nodules in IA Nodule Nodule with halo Neutropenia 97%
82%
Non-haematological disorder
24%
Maertens J. ICAAC 2006

33. Can we rely on the ‘halo sign’?
Aspergillus causing VAP (ICU)
Halo sign: 0%
Meersseman W, et al. Am J Respir Crit Care Med 2008; 177: 27-34
Singh N, Husain S. J Heart Lung Transplant 2003; 22:
Bulpa P, et al. Eur Resp J 2007: 30:

34. Can we rely on the ‘halo sign’?
Aspergillus causing VAP (ICU)
Halo sign: 0%
Lung transplant recipients
No specific sign at chest CT
IA in COPD
Non-specific consolidation
Meersseman W, et al. Am J Respir Crit Care Med 2008; 177: 27-34
Singh N, Husain S. J Heart Lung Transplant 2003; 22:
Bulpa P, et al. Eur Resp J 2007: 30:

35. Yield of other dx methods
Lower sensitivity of respiratory cultures
Lower fungal burden
Lower PPV
Haematological patient 77%
Steroid-treated patient 58%
Horvath JA, Dummer S. Am JMed 1996; 100: 171-8

36. Meta-analysis of GM testing
Proven or probable IA
Sensitivity
Specificity
Haematological malignancies
0.58 (52-64)
0.95 (94-96)
Solid organ transplantation
0.41 (21-64)
0.85 (80-89)
GM testing has been approved for use in serum in neutopenic patients
Pfeiffer CD, et al. Clin Infect Dis 2006; 42:

37. Clinical case 56 year-old COPD on steroids
ICU for respiratory tract infection
CRX: diffuse infiltrate
Meersseman W. In: Aspergillosis: from diagnosis to prevention. Pasqualotto AC, ed. Springer 37

38. Clinical case BAL H. influenzae Negative for fungi
Meersseman W. In: Aspergillosis: from diagnosis to prevention. Pasqualotto AC, ed. Springer 38

39. Clinical case BAL Galactomannan Died despite caspofungin H. influenzae
Negative for fungi
Galactomannan
Serum was negative
2.6 ng/ml in BAL
Died despite caspofungin
Meersseman W. In: Aspergillosis: from diagnosis to prevention. Pasqualotto AC, ed. Springer

40. Clinical case BAL Galactomannan Died despite caspofungin
H. influenzae
Negative for fungi
Galactomannan
Serum was negative
2.6 ng/ml in BAL
Died despite caspofungin
Necropsy confirmed IPA
There is a debate on the best cut-off for BAL GM testing
Meersseman W. In: Aspergillosis: from diagnosis to prevention. Pasqualotto AC, ed. Springer

41. Which patient has neutropenia?
Maertens J. ICAAC 2006

42. > 50 days of neutropenia Persistent fever GM OD index: 2 x >0.5
35 year old male
Relapsed AML
> 50 days of neutropenia
Persistent fever
GM OD index: 2 x >0.5
64 year old male
Hypoplastic MDS
High dose steroids (aGvHD III)
Cough and pleuritic chest pain
GM OD index: 2 x >0.5
Maertens J. ICAAC 2006

43. has higher serum GM levels?
Which patient
has higher serum GM levels?
Maertens J. ICAAC 2006

44. Max GM: 7.8 Max GM: 0.8 64 year old male 35 year old male
Relapsed AML
> 50 days of neutropenia
Persistent fever
GM OD index: 2 x ≥ 0.5
64 year old male
Hypoplastic MDS
High dose steroids (aGvHD III)
Cough and pleuritic chest pain
GM OD index: 2 x ≥ 0.5
Maertens J. ICAAC 2006

45. IA in a neutropenic patient
50-yo male
AML on cycle 2, D27 of clofarbine/idarubicin
ANC of 0
High fever
R-sided pleuritic chest pain (2 days duration)
Serum GM 1.2
Lewis RE, Kontoyiannis DP. Med Mycology 2008; S1-11

46. IA in a steroid-treated patient
52-yo female
D45 allo HSCT (AML)
ANC of 1800
GVHD on tacrolimus and steroids
No fever
BAL: A. fumigatus and P. aeruginosa
Negative serum GM
Lewis RE, Kontoyiannis DP. Med Mycology 2008; S1-11

47. Same response to
antifungal drugs?

48. Antifungal treatment Latest IDSA guidelines
No distinction regarding underlying disease
Walsh TJ, et al. Clin Infect Dis 2008; 46:

49. Dominant mechanisms Steroid-induced IA Neutropenia
Adverse host response
Neutropenia
Fungal development
Berenguer J, et al. Am J Resp Crit Care Med 1995; 152:

50. Effects on the immune system
d-AmB
Pronounced pro-inflammatory activity
Release of inflammatory cytokines, chemokines, NO, prostaglandins and others
Fever, chills, myalgias and rigors
Beyond their direct mechanisms of inhibiting fungal growth, systemic antifungal agents have been shown to
have varying degrees of immunomodulatory activity that could be either beneficial or deleterious in the
glucocorticoid-immunosuppressed host
Lewis RE, Kontoyiannis DP. Med Mycology 2008; S1-11

51. Effects on the immune system
d-AmB
Pronounced pro-inflammatory activity
Release of inflammatory cytokines, chemokines, NO, prostaglandins and others
Fever, chills, myalgias and rigors
Potential deleterious effects in steroid-treated hosts with IA
Theoretically, pro-inflammatory antifungals such as AMB-d would be expected to have potentially deleterious effects in the treatment of IA in the glucocorticoid-immunosuppressed host. This concern has been substantiated in animal models where AMB-d has shown limited efficacy in the prevention or treatment of IPA in the glucocorticoid-immunosuppressed animals.
Lewis RE, Kontoyiannis DP. Med Mycology 2008; S1-11

52. Effects on the immune system
Animal models
d-AmB reduces mortality and fungal burden in neutropenic mice with IA
Ineffective in steroid-immunosuppressed mice
Balloy et al. found that treatment with AMB-d reduced mortality in chemotherapy- treated (neutropenic) mice with IPA, but was ineffective in glucocorticoid-immunosuppressed
mice. Similarly, studies from our laboratories have demonstrated that while AMB-d significantly reduces
Aspergillus lung fungal burden in neutropenic animals, the drug appeared to have minimal efficacy in
the cortisone-immunosuppressed mice.
Balloy V, et al. Infect Immun 2005; 73:
Lewis RE, et al. Antimicrob Agents Chemother 2007; 51:

53. Empty liposomes Potent immunomodulating effects
Pre-treatment of steroid-immunosup. mice with empty liposomes
Reduces inflammatory pathology
Improves fungal clearance and survival
Similar efficacy than 10 mg/kg L-AmB and 1 mg/kg of d-AmB
Independent of their role as drug carrier, liposomes are known to have potent immunomodulating effects in
phagocytic cells. Bellochio and colleagues found that incorporation of AMB into liposomes diverts
PMN TLR-2 activation to TLR-4, resulting in reduced production of TNF-a and ROI release and enhancement of non-oxidative killing mechanisms against Aspergillus hyphae. We have recently shown that pre-treatment of glucocorticosteroid-immunosuppressed mice with empty liposomes reduces inflammatory pathology and improves fungal clearance and survival following intranasal inoculation of A. fumigatus. Remarkably, the efficacy of empty liposomes in
the glucocorticoid-immunosuppressed mouse model approached that of 10 mg/kg body weight of L-AMB, highlighting the impact of inflammation on antifungal pharmacology for aspergillosis in the glucocorticoid
immunosuppressed host [26]
Lewis RE, et al. Antimicrob Agents Chemother 2007; 51:

54. Other antifungal drugs
Echinocandins
Immunostimulatory effects
β-glucan unmasking
Triazoles
Few direct effects on mononuclear and PMN
Lewis RE, et al. Antimicrob Agents Chemother 2007; 51:

55. Drug-drug interactions
Itraconazole and steroids
3-4x  in steroid AUC
15-30%  in t½
Voriconazole
 prednisolone Cmax and AUC by 11% and 34%, respectivelly
Lewis RE. AAA 2006

56. Any influence on
disease prognosis?

57. Non-myeloablative allo HSCT Steroid dose to treat GVHD
Overall survival after diagnosis of invasive mould disease
Fukuda T, et al. Blood 2003; 102:

58. Conclusions
Steroids are important risk factors for IA

59. Conclusions Steroids are important risk factors for IA
Steroid-induced changes in immunobiology of IA mandate different approaches to diagnosis and management compared to neutropenia-associated

60. Conclusions Steroids are important risk factors for IA
Steroid-induced changes in immunobiology of IA mandate different approaches to diagnosis and management compared to neutropenia-associated
Prognostic importance

61. Acknowledgments CNPq Teresa Sukiennik Luiz Carlos Severo
Arnaldo L Colombo / Infocus scientific committee