FUNGAL SPORES Michel Thibaudon Samuel Monnier Julien Clertant

FUNGAL SPORES Michel Thibaudon, Samuel Monnier, Julien Clertant In collaboration with Bernard CLOT and with slides from Dr. M. Muilenberg, Cambridge U. , USA Dr. D. Job, U. of Neuchâtel, CH Inspired from « Mycology of the air » Michel Thibaudon – March 2018

Disclosure In relation to this presentation, I declare the following, real or perceived conflicts of interest: Type Company Employment full time / part time Resau National de Surveillance Aérobiologique Spouse / Family member employment / engagement None Research Grant (P. I. , collaborator or consultant; pending and received grants) None Other research support None Speakers Bureau / Honoraria None Ownership interest (stock, stock-options, patent None or intellectual property) Consultant / advisory board None A conflict of interest is any situation in which a speaker or immediate family members have interests, and those may cause a conflict with the current presentation. Conflicts of interest do not preclude the delivery of the talk, but should be explicitly declared. These may include financial interests (e. g. owning stocks of a related company, having received honoraria, consultancy fees), research interests (research support by grants or otherwise), organisational interests and gifts. EAACI dedicated to Allergy Science, committed to your Health 2

1 Classification Michel Thibaudon – March 2018

Michel Thibaudon – March 2018

Eukaryotes: 5 kingdoms MONERES PROTISTS PLANTS FUNGI ANIMALS Fungi: eukaryotes, heterotrophic, filamentous (hyphae mycelium), cell wall made from chitin, sexual and asexual reproduction by spores. Michel Thibaudon – March 2018

Michel Thibaudon – March 2018

Fungi ~1. 500. 000 species, or more ~160. 000 species described Saprophytic or parasitic Michel Thibaudon – March 2018

CLASSIFICATION Domain Kingdom Divisions Subdivisions Basidiomycetes Eumycètes Eucaryote Fungi or Mycetes Ascomycetes Myxomycètes ? ? Deuteromycetes Zygomycetes Mastigomycetes Michel Thibaudon – March 2018

Fungal spores primary functions: -to tide over unfavorable periods -to disperse the fungus to new food sources - (meiospores) genetic recombination Fungal spores take-off is often an active mechanism Michel Thibaudon – March 2018

Basidomycetes Basid 4 basidiospores meiospores, exospores -never septate -never branched -bilateral symetry -apiculus (attachment point) -usually short (lenght / width ratio smaller than 4: 1) -sometimes a pore Michel Thibaudon – March 2018

Basid and basidiospores Michel Thibaudon – March 2018

Ascomycetes Ascus 8 ascospores meiospores, endospores -septate or not -groups of 8 (sometimes) -often long (more than 4 times longer than wide) -never birth scars -never germination pores -never branched Michel Thibaudon – March 2018

Michel Thibaudon – March 2018

Ascospores No attachment points; variable size, shape, septation B. Kendrick drawing Michel Thibaudon – March 2018

asci Michel Thibaudon – March 2018

(Deuteromycetes) = (Fungi imperfecti) = Mitosporic fungi (~moulds) mitospores = conidia (sing. conidium) (asexual) -produced at the end of specialised hyphae -always a central birth scar -frequently septate -can be very large (or very small) -can form chains Michel Thibaudon – March 2018

mitosporic fungi often responsable of: -biodeterioration -biotechnology (enzymes, hormones, etc production) -food industry (beer, wine, cheese, …) -pharmaceutical (allergen extract. , antibiotic prod. , …) -mycoses -mycotoxins -allergies -infections (A. fumigatus) -sick building syndrom often associated with moisture and molds -plant pathogens Michel Thibaudon – March 2018

Fungal allergens -not enough data and research -one problem: lack of standardized extracts well known: -Cladosporium (in particular children) -Alternaria (and other cross-reactive) -Stachybotris (indoor, big story in the USA) -occupational allergies in the mushroom industry (eg Pleurotus) Michel Thibaudon – March 2018

Michel Thibaudon – March 2018

2 How to collect fungal spores Material and method Michel Thibaudon – March 2018

Today, we will discuss two very important topics in aerobiology: -viable spores sampling, problem of the substrate (growing medium) … -indoor and outdoor aerobiology Michel Thibaudon – March 2018

1 Outdoor fungal spores: Hirst method - Continuous detection Michel Thibaudon – March 2018

1 Outdoor fungal spores: Hirst method - Continuous detection Collection of the drum band Dividing the band into daily sections 1 slide by day Michel Thibaudon – March 2018 Counting with a vocal recognition system spores count

1 Specific or non-inert particles Cyclonic method optical microscopy Coriolis Air Liquid Particles il /f n tio n io t tra a or 300 l/min 3 m 3 in 10 min l Co Liquid sample for rapid analysis PCR Ch em sc an Method for specific identification LAL Transfert of biological particles from the air to a collect LIQUID medium BROADBAND Easy and rapid method Endotoxins Mycotoxins Method with high sensibility Michel Thibaudon – March 2018

2 Impaction on culture medium Culture Be careful to the quality of culture medium Michel Thibaudon – March 2018

3 Filtration method Transfer gelatin membrane on petri dish Air filtration on dense gelatin membrane n u c n b I Be careful to the quality of culture medium Michel Thibaudon – March 2018 io t a

4 Equipment used for this study: Continious impaction : pollens and molds trap : LANZONI VPPS 2000 Impaction on culture medium : MAS 100 Filtration on gelatin membrane : AIRScan Cyclonic sampler : Coriolis®µ Michel Thibaudon – March 2018

4 Analysis methods used: ØCulture medium : Trypto casein soy, Sabouraud MEA Incubation temperatures between 20°C and 25°C. ØIdentifications : Macroscopic Microscopic by transfer on transparents supports, coloration and placed on glass slides. ØNumeration realised when it is possible (invasions limiting quality counts in some samples) Michel Thibaudon – March 2018

4 Analysis methods used: Detection method by optical microscopy (for liquid samples from cyclonic sampler and gelatin membranes gélatines whole or fractionned) : Analysis by optical microscopy, magnification x 400 Michel Thibaudon – March 2018

4 Spore Identification Important Characteristics • Color • Size • Shape • Septation • Attachments Michel Thibaudon – March 2018

4 Study period: Sampling from 2010 to 2012 in very different environments (during 3 years): Indoor air Industries facilitise Offices Hotel House Laboratories Clean room Michel Thibaudon – March 2018

3 Results Michel Thibaudon – March 2018

3 Results Moulds Hirst trap MICROSCOPIC OBSERVATION Absidia Acremonium Alternaria Ascospores Aspergillacea Aspergillus Penicillium Aureobasidium Basidiospores Botrytis Cercospora Chaetomium Cladosporium Didymella Entomophthora Epicoccum Erysiphe Fusarium Fusicladium Helicomyces Helminthosporium Mucorales Myxomycetes 0 0 +++ +++ 0 0 0 +++ +++ +++ +++ 0 +++ Easy detection +++ Impaction / filtration on dense gelatin membrane Culture on : TSA Sabouraud MEA + ++ +++ 0 0 0 + ++ +++ 0 + ++ 0 0 0 + ++ +++ 0 0 0 0 + ++ +++ 0 0 0 0 0 + ++ +++ 0 0 0 Possibility of detection Michel Thibaudon – March 2018 ++ + Cyclonic trap/ filtration on dense gelatin membrane MICROSCOPIC OBSERVATION 0 0 +++ +++ 0 0 0 +++ +++ 0 + +++ 0 +++ No possibility of detection 0

4 3 Results Moulds Hirst trap MICROSCOPIC OBSERVATION Impaction / filtration on dense gelatin membrane Culture on : TSA Sabouraud MEA 0 0 0 Cyclonic trap/ filtration on dense gelatin membrane MICROSCOPIC OBSERVATION Peronospora +++ Pithomyces +++ 0 + ++ +++ Pleospora +++ 0 0 0 +++ Polythrincium +++ 0 0 0 +++ Sporidesmium +++ 0 0 0 +++ Sporobolomyces +++ 0 + ++ +++ Stemphylium +++ 0 0 0 +++ Stachybotrys +++ 0 0 + +++ Taeniolella +++ 0 0 0 +++ Tilletiopsis +++ 0 0 Torula +++ 0 + ++ +++ 0 0 + ++ 0 Trichothecium +++ 0 + ++ +++ Uredospores +++ 0 0 Ustilago +++ 0 0 Trichoderma Easy detection +++ Possibility of detection Michel Thibaudon – March 2018 ++ + +++ No possibility of detection 0

4 3 Results Air contamination Spores small quantity Spores high quantity Mycelium small quantity Mycelium high quantity Easy detection +++ ++ Optical microscopy detection +++ Detection + +++ Counting +++ ++ Identification ++ +++ Détection ++ (invasion problem) +++ Counting ++ (invasion problem) ++ Identification ++ +++ Detection + +++ Counting +++ 0 Identification ++ +++ Detection ++ (invasion problem) +++ Counting ++ (invasion problem) 0 Identification Culture Possibility of detection Michel Thibaudon – March 2018 ++ + No possibility of detection 0

4 3 Results Michel Thibaudon – March 2018

4 3 Results Moulds Absidia corymbifera Infectious impacts Allergic impacts YES Acremonium Alveolit Toxic impacts YES NO DATA Alternaria alternata YES Aspergillus flavus YES Aspergillus fumigatus YES Aspergillus glaucus YES Aspergillus niger YES Aspergillus versicolor YES Aureobasidium YES Chaetomium YES Cladosporium sphaerospermum YES Epicoccum YES YES YES Fusarium YES YES Mucorales YES YES Penicillium sp. YES YES Stachybotrys chartarum YES Trichoderma YES Trichothecium YES Michel Thibaudon – March 2018

4 3 Results Annual variation of Alternaria and cladosporim spores in France Alternaria Cladosporium 25000 700000 600000 500000 15000 400000 300000 10000 200000 5000 100000 0 0 2008 2009 2010 2011 2012 2013 Michel Thibaudon – March 2018 2014 2015 2016 2017 Cladosporium - Annual index Alternaria - annual index 20000

4 3 Results Outdoor fungal spores 2017 Michel Thibaudon – March 2018

4 3 Results MOST FREQUENT FUNGAL SPORES IN THE OUTDOOR AIR IN PERCENTAGE MOST FREQUENT FUNGAL SPORES IN THE INDOOR AIR IN PERCENTAGE- CMEI Cladosporium Ascospores Basidiospores Tilletiopsis Alternaria Didymella Aspergilacées Ganoderma Helicomyces Ustilago Botrytis Sporobolomyces Autre moisissures Erysiphe Epicoccum Myxomycetes Torula Peronospora Uredospores Cercospora Polythrincium Pithomyces Fusarium Fusicladium Helminthosporium Pleospora Entomophtora Chaetomium Nigrospora Stemphylum Tricothecium Sporidesmium Taeniole Cladosporium sp. Aspergillus sp. Ulocladium sp. Stachybotrys sp. Sedosporium sp. Chaetomium sp. Alternaria sp. Acremonium sp. Penicillium sp. Scopulariopsis sp. Mucorale sp. Trichoderma sp. Epicoccum sp. Pithomyces sp. 0 10 20 percentage 30 40 50 Merule sp. Michel Thibaudon – March 2018

4 Example of recommendation Michel Thibaudon – March 2018

4 3 Results Pollens Moisissures We must destroy the wall Bactéries Extérieur Virus Air intérieur Michel Thibaudon – March 2018

4 Seasonality and risk of contamination Evolution of the number of moulds spores per cubic meter of outside air and in clean room (2012) 10000 Number of mold spores per cubic meter of air contamination exterieure contamination dans la salle propre 1000 10 1 Dec Jan Feb Mar Apr May Jun Jul Aug Implentation of Action plan Michel Thibaudon – March 2018 Sep Oct Nov Dec

5 Conclusion Michel Thibaudon – March 2018

• All methods of sampling and analysis based on detection thanks to culture of molds viable and culturable, leave aside a considerable number of nonculturable fungi. • It is important to associate classic analysis culture and methods of analysis by direct detection (optical microscopy) and qualitative methods (PCR…) • The future development of standards, including the ISO 14698, must consider these issues on the mastery of fungal contamination in cleanrooms Michel Thibaudon – March 2018

Happy end Thanks for your attention Michel Thibaudon – March 2018