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1 PEG Frühjahrstagung Sektion Antimykotische Chemotherapie Analysis of Host Response signatures for diagnosis of invasive fungal infections Immunreaktionen zur Diagnostik I Mark Reinwald III. Med. Klinik Hämatologie und Onkologie Universitätsmedizin Mannheim
2 Aspergillus species most frequent fungal pathogen in hematological high risk pts Aspergillosis Candidiasis 8 44 Zygomycosis Fusariosis Pneumocyst. Endemics 30 Other/unspec Molds Other Yeasts Distribution of organisms causing invasive fungal infection (n=995) 1. Kontoyiannis D et al., Clin Infect Dis 2010; 15, 50(8),
3 Diagnosing IA The EORTC/MSG Criteria Falldefinition Wirtsfaktoren Clinical Features Mycological criteria Proven (=bewiesene Aspergillose) Probable (=wahrscheinliche Aspergillose) Possible (=mögliche Aspergillose) No IA +/- +/- + + De Pauw et al., Clin Infect Dis 2008 Histopathologischer oder kultureller steril gewonnener Nachweis aus einer normalerweise sterilen Lokalisation Entweder a) Histopathologischer oder kultureller gewonnener Nachweis aus einer nicht-sterilen Lokalisation (z.b. BAL, Sputum, Nasenspülflüssigkeit) b) Positivität für GM c) Positivität für BDG (=kein Hinweis auf IA) +/- +/- -
4 Invasive aspergillosis in hematological pts is a life-threatening and underdiagnosed complication in patients at high risk receiving intensive chemotherapy and/or undergoing allo hematopoietic stem cell transplantation 2 Prevalence of IFI 31 % ( 314/1017) Diagnosis Post-Mortem 75 % (235 /314) Diagnosis Pre-Mortem 25 % (79 /314) Only 79/314 pts = 25 % with autopsy-proven IFI diagnosed premortem 2. Chamilos et al. Haematologica 2006; 91:
5 Lewis R, Mycoses 2010 But we are getting better. 20-yr single center autopsy study declining autopsy rates, but fewer IFI after 2003, moulds majority of disseminated IFI at autopsy; IA decrease with mould active therapy increased prevalence of Candida in most recent time period improved antemortem diagnosis: 16 to 51%
6 Invasive aspergillosis mortality rates still high 3 Over the years decrease of mortality rates in IA pts Improvement possibly based on: a) increase of nonmyeloablative conditioning None b) faster WBC recovery by use of peripheral blood stem cells c) improvement in diagnosing of IA d) new antifungal drugs 3. Upton et al. CID 2007; 44:
7 Diagnostics in IA What to detect in IA? Host Evidence?? Host + Pathogen Evidence (Radiology) Pathogen evidence Aspergillus Antibodies?? Host Response Histology Culture GM BDG Asp PCR Host Response: The characteristic response pattern of an organism to an invading pathogen
8 Biomarkers in IA derived from the pathogens
9 Host Response Diagnostics comprehensive analysis of the host response to the infection caused by different microbial pathogens different classes of pathogens trigger specific pattern-recognition receptors (PRRs) differentially expressed on leukocytes a comprehensive molecular phenotype of these cells can be obtained using mould-specific T-cells, gene expression profiling (GEP) microarray analysis Advantages: a) Pathogens difficult to grow/detect might be detected (e.g. Mycobacteria, Moulds) b) Host response analysis might elucidate further potential diagnostic targets
10 Host Response Diagnostics Already present in routine clinical practice? Yes, they are.. In the diagnosis of M. tuberculosis infections host-response-based analysis is the mainstay of non-invasive diagnostics with the TB-Elispot and the quantiferone assay Lai CC, Diagn Microbiol Infect 2011
11 Detection of an IMD specific host response Camargo, CID 2014
12 Genetic Factors in PRR affect IA incidence and survival Polymorphisms in TLR4 Hematopoietic stem cell recipients transplanted with a specific TLR4 polymorphism (S4+) show increased rates of IA and have impaired outcome Bochud P, NEJM 2008
13 Genetic Factors in Dectin-1 and DC-SIGN affect IA incidence Analysis of 182 pts with HM, 57 with proven/probable IA for SNPs in DC- SIGN, Dectin-1, Dectin-2, CCR2 and CCL2 Significant association of SNPs with IA incidence was observed for DC-SIGN and Dectin-1 Synergistic genetic effects were observed for SNPs in different PRRs Dectin-1 Sainz, PLOSone 2012
14 Genetic Factors in PRR affect IA incidence and survival Polymorphisms in Pentraxin Polymorphisms in PTX3 affect IA incidence and outcome Cunho C, NEJM 2014
15 Genetic Factors in PRR can also be protective IL10- Polymorphisms In 105 BMT pts a protective effect was found for the IL-10 ACC haplotype regardless of chronic GvHD or HLA-disparity Seo KW, BMT 2005
16 Analysis of Host factor PRRs for profiling risk of IA Camargo J, CID 2014
17 Host response Background in IA Lass-Floerl C, Mycoses 2013
18 Host response Background in IA II Lass-Floerl C, Mycoses 2013
19 Host response Background in IA III Gressnigt MS, Ann N.Y. Acad Sci 2011
20 Host response Background in IA III Gressnigt MS, Ann N.Y. Acad Sci 2011
21 Host response Using mould-specific T-cells for diagnosis Using an established assay 69 pts were assesed for mould (Aspergillus/mucorales) reactive T-cells based on upregulation of CD154 ROC analysis revealed a cutoff of 0.39 % CD154+ among CD4 cells as being suggestive of IA for Mucorales a cutoff of 0.16 % was found Sensitivity 90%, specificity 80 % Bacher P et al, Am J Resp Crit Care 2015
22 Host response inoculation with Aspergillus leads to increase in mirna 132 expression In a preclinical model confrontation of monocytes and DC cells leads to increase in microrna 132 expression Inoculation with LPS did not lead to increase suggesting a fungal-specific reaction Das Gupta M, Int J Med Microbiol 2014
23 In bacterial brain abscesses differential expression of inflammatory cytokines is observed for different species In 90 pts with bacterial brain abscesses expression of inflammatory cytokines was measured by ELISA and qpcr Differential expression was observed for different bacterial species Local expression was higher than systemic concentrations Bajpai A, J Neuroimmun 2014
24 Significant increase in IL17a and Kynurenine in Candidemia compared to non-candidemia infections Dectin-1, TLR2, TLR4, Tregs, IL-6, IL-10, IL17A, IL- 22, IL-23, IFN-ɣ, Kynurenine and tryptophane were quantified in ICU pts (n=131) comparison of candidemia and non-candidemia in ctrls 6/44 had other positive blood cultures Krause R, J Infection 2015
25 Transcriptional Profiling of Host response Mejias A, J Infect 2014
26 Transcriptional Profiling of Host response using GEP GEP is able to discriminate between different pathogen species based on specific expression signature of blood leukocytes Ramilo O, Blood 2007
27 Transcriptional Profiling via GEP is superior to PCT for differentiating viral from bacterial LRTI Prospective study in adult pts hospitalized for LRTI (118 pts, 40 ctrls) Immunocompromised pts and pts under antibiotics prior to admission were excluded GEP was superior in discriminating between bacterial and viral lower respiratory tract infections compared to procalcitonin Suarez NM, J Infect 2015
28 Transcriptional Profiling of Host response in invasive fungal infections - Candidemia Preclinical data show that candidemia has a specific gene expression signature compared to healthy controls and S. Aureus bacteremia Zaas AK, Sci Trans Med 2010
29 Transcriptional Profiling of Host response in invasive fungal infections - Candidemia Candidemia shows characteristic GEP patterns that change dynamically over time in relation to treatment or progressive disease Zaas AK, Sci Trans Med 2010
30 Transcriptional Profiling reveals differentially expressed genes involved in immunoprocesses Candidemia shows gene expression patterns that upregulate specific signatures involved in immunologic processes Construction of a GEP model that predicted 98 % of Candida infections in a validation cohort, Performance characterístics showed a sentitivity of 96 % and specificity of 100 % Zaas AK, Sci Trans Med 2010
31 Transcriptional Profiling of Host response in sepsis High TNF Low MIP2 + low TNF High MIP2 GEP is able to define subgroups with higher mortality rates and prone to suffer from severe sepsis McConnell KW, Crit Care 2010 Maslove et al, Crit Care 2012
32 Transcriptional Profiling of Host response TBC patients GEP discriminates phases of TBC treatment in patients Ottenhoff THM, PLOS One 2012
33 Transcriptional profiling of GEP for inhalative pathogens Evans, Resp Res 2010
34 Transcriptional profiling of GEP for inhalative pathogens Specific signature for A. fumigatus Preclinical data from murine models suggest a specific gene signature for Aspergillus detected from homogenized lung tissues Evans, Resp Res 2010
35 Transcriptional profiling of GEP for IA Clinical Data Murgolo NJ, ICAAC 2012, Abstract M-1695
36 Host Response Profiling in IA Our Approach Accumulation of GEP Signatures of proven/probable IA patients from Blood and BAL Assessment of feasibility of performing host GEP Analysis on BAL Identification of a specific gene expression signature for patients with proven/probable IA as opposed to other infectious agents Clinical trial will hopefully start sometime in the future (given financing)
37 Host Response Profiling in IA Open Questions? Analysis feasible in leukopenic patients or patients under therapeutic immunosuppression? GEP Analysis feasible in BAL given the lack of standardized BAL volume? Should an enriched cell population be assessed for GEP (e.g. macrophages, T-cells? GEP analysis probably best in biopsy samples, yet rarely obtained in the hematologic patient population
38 Vielen Dank fürs Zuhören
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