Biomarkers for the diagnosis of emerging invasive fungal infections :
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1 Biomarkers for the diagnosis of emerging invasive fungal infections : Challenges, Perspectives Example of Mucorales infections Laurence Millon Parasitologie-Mycologie, CHU Besançon UMR CNRS 3249 Chrono Environnement, Université de Bourgogne Franche-Comté Journée Infections Nosocomiales Résistance & Innovation Innovations diagnostiques -Lyon 10 decembre 2015
2 Invasive fungal infections Yeast (Candida - Cryptococcus) Filamentous fungi (mold) Aspergillosis (85-90%) Emerging fungal infection Mucorales ( incidence) Scedosporium, Fusarium Bitar, Emerg Infect Dis 2014
3 Lanternier, Clin Infect Dis, 2012 Which species? Mucorales Rhizopus spp, (55%) Lichtheimia sp, (29%) Rhizomucor sp (7%) Mucor sp (3%) Lichtheimia sp Mixed infection? Aspergillus + Mucorales Rhizopus sp Rhizomucor sp
4 Pathology Mold = ubiquitous contaminant Inhalation of airborn spores (3-10µ) or passage through injured skin
5 Immunosupressed patient Defense fungi Virulence factors mucosal barrier phagocytosis Development of the fungi in tissue Infection Pulmonary, rhinocerebral, disseminated, cutaneous
6 Treatment and outcome Surgery + antifungals Mortality rates Disseminated:79% Pulmonary:48% Rhinocerebral: 25% Cutaneous: 22 % Hematological diseases: 60% Diabètes: 30%
7 Mucormycosis / invasive aspergillosis Similar underlying conditions, similar radiological and clinical signs Different antifungal treatment Benefit of early diagnosis/early directed treatment Rhizomucor sp Aspergillus fumigatus
8 Benefit of early diagnosis of mucormycosis Early treatment Delayed treatment Distinguishing between Mucormycosis and Aspergillosis as early as possible is essential Chamilos G, Clin Infect Dis, 2008 Walsh TJ, Clin Infect Dis, 2012
9 Biological diagnosis of mucormycosis Biopsy - BAL Blood sample Histo pathology Mycology Culture Molecular biology Biomarker macro micro PCR sequen cing MS Maldi TOF PCR sequencing PCR/ ESI- MS x antigène Circulating DNA qpcr
10 QPCR detection of circulating DNA for diagnosis of Mucormycosis Retrospective study (10 patients -Besançon ) 2 case reports (2 patients Japan 2014) Multicenter retrospective study (44 patients- 10 french hospitals -2015) Millon, Clin Infect Dis 2013 Shigemura, Int J Hematol 2014; Shigemura Int J Infect Dis 2014; Millon et al, Clin Microbiol Infect, in press
11 Retrospective analysis 10 patients (mucormycosis/ ) 51 serum (-20 C) : 2-9 serum per patient DNA Extraction - 1mL serum Kit Large volume MagnaPure (Roche Diagnostics) Combinaison of 3 qpcr (hydrolysis probes) : 3 targets Lichtheimia Rhizomucor Mucor/Rhizopus (Haugland et coll, Environmental Protection Agency Millon, Clin Infect Dis 2013
12 QPCR Non invasive Sensitive 9/10 patients PCR-positive Early positive 10 days [68-3 days] before mycological diagnosis Positive at the time of 1st symptom (fever) (7/12patients) Specificity 19 fungal species (Other Mucorales, Aspergillus, Fusarium, Scedosporium, Candida) 31 serum (aspergillosis (n=17), pneumocystis pneumonia (n=14) Help in diagnosing mixed infection? Millon, Clin Infect Dis 2013
13 3-year-old boy Chronic granulomatous disease/ allohsct Post mortem diagnosis of disseminated mucormycosis (kidney necropsy) Retrospective analysis - Stored serum samples ( D-9 to D49 after HSCT) - QPCR targeting Rhizomucor 18S rdna (adapted from Millon, 2013) Shigemura T, Int J Hematol 2014
14 allohsct fever Skin rash Radiological signs (lung, kidney) Radiological signs (brain) Death Detection of circulating DNA (D14) 2 days before radiological signs (D16)
15 Fluctuation of DNA load related to levels of CRP Death
16 Increase in DNA load 7days after increase in methyl prednisolone (mpsl) dose
17 Early diagnosis and monitoring of mucormycosis by detection of circulating Mucorales DNA in serum Retrospective analysis of 44 cases collected through the French Surveillance Network of Invasive Fungal Infections (RESSIF) Millon et al, Clin Microbiol Infect, in press
18 Objectives To assess the diagnostic performance of Mucorales qpcr assay in a retrospective multicentre study (RESSIF*) To assess the correlation between DNA load and treatment efficacy and outcome * RESSIF French surveillance network of invasive fungal infection (Centre National de Référence Mycoses Invasives et Antifongiques Institut Pasteur, Paris) Millon et al, Clin Microbiol Infect, in press
19 Patients -Methods 44 mucormycosis (RESSIF ) 19 probable-25 proven 194 stored serum (1mL / -20 C) (4 serum/patient [1-13] D-30/D+30) Pulmonary (17) Disseminated (14) Rhinocerebral (8) Cutaneous (4) Digestive (1) Extraction DNA Large Volume MagNa Pure Nucleic Acid Isolation Kit (Roche Diagnostics) Combinaison of 3 qpcr assays * (target 18S-rDNA) Hematological malignancies (34) Diabetes (3) Solid organ transplants (4) Others (5) Rhizomucor Lichtheimia Mucor/ Rhizopus * Millon et al, Clin Infect Dis, 2013
20 Circulating DNA / Diagnosis Concordant 100% PCR results and culture/molecular identification 16 Rhizopus/1 Mucor 14 Lichtheimia 12 Rhizomucor 1 Syncephalastrum Sensitive High DNA load (Cq= 34 cycles [23-41] 81% 36/44 patients 92% 34/37 patients serum > 1mL & targeted species only Early 9 days before diagnosis (histology or mycology) (0-28 days) 8 days before Initiation of Liposomalamphotericine B (L-AMB) (26/36 cases) Millon et al, Clin Microbiol Infect, in press
21 Circulating DNA / Monitoring qpcr results / Outcome 36/44 patients with positive PCR 3 patients : not evaluable 14 patients : PCR patients : PCR ++ P <10-6 PCR + - PCR + + At least one positive PCR becoming negative after treatment initiation At least one positive PCR remaining positive Millon et al, Clin Microbiol Infection, in press
22 qpcr results / Treatment PCR + - PCR PCR + - survival D84 = 48 % All treated by L-AMB fungal load (> 2 cycles) : 5 days after initiation of L-AMB [1-17 days] complete negativity : 7 days after initiation of L-AMB [3-19 days] 19 PCR ++ survival D84 = 4% 12/19 treated by L-AMB but no fungal load 7/19 not treated by L-AMB Millon et al, Clin Microbiol Infect, in press
23 Species detected by qpcr / Survival Positive PCR Muc (Rhizopus/Mucor) Positive PCR Acory (Lichtheimia) Positive PCR Rmuc (Rhizomucor) Millon et al, Clin Microbiol Infect, in press
24 Conclusion - Perspectives High DNA load of circulating DNA in patients with mucormycosis Early detection Anticipation of diagnosis Accurate quantification follow-up of DNA load : adjunct for treatment monitoring Acceptable cost, easy implementation in microbiology labs of tertiary care hospitals PCR = microbial criteria for Probable Aspergillosis (EORTC-MSG nov 2015) Aspergillus + Mucorales PCR screening in high risk patients?
25 Multicenter Prospective Study Objectives 1/ To assess the performance of the DNA detection test for diagnosing mucormycosis in a prospective study 2/ To evaluate the time saved before treatment in comparison to other diagnosis tools Initiated January patients with probable or proven mucormycosis have to be included 8 University Hospitals in France (Besançon, Strasbourg, Dijon, Amiens, Paris Saint Louis, Paris Necker, Créteil, Nantes; + Nancy, Lyon in 2016) Funding / PHRC National (French Ministry of Health)
26 Timing of the study
27 Data from E-CRF analysis 25/09/ patients recruited Hematological malignancies (AML(33), allohsct (4), ALL (4), lymphoma and other (5)) Solid organ transplant (4) Burn (1) Diabete s(1) 27 completed files 22 Invasive Fungal Intection 11 probable or proven Mucormycosis 10 robable or proven Aspergillosis 1 Fusariosis 1 december 2015 : 95 patients recruited
28 Technical improvment External quality controls September 2014 (6 labs) January 2016 (9 labs) Accurate quantification (copies /ml) Other Mucorales targets (Cuninghamella) Duplex qpcr
29 Many thanks to A Berceanu, F Larosa, F Grenouillet, E Scherer, AP Bellanger, S Roussel, S Rocchi, M Vacheyrou, FE Grenouillet, A Crespo, E Grisot - CHU Besançon R Herbrecht, V Letscher-Bru - CHU Strasbourg O Lortholary, F Lanternier, ME Bougnoux - APHP Hôpital Necker Paris JM Mollina, B Denis, S Bretagne, A Alanio- APHP Hôpital Saint Louis Paris C Cordonnier, F Botterel APHP Hôpital Mondor Créteil D Caillot, F Dalle - CHU Dijon JP Marolleau, A Charbonnier, T Chouaki - CHU Amiens F Morio - CHU Nantes S Cassaing - CHU Toulouse P Poirier - CHU Clermont Ferrand C Kauffmann Lacroix - CHU Poitiers D Toubas - CHU Reims D Poisson - CHU Orléans <a href=" et Images</a> F Dromer, O Lortholary, S Bretagne - Centre National de Reference Mycose Invasive et Antifongique, Institut Pasteur Paris
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