In vivo response in a mixed infection model of azole susceptible and resistant Aspergillus spp. strains Laura Alcazar-Fuoli, CNM, ISCIII

In vivo response in a mixed infection model of azole susceptible and resistant Aspergillus spp. strains Laura Alcazar-Fuoli, CNM, ISCIII Pre-Advances Against Aspergillosis Workshop: Aspergillus Speciation in the 21st Century - Implications for Laboratory and Clinical Practice Meliá Castilla Hotel and Conference Centre Madrid, Spain

Genetic relatedness Introduction: Mixed infections Definition: Denotes diseases involving several microorganisms. Kingdom A + Kingdom B Genus A+ Genus B Species A + Species B Strain A + Strain B Substrain A + Substrain B

Types and sites of polymicrobial diseases Medically important bacterial fungal interactions. Anton Y. Peleg, Deborah A. Hogan & Eleftherios Mylonakis Nature Reviews Microbiology 8, 340-349.

Relations required for development of human disease William Costerton and Mark E. Shirtliff Brian M. Peters, Mary Ann Jabra-Rizk, Graeme A. O'May, J. Polymicrobial Interactions: Impact on Pathogenesis and Human Disease. Clin. Microbiol. Rev. 2012, 25(1):193.

Relations required for development of human disease William Costerton and Mark E. Shirtliff Brian M. Peters, Mary Ann Jabra-Rizk, Graeme A. O'May, J. Polymicrobial Interactions: Impact on Pathogenesis and Human Disease. Clin. Microbiol. Rev. 2012, 25(1):193.

Clinical challenges of mixed infections by fungi: How the host responds to polymicrobial infection compared to monomicrobial infection?. Composition of microbial populations will modify the clinical course and severity of the disease. Diagnostic tools. Impact on the selection of antimicrobial therapy and response to treatment, especially when it involves pathogens exhibiting antimicrobial resistance.

Cases of mixed infections by fungi Case 1: A 29-year-old female with acute nonlymphoblastic leukemia who received a bone marrow transplant in 1985. Surveillance of yeast colonization and drug regimen. AMB resistant D R Soll, M Staebell, C Langtimm, M Pfaller, J Hicks, and T V Rao. J Clin Microbiol. 1988 August; 26(8): 1448 1459. Multiple Candida strains in the course of a single systemic infection.

Cases of mixed infections by fungi Case 2: A 33 year-old man that had a right lung transplantation procedure in 1990. Treated with azathioprine, cyclosporine and prednisone. Four months after transplantation, the patient was diagnosed with cytomegalovirus pneumonia. Six weeks later he presented with productive cough and hyaline septate branched hyphae were seen in the BAL. AMB and Aspergillus flavus was recovered. The patient died 15 days later and deep pulmonary nodule, and culture showed growth of both A. flavus and Aspergillus fumigatus.

Case 3: A 48-year-old caucasian woman with leukaemia. Infection by several Aspergillus species. Cases of mixed infections by fungi MCF CPF +AMB

Cases of mixed infections by fungi Infection by several cryptic species of A. fumigatus complex Case 4: A 68-year-old male smoker with COPD and long term inhaled and systemic corticosteroids. ICU Day 13: BA pure A. fumigatus VRZ Day 16: BA pure A. fumigatus. Day 17: A. fumigatus and white colonies of a mycelial fungus of slow sporulation identified as A. lentulus. Died

Cases of mixed infections by fungi Infection by several cryptic species of A. fumigatus complex Case 5: A 43-year-old Argentine woman that underwent kidney transplantation in May 2005. Immunosuppressive regimen comprised a combination of mycophenolate mofetil hydrochloride, cyclosporine and prednisolone. Three graft rejection episodes. AMB Characteristic morphological and phylogenetic analyses demonstrated the presence of A. fumigatus and A. lentulus. Died

Infection by several cryptic species of A. fumigatus complex It is important to study several isolates per patient in order to reveal the presence of cases of co-infection by cryptic species

Mix infections are complex Bad response High mortality rates Diagnosis is difficult Even more complicated when cryptic species are involved

Mix infections are complex Bad response High mortality rates Diagnosis is difficult Even more complicated when cryptic species are involved Model of mix infection with A. fumigatus and A. lentulus

Aspergillus fumigatus and Aspergillus lentulus

Design of a Multiplex Real Time PCR (MN- PCR) for simultaneous detection of A. fumigatus and A. lentulus

Design of One-step Nested Multiplex Real Time PCR (MN-PCR) for the simultaneous detection of A. fumigatus and A. lentulus

Molecular identification of Aspergillus section Fumigati β-tubulina: Clustal V 30.2 30 25 20 15 10 5 0 CM3538Tub.seq CM4330_T ub.seq CM3599Tub.seq CM3134Tub.seq CM3583Tub.seq AY738520.SEQ CM1290TuB.seq AY738513.SEQ AY738517.SEQ CM3537Tub.seq AY738523.SEQ CM4370_T ub(sens).seq CM3364TUB.SEQ CM4428Tub2.seq CM4426TUB.s eq CM4420TUB.s eq CM4387Tub.seq CM4415_T UB.seq CM4428Tub.SEQ AB248076_ As pergillus fumis ynnematus.seq CM4063bT ub.s eq AB248077_ As pergillus fumis ynnematus.seq AB248078_ As pergillus fumis ynnematus.seq CM4063bT ub(sens).seq CBS109890bT ub(sens).s eq CM237T ub.s eq A. fumigatus CBS525Btub.seq CBS544T ub.s eq Nlaciniosa(AY870748).SEQ N. fischeri Nspinosa(AF057329).s eq Nspinosa(AY870765).seq Nspinosa(AY870761).seq Nspinosa(AY870759).seq Nspinosa(AY870762).seq Nspinosa(AY870760).seq CBS111_55T ub.seq AY870757_ Nc oreana_ibt 22051.s eq AY870758_ Nc oreana_kacc41659.s eq CM2280Tub.seq CM3227Tub.SEQ DQ094884_ Afumigatiaffinis _ IBT 13131.seq DQ094885_ Afumigatiaffinis _ IBT 12703.seq DQ094886Anovofumigatus _IBT 16806.s eq DQ094887Anovofumigatus _IBT 16755.s eq CBS208T ub.s eq CM3914Tub.seq CM4060bT ub.s eq CBS404T ub.s eq CM3769Tub.seq Npseudofisc heri(af057325).seq CM2270TuB.seq Npseudofisc heri(ay870742)cbs208_ 92.SEQ AY590130_ Aviridinutans _MK284.seq CM3147Tub.seq AY590129_ Aviridinutans _MK246.seq CM4518Tub.seq A. AF134780_Aviridinutans_ IMI280490.s eq CBS114218T ub.seq CM3740Tub.seq Nhirats ukae(af057324).seq CM3305Tub.seq CM3764Tub.seq CM4328_tub.seq CBS117067T ub.seq N. hiratsukae CM3303Tub.seq CBS109356T ub.seq Nglabra(AY870734).seq Nglabra(AY870735).SEQ Nglabra(AY870736).SEQ CBS165_63T ub.seq CBS113_64T ub.seq Naurata(AF057318)CBS466_65.SEQ Nspinosa(AY870763).SEQ AfumigatusXM747363.s eq A. lentulus A. fumisynnematus A. fumigatiaffinis N. pseudofischeri viridinutans N. glabra

20 15 10 5 0 Molecular identification of Aspergillus section Fumigati RodletA: Clustal V 22.6 CM4387_NRodA.s eq CM4420RodA.s eq CM4426RodA.s eq CM4415_RODA.s eq CM4370_Rod.seq CM4428RodA.s eq CM4415RodA.s eq CM4426RodA3_049.seq AY738519AL.seq DQ439776 Alentulus_FH220.s eq AY738525AL.seq AY738514AL.seq CM3537Rod.seq CM3538Rod.seq CM1290Rod.seq AY738522AL.seq CM3583Rod.seq CM4330_RodA.seq CM3364ROD.s eq CM3599Rod.seq CM3134Rod.seq AB249898_Afumisynnematus.seq AB249899_Afumisynnematus.seq CM4063Rod.seq AB249897_Afumisynnematus.seq AB249897_Afumisynnematus.seq CM237rod.s eq N.udagawae(DQ439767).SEQ M0742_RODA.seq CBS109890Rod.seq CM3622ROD.s eq CM3663ROD.s eq CM3550ROD.s eq N.fischeri(DQ439770).SEQ CM3587ROD.s eq A.neoliptic us(dq439771).seq CBS525RodCON.seq CBS544_65Rod.seq CM2280Rod.seq CM3227ROD.s eq CBS111_55Rod.seq AB250103Aviridinutans.seq CM4518_RODA.s eq A. CM3147Rod.seq N.udagawae(DQ058375).SEQ N.udagawae(DQ439769).SEQ CBS114218Rod.seq AF057344_Neosartorya hirats ukae.s eq CM3740Rod.seq CM3764Rod.seq CM4328Rod.seq CBS117067Rod.seq CM3305Rod.seq CM3303Rod.seq CBS109356Rod.seq CBS113Rod.seq CBS165_63Rod.seq N. CBS208ROD.seq CBS404_67RODA.s eq CM2270Rod.seq CM3769Rod.seq CM4060Rod.seq CM3914Rod.seq RodEmericelaNidulans.seq A. lentulus A. fumisynnematu A. fumigatus N. fischeri A. fumigatiaffinis viridinutans N. hiratsukae glabra N. pseudofischeri

Design of a Nested Multiplex Real Time PCR (MN-PCR) for the simultaneous detection of A. fumigatus and A. lentulus cyp51a A. fumigatus A. lentulus A. fumigatiaffinis N. fischeri A. viridinutans N. pseudofischeri 1.2 > 97% Nucleotide Substitutions (x100) 0 CM 3673 CM 3764 CM 5280 CM 4387 CM 4063 CM 4909 CM 5441 CM 4635 CM 3147 CM 3914 CM 4060 A. lentulus A. fumisynnematus N. udagawae N. hiratsukae A. fumigatiaffinis A. viridinutans N. pseudofischeri

Design of a Nested Multiplex Real Time PCR (MN-PCR) for the simultaneous detection of A. fumigatus and A. lentulus A. fumigatus CYP51A A. lentuluscyp51a A. fumigatus CYP51A A. lentuluscyp51a A. fumigatus A. fumigatus CYP51A A. lentuluscyp51a A. fumigatus CYP51A A. lentuluscyp51a A. fumigatus CYP51A A. lentuluscyp51a A. lentulus Samson et al. Studies in Mycology 59: 147 203. 2007 A. fumigatus CYP51A A. lentuluscyp51a 71 bp

Design of One-step Nested Multiplex Real Time PCR (MN-PCR) for the simultaneous detection of A. fumigatus and A. lentulus Fungal species and strains Aspergillus flavus (CM-2669) Aspergillus terreus (CM-2013) Fusarium verticillioides (CM-2975 Fusarium oxysporum (CM-2914) Scedosporium prolificans (CM-1627) Scedosporium apiospermum (CM-3169) Cryptococcus neoformans (CL-2132) Rhizopus oryzae (CM-3020) Rhizomucor variabilis (CM-2437) Candida albicans (ATCC64551) Candida glabrata (CL-5533) Human Mice Fungal species and strains section fumigati A. fumigatiaffinis (CM-2280) N. hirasukae (CM-5817) N. pseudofischeri (CM-5593) A. novofumigatus (CM-6098) A.viridinutans (CM-3147) N. udagawe (CM-5416) A. fumisynnematus (CM-4063) A. fumigatus (20 strains) A. lentulus (20 strains)

Design of One-step Nested Multiplex Real Time PCR (MN-PCR) for the simultaneous detection of A. fumigatus and A. lentulus FAM HEX A. fumigatus strains A. lentulus strains Sensitivity Reproducibility Specificity A. fumigatus 100 fg/ul 96,3 % 100 % A. lentulus 100 fg/ul 97,6 % 100 %

Validation of the MN-PCR in a murine model of aspergillosis Cyclophosphamide model 10 5 conidia spores in 30 µl of saline solution Hydrocortisone acetate 10 5 conidia spores in 30 µl of saline solution A. lentulus A. fumigatus A. lentulus A. fumigatus

ng of fungal DNA / l ng of fungal DNA / l Validation of the MN-PCR in a murine model of aspergillosis Cyclophosphamide model 10 5 conidia spores in 30 µl of saline solution Hydrocortisone acetate 10 5 conidia spores in 30 µl of saline solution 10 100 % (8/8) 92 % (12/13) 10 100 % (10/10) 100 % (10/10) 8 8 6 6 4 4 2 2 0 A. fumigatus A. lentulus 0 A. fumigatus A. lentulus

ng of fungal DNA/ mg of murine DNA Validation of the MN-PCR in a murine model of aspergillosis Cyclophosphamide model 10 5 conidia spores in 30 µl of saline solution 0.4 60 % (3/5) 0.3 0.2 60 % (3/5) 5 BALs 72h post infection 0.1 Control not infected 0.0 Control NI A. fumigatus A.lentulus

In vivo model of mixed infection with azole susceptible and resistant Aspergillus spp. (A. fumigatus and A. lentulus) Antifungal response

Mixed infection model of R. oryzae-a. fumigatus. Corticosteroid-immunosuppressed mice to explore whether in vivo exposure with other antifungals used in the treatment of invasive aspergillosis provoke selection pressure for breakthrough mucormycosis during infection.

Voriconazole pre-exposure in vitro accelerates breakthrough infection in vivo with R. oryzae Lewis RE, Liao G, Wang W, Prince RA, Kontoyiannis DP. Voriconazole pre-exposure selects for breakthrough mucormycosis in a mixed model of Aspergillus fumigatus-rhizopus oryzae pulmonary infection. Virulence. 2011 Jul-Aug;2(4):348-55. Epub 2011 Jul 1.

Voriconazole accelerates mortality due to R. oryzae in a mixed infection model of pulmonary aspergillosis and mucormycoses Lewis RE, Liao G, Wang W, Prince RA, Kontoyiannis DP. Voriconazole pre-exposure selects for breakthrough mucormycosis in a mixed model of Aspergillus fumigatus-rhizopus oryzae pulmonary infection. Virulence. 2011 Jul-Aug;2(4):348-55. Epub 2011 Jul 1.

Alternative host model to study fungal infections Galleria mellonela Greater wax moth Insect model, although we use the larval phase Lepidoptera

Alternative host model to study fungal infections Galleria mellonela structure (Larval stage)

Galleria mellonela Immunity Haemocytes Antimicrobial Peptides ROS Melanization

Alternative host model to study fungal infections Role of virulence factors (mutant screening) Host-pathogen interations Antifungal efficacy and resistance detection Host response

Alternative host model to study fungal infections

Alternative host model to study fungal infections Galleria mellonela Disadvantages Lacks adaptative immunity Genome not known Expertise required to breed in the laboratory Advantages Allows different temperatures incubations Controlled inocula Easy to monitor survival Easy to implement in the laboratory Suitable for antimicrobial efficacy testing

Aspergillus fumigatus Aspergillus lentulus Aspergillus fumigatus and Aspergillus lentulus A

Radial growth (cm) Model of A. fumigatus and A. lentulus; in vitro assays Microscopic morphology (RPMI) Macroscopic morphology (MM) AkuB KU80 AkuB KU80 10 4 10 3 10 2 10 1 A.lentulus CM1290 A.fumigatus akub KU80 CM1290 CM1290 3.00 2.50 2.00 akub CM-1290 1.50 8 hr 24 hr 1.00 0.50 0.00 24h 48h 72h 4d 5d Time

Correlation between virulence of A. lentulus and A. fumigatus in mice and G. mellonella Cyclophosphamide model A. lentulus A. fumigatus A. fumigatus A. lentulus

Histopathology of infected G. mellonella Aspergillus fumigatus Aspergillus lentulus 20 x magnification, PAS stain

% Hemocites relative to PBS Haemocyte density by obtaining the hemolymph from infected larvae 160 140 120 100 80 60 A. fumigatus A. lentulus 40 20 0 0 50 100 150 200 250 300 Time (minutes)

Survival rate of G. mellonella infected with A. fumigatus and A. lentulus Untreated Treated with voriconazole

Histopathology of infected G. mellonella treated with voriconazol A. fumigatus A. lentulus 20 x magnification, PAS stain

Fungal burden by qpcr of infected G. mellonella treated with voriconazol Aspergillus fumigatus Aspergillus lentulus

Fungal burden by qpcr of infected G. mellonella treated with voriconazol Aspergillus fumigatus 15 X p < 0,5 Aspergillus lentulus 3,5 X

Correlation between virulence of a mixed infection in mice and G. mellonella Cyclophosphamide model Inoculum: 10 5 Mixed infection: A. fumigatus + A. lentulus: 5 x 10 4 each A. fumigatus A. lentulus Mixed

Survival rate of G. mellonella infected with a mixed infection of A. fumigatus and A. lentulus A Untreated B Treated with voriconazole

Survival rate of G. mellonella infected with a mixed infection of A. fumigatus and A. lentulus A Untreated B Treated with voriconazole

Fungal burden of G. mellonella infected with a mixed infection of A. fumigatus and A. lentulus Untreated Treated with voriconazole A. fumigatus A. lentulus A. fumigatus A. lentulus

Fungal burden of G. mellonella infected with a mixed infection of A. fumigatus and A. lentulus A. lentulus is selected by voriconazole in a mixed infection A. fumigatus A. lentulus

Fungal burden of G. mellonella at 4 day post- infection p < 0,05 Fungal burden of infected larvae with A. fumigatus is reduced in the mixed infection but not for A. lentulus Single infection Mix infection

Conclusions Mixed infections are complex and challenge fungal diagnosis and treatment. We were able to simultaneously detect two species closely related such as A. fumigatus and A. lentulus by qpcr showing a promising target for clinical detection of Aspergillus section Fumigati in single or mixed infections. Microbial virulence and correlation between antifungal susceptibility data in vitro/in vivo can be explored in the Galleria mellonella model of Aspergillus spp infection. Aspergillus infected larvae did not respond to VCZ treatment when they were infected with A. lentulus only or in combination with A. fumigatus. The presence of antifungal resistant species would likely complicate the management of fungal infections.

Knowledgments Members of the Mycology Reference Laboratory Spanish National Center for Microbiology Manuel Cuenca Estrella Alicia Gomez Lopez Oscar Zaragoza and Rocio Garcia Emilia Mellado Maria Jose Buitrago Gema del Rio