Invasive aspergillosis in patients taking steroids Alessandro C. Pasqualotto pasqualotto@santacasa.tche.br Santa Casa de Porto Alegre

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

Aspergillus love steroids A fact: Aspergillus love steroids

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: 1828-38

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: 1828-38

Steroids enhance Aspergillus growth 30-40% increase in growth rate Ng TTC, et al. Microbiology 1994; 140: 2475-9

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

What about clinical data?

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: 4358-66

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

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: 2292-36

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: 1060-76

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: 2300-2

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

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)

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

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: 175-83

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

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: 954-63 Lionakis MS, Kontoyiannis DP. Lancet 2003; 362: 1828-38

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

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

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

Differences in pathogenesis?

Pathogenesis of IA Dagenais TRT, Keller NP. Clin Microbiol Rev 2009; 447-65

Steroids vs. neutropenia Chemotherapy BAL fluid Rapid PMN influx No PMN influx Balloy V, et al. Infect Immun 2005; 73: 494-503 Chamilos, et al. Haematologica 2006; 91: 986

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: 494-503 Chamilos, et al. Haematologica 2006; 91: 986

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: 494-503 Chamilos, et al. Haematologica 2006; 91: 986

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: 494-503 Chamilos, et al. Haematologica 2006; 91: 986

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

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

Typical CT findings in IA Day 0: Halo Day 4:  nodule, halo Day 7: Air crescent Caillot, et al. J Clin Oncol 1997; 15: 139-47

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

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: 258-66 Bulpa P, et al. Eur Resp J 2007: 30: 782-800

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: 258-66 Bulpa P, et al. Eur Resp J 2007: 30: 782-800

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

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: 1417-27

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

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

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

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

Which patient has neutropenia? Maertens J. ICAAC 2006

> 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

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

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

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

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

Same response to antifungal drugs?

Antifungal treatment Latest IDSA guidelines No distinction regarding underlying disease Walsh TJ, et al. Clin Infect Dis 2008; 46: 327-60

Dominant mechanisms Steroid-induced IA Neutropenia Adverse host response Neutropenia Fungal development Berenguer J, et al. Am J Resp Crit Care Med 1995; 152: 1079-86

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

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

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: 494503 Lewis RE, et al. Antimicrob Agents Chemother 2007; 51: 1078-81

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: 1078-81

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: 1078-81

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

Any influence on disease prognosis?

Non-myeloablative allo HSCT Steroid dose to treat GVHD Overall survival after diagnosis of invasive mould disease Fukuda T, et al. Blood 2003; 102: 827-33

Conclusions Steroids are important risk factors for IA

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

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

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