Environmental fungal risk during respiratory diseases:
diagnostic and treatment
11:00 – 11:30 Asthma and COPD
Malcolm Richardson, UK
11:30 – 12:00 Cystic fibrosis and fungal disease
Riina Richardson, UK
12:00 – 12:30 Bacterial and viral associated respiratory fungal infections
Françoise Botterel, France
You have we try to to keep the time okay time difference okay so now I’m very happy to to introduce Malcolm Richardson Malcolm Richardson is going to talk on asthma and COPD after the first session this morning on the environment this afternoon we will talk on uh we are not the afternoon but
Still in this second part of the morning on diseases is of course every note everybody knows Malcolm he was the former president of ishan the international Society for medical mycology he is now the new president of the British Society for medical mycology and he comes from Manchester with many many Works many
Experience so thank you very much Malcolm well thank you very much for inviting me it’s a great pleasure to come back here to Turin we were here last year but um I’ve been charged with uh talking about asthma and COPD but of course behind this I have to mention a little bit
About exposure and one of two cases where uh asthma and COPD um were implicated in in what I’m going to be talking about in terms of diagnosis and um treatment so I come from the mycology reference Center Manchester which is a National Health Service reference Center
And also we became an ecmm center of excellence in 2017 but also I still have a teaching affiliation with the University of Manchester but desperately trying to retire but not very successfully okay so these are my conflicts of interest nothing really of any significance or note for this um for
This particular meeting and the topic so of course we’ve heard already and we’re going to hear more about this this afternoon patients do live in Maori houses they certainly do in the UK and so the the top left panel here is a mycologist’s dream um I could ask you please can you
Identify by looking at the the colonies on this ceiling what they are well I can tell you what they are there’s stachypotris the suspicious Niger of course there’s penicillin that’s cladasporin and so on and so on and so on why why do we have this beautiful picture of different fungal species the
Reason is if you look carefully through that door is the kitchen and very humid a lot of cooking and drying clothes inappropriately maybe a tumble dryer which was not vented properly and so on and so on so this is a vapor trail coming out of the kitchen
And of course saturating the um the wallpaper or the ceiling paper the plasterboard on the ceiling so we’re familiar with aspergillus fumigators and we’ll talk a little bit about this in terms of deposition that’s aspergillus Canadia in the upper Airways um bottom left we have Alton area and we
Associate this with thunderstorm asthma and we’ve had a lot of thunderstorms in the UK recently let’s see whether or not we do get cases of of clinically diagnosed and laboratory diagnosed thunderstorm asthma now this is an interesting picture this reminds me the bottom right hand side this
Reminds me of when I was a student in Leeds in the early 1970s all right in my PhD on on Candela but here we have um mold on the wall of this of this Flats probably and it says happy birthday those of you sitting in the
Front rows might be able to read this it says happy birthday and there are some lovely snow-capped mountains and in front of these mountains there’s a choir singing happy birthday to you no but anyways is this before or after the party a nice um assemblage of beer cans and wine
Bottles anyway so this is penetrating damp this is not because of high relative humidity this is penetrating damp I’m sure we hear more about this um this afternoon in terms of mycology of the indoor environment and the reasons for this and of course thus we associate dust with in our experience
Investigating and I probably haven’t investigated over 400 houses now both in the UK the United States and and also in um in the Nordic countries um more dust the more aspergillus you find what is dust it’s a matrix of of corneocytes of hair clothing fibers etc etc it’s a perfect environment and we
Know that aspergillus grows on the Keratin of corneocytes from the stratum corneum so we have Asperger’s fascicular here and aspergillus fumigators and here we have dust sprinkled onto a sad plate and a nice colony of aspergillus fumigators right this was mentioned previously and obviously this had appeared in the
French press or on the French TV this was a national scandal in December 2020 uh this little boy died he was two years old his parents were from the Sudan he was born in the UK and um here we have a very brief history and sort of presenting you with this because there
Was a suggestion of a background of asthma and fungal centralization and that’s what I’m going to be talking about so no real significant medical history he had flu-like symptoms and he had the stride or this noisy breathing no covert positive contacts he had difficulties with breathing he was
Treated with nebulizers and there was some partial relief finally ended up in an urgent care center this is in a city called Rochdale which is about 20 miles north of of Manchester and um intubated Cardiac Arrest threading around the the glottis and Airway edema and they were unable to resuscitate this
This little boy so pediatric post-mortems as we’ve learned and we I have been involved now in four similar cases since this because of heightened awareness within the government and within the medical community and of course within the police so I was asked to go and investigate this house with the police
Because these become incidents as a possibility of a criminal event but not in this case or the other ones so he had significant granulomatous Trucking or bronchitis um we have learned a lot what sort of samples to be sent for investigations whether it’s biomarkers like Alexa Manan
How to get how how can the the Pediatric Pathologists get appropriate samples when they do the post-mortem there are some value for the laboratory or all sorts of issues there so there’s a group of people actually within estimate of forensic microbiology it’s a study group it’s a fascinating group of of people if
You’re interested all of these cases those extensive candidate colonization in the upper OAS and in the lungs um there was this focal increase in eosinophils a marker of asthma and um so the question was raised by the pathologist was this severe asthma with fungal centralization and the pathologist’s opinion there were two PM
Reports um acute exacerbation of an asthma-like airway disease in part or in full due to mold exposure so here we are this is not a pleasant environment to live in I think you would agree with me we have a cupboard here that was um there’s a little flat one
Bedroom flat this could be because of water Ingress from a flat upstairs or it could be of course um just the environment we have the the bathroom here with this level of mold on the wall and if you look carefully here the blue arrow points out where the
Parents were told or just go away clean the mold off the wall everything will be fine so you can see sort of water drips here where this cleaning had taken place and they’re also advised just go away and buy some paint without actually saying using an antifungal paint to try
And cover up all of this this mess and this is the the kitchen so the defense lawyers because I had to go to the inquest and that was December last year try it out and make out a case this was due to Lifestyle nothing to do with
Defects in the in the property so this is one of maybe 200 Flats in a big complex of flats uh within the the city of Rochdale so that’s just to set the scene from what I’m going to be talking about and of course you’ll hear a lot more
About this this afternoon I’m not really going to but this was mainly um this is air samples and contact samples of the the visible mold um penicillium Niger uh quite a lot of clavasporium as we can see here and so on and so on but note no aspergillus was
Found now these are using grab samples using this type of sample this is what we return to use you can have a choice of media you can incorporate your antifungals into my kitchen door into one of the sampling heads and so on so this was the the scope uh the profile of
Molds isolated in that apartment now because of this so one year um well actually seven months after the inquest now we have this new directive from central government this became a scandal it was in the Press on the TV and we now have the formation of this group so the
Office for health Improvement and disparities has been charged with a number of tasks to outline the health risks associated with damper mold in the home as if we don’t know what these are already identify vulnerable groups um this is all to do with guidance for the landlords so in this particular case
This was a city council of the landlord it’s really the private sector landlord so this is all social housing and how to provide guidance to assess damp mold related hazards we’ll hear more about that this afternoon and what interventions can landlords take to tackle and reduce the risk of damp and
Mold these are big big tasks to address so let’s see how many years it takes before we can that’s answer some of these tasks that people have been charged with to um so so we’re part of this of this exercise in trying to improve social housing
Okay so a few years ago a former head of respiratory medicine Ashley Woodcock and people from the mycology reference Center thought they would look into our pillows a source of molds so how often do you change your pillow how often do you wash your pillowcase probably very
Frequently but how often do you change your pillow what’s inside your pillow what’s your pillow made of synthetic materials or or down um so they took a lot of pillows and as you do you beat the pillow to death with a map beater and you um you analyze what’s
In the air we’ve talked about dispersal of fungal Canadia and we hear more about that this afternoon so um they found some fumigators we’ve heard from from Esther about rhodo turola that’s um this wasn’t mentioned but we have found out that rotatora is a driver of hypersensitive pneumonitis and we
Have isolated rotatora from bagpipes and we published this bag pipe players long a new source of interstitial lung disease so that’s just significant finding from pillows so um both synthetic and feather pillars so and look at these other molds the these some common some not so common molds found in these pillows
So fungal spores um we have large spores we have small spores we have alternaria and so on and so on different sizes and of course it’s the size of the spool that dictates the deposition of the spores in the upper Airways so here we have this um this nice graphic here
Showing you depending on the size of the Spore where uh fungal candidia-like altered area or right down here very small spores aspergillus where are they deposited in the Airways and after how quickly does it take for a small sport to reach right down is the alveoli of the lungs
Okay and then this is a familiar this is a familiar graph that um we we show quite often to to make a number of points and this has been adapted by my former colleague David Denning in Manchester so we have on the left hand side we have invasive pulmonary
Aspergillosis and then we have in the middle here chronic pulmonary aspergillosis this really is what we’re going to be talking about this morning to a certain extent and then we have fungal allergy abpa and and saphs so that’s again just introducing the whole background of asthma and and COPD hmm
So some more work from uh from from us in in Manchester looking at the uh the microbiome of um the bals taken from different groups of patients we can see abpa healthy people and so on severe asthma and saphs and we can see here the distribution of various
Species and of course the green bars the note aspergillus species complex that’s going to be mainly fumigators but other species as well and some work that um one of our PhD students from Saudi Arabia has been doing still to be published looking at NGS comparison of dust and sputum so
Dust from patients with clinically diagnosed CPA dust from their vacuum cleaners and we had to get ethics permission for this it was a hell of a job to get this experiment for patients to bring in little samples of dust from their vacuum cleaners but that’s the
System you work in and also to analyze sputum samples because we get regular sputum samples from patients who come to the CPA clinics in Manchester but I just want to point out the bars here Asperger’s penicilloides now this was actually mentioned is the speaker from the previous session here
She mentioned it in her list of xerophilic molds see okay you did now this is an interesting because you will not detect aspergillus penicilloid he’s using regular media it’s a halophile and so yeah you would miss it but look it’s here it’s present in dust and it’s
Present in dispute en samples and when you look at the literature so there’s some details about this is a very low water availability as as you showed this morning very diverse habits for the Dead Sea for example solar Sultans grains dried fruits so basically dried environments but you need 10 salt
In your regular media to isolate it and a long time ago so no 1978 Rob Sampson and and colleagues published on this a very common it’s very common in household dust samples but you wouldn’t know this unless you use the appropriate isolation media and um it’s an interesting organism and we
Think this is a very powerful allergen so we’re sort of carrying on on this work on aspergillus appendicularities but this is interesting it can stimulate the population growth of house dust mites we’re not here to talk about allergy to house thus might but there’s a very close relationship between
Aspergillus and a house that’s mites okay so here we have the spectrum of um allergic diseases that we associate with various fungal spores we have Alton area aspergillus cladisporian penicillium and we have the various clinical manifestations allergic asthma rhinitis there’s a massive amount of overlap between these various clinical
Presentations we have abpa or abpm allergic bronchopulmonary mycosis we have this hypersensity pneumonitis I mentioned a few minutes ago we have mycotoxicosis if we believe in the impact of of vocs volatile organic compounds on respiratory health and and so on so a vast range of different types of allergic reactions so what is
Fungalasma run again using this review from my former colleague David Denning defines the presence of fungal centralization or fungal allergy in patients with asthma sensitization immune mediated response to a fungus without evidence of informational tissue damage and then fungal allergy um this is an immune mediated response to a fungus sometimes associated with
Tissue damage so was this the background to that little boy dying in that apartment then we come on to COPD this is where we do have big alterations in the long architecture patients are on steroids frequent Hospital admissions this is a serious disease so fungal exposure on
Top of this is is bad news and also there’s a link with malnutrition so the incidents over the years shown here and this table could go on and on with sort of more and more reports showing that aspergillosis and COPD are very very closely linked in different groups of patients hemiology patients patients
With AIDS and and so on so quite quite large numbers in these various surveys and from Madrid okay an old paper but here we have COPD patients um 22 of them had invasive aspergillosis and we can see here now the admissions as a result of aspergillosis and COPD so
It this seems to be on the on the increase and again the same study that was carried out in Spain so fungal allergy then in terms of management now we’re going to talk about diagnosis and treatment because that’s really what I’ve been charged to talk about so we have these aspects of the
Management of fungal allergy so avoidance of the fungus that’s impossible um control of the inflammatory process that is possible improvements of the airway airflow reducing the amount of mucus and obstruction trying to reduce the the fungal burden and also of course control of bacterial infection on top of this
So what about antibody tests and there are people in the audience here who are much more expert than I am in terms of um understanding and developing and using antibody tests of various sorts and than I am but um the the size of the circle here denotes the numbers of of patients of
Abpa ige determinations are very useful as are IGG determinations to a much lesser extent CPA we use antibody testing very effectively because that’s one of the the still works of of diagnosis in conjunction with a sputum culture for example and imaging and then of course we have invasive aspergillosis and
That’s what we’re talking about with COPD patients of course we’re all familiar with the aspergillus galactom and test but that’s a panthel test of course and over the years and maybe other people can comment on this there’s an ongoing interest in the use of of IGG and IGM detection in patients with
Supposed invasive aspergillosis so there’s a lot of detail here I’m not going to go through this line by line but if we look at fumigators Associated asthma we this is controlled asthma we have elevations in ige specific ige we have this level of total ige and then we have
Saf so this is severe asthma with fungal centralization we get this elevation of ige high levels of total ige and um interestingly enough though normal levels of IGG whatever the normal level is and then we have abpa that’s all part of this overall spectrum of allergic diseases um asthma
Specific ige total ige elevated levels of of IGG we’re not too sure what the threshold is here and and depending on the uh the method of detection and then we have um an increase in the eosinophil count and that’s what we did see in that little boy from from Rochdale in in Greater
Manchester and of course we have these other investigations that he carried out as well okay so over the years I’ve been a lot of work done here in in France even before I started my my PhD in Leeds in the early 1970s a lot of literature especially the French literature but
Then more recently um in in international journals we’ve had all sorts of methods for determining IGG um antibody levels so from precipitants uh right through to Eliza and and Beyond and um still to this very day we do have in-house immunoassays these are not particularly well standardized perhaps
More in France than in the UK and and elsewhere we have the immunocap method and here I’ve given you a range of cut offs we tend to use 40 milligrams per liter as a cut off but that was really specifically um described by by Richard Barton from
Leeds in the UK in relation to cystic fibrosis patients but we do use that in our CPA population and we have of course the New Alternatives and this is the shortlister about 21 different companies now that make Asperger’s IGG assays manualizer and the point here is a variety of
Antigens employed from very crude soups if you like of the cytoplasmic somatic so all antigens through to recombinant um antigens and I’ll just touch upon those very briefly in a few minutes so slides from my own collection This was um from many many years ago ottoloni double effusion or immunodefusion count
Immunelectrophoresis and then our own work back in the early 70s using two-dimensional crossed immunelectric free says very powerful technique and then ride through now to this highly automated system and this is the system that is used in in the UK at least in big Departments of clinical immunology
And looking at the variety of immunocaps that you can you can use it’s a very powerful machine so that’s where our samples end up from patients with CPA and and saphs and allergic aspergillosis so nicely all summarized here and the author the first author um
Two if not more of the authors of this um article that appeared in medical mycology a few years ago now and I understand this may be be revised um but a very useful sort of guidance for um for the use of antibody detection and something we should do as a British
Society as well so we have now um this is a French company called LD bio and I’m sure some of you are familiar with this point of care test um so this Hunter was a postdoc that we had myself and David Denny and we had um and published a few papers using this
But this is just the paper looking at the utility of this particular type of technique um in the setting of chronic pulmonary aspergillosis the point is here that um pretty good comparison with the immunocat so where you have the immunocap sample 40 or greater milligrams per liter pretty good
Concordance with the the results the positive test result with this with this particular device so this is just a few Euros we’ve introduced this type of technique into CPA clinics in in China where we’ve been working recently and um I’m not sure exactly how pop or what
The uptake of this particular poct has been but in our hands it’s like a screening test and then if you get a positive that can be then sent to our Central department of clinical Immunology for the immunocap type of investigation so here we have this uh this paper here um
Now we’re talking about specific allergens of aspergillus as markers for genuine sensitization so here we have a paper describing 23 allergenic proteins mostly cross-reactive so this is now focusing on ASP F1 some details about this um belongs to the Michael Julian family and so on and so on there’s a lot of
Detail about this no homology with any known fungal Gene this is a work done by by Paul Boyer in in our in our group um it’s a very specific halogen for fumigators interesting almost undetectable in-house dust that’s an interesting observation from this paper so there are many more other specific
Allergen components are being described and the utility of using these in very specific amino acids and the Japanese also have been um working on this for many years this now gets extremely complicated you have asthmatic patients with positive ige antibody to fumigators and then if you use recombinant antigens you can come up
With yes abpa for example is is highly likely if not if you get a negative result it’s less likely so this is now really really drilling down into using very specific allergens from Asperger’s fumigators so we end up with um with treatments this very recent review in clinical respiratory journal and three slides
Showing you um Hospitality says well first of all in COPD patients so we have um this near we have patients who are antibiotic or corticoid resistance um radiology and then we have a combination of culture and we’ve been developing what we call high volume culture in Manchester where we may plate
Out five milliliters of of sputum or a BAL not a small smaller volume as until recently described and advocated in various guidelines collectamana and of course and also IGG and IGM using either Elisa or the LD bio ICT type of poct and of course then
NGS what does NGS does it really help us some of you here may have been developing and have experience of NGS we see NGS at the moment as the same as sputum culture it gives you glass variety and profile of molds in that type of specimen how on Earth do you
Distinguish between what is colonization and what is really really significant in terms of Allergic Disease or disease so then you’ve got all these combinations and proven IPA at the left-hand side and then the treatment that’s used a variety of Hazel drugs foreign we now have pc9945 and inhaled a very potent um Azo
Or uh occasionally of course depending on the host vulnerability liposomal amphoterosene B and then we have CPA chronic pulmonary aspergillosis in COPD again it’s a problem and of course can then progress into into invasive disease again similar clearing presentations on various clinical investigations that are carried out the same Spectrum profiling of
Diagnostic methods and then we range from nodules right through to Chronic necrotizing pulmonary aspergillosis and the various antifungals or whether or not antifungals are used or we just observe the patient and then find the centralization in COPD patients here the patients are high for immune breathing difficulties and
Um again the spectrum of culture and and biomarketype investigations and then we have the use of corticosteroids and then intracons of Oregons are for quite long treatment periods so um finally then we have this and I’m sure this will be mentioned or this concept will be mentioned uh this
Afternoon here we have 62 patients and this is a patient from some of the people we do know here um this is from France I know one or two people it could be one or two people in the audience here who are authors of this uh of this
Paper anyway they um looked over 60 patients with COPD they use an electrostatic dust collector and of course we may hear more about that this afternoon and an alternative way of collecting dust sort of pinching the dust from a vacuum cleaner bag is using quite a nice sophisticated way of collecting dust and
In summary then um fumigators or aspergillus more frequently detected use during the exacerbation um high levels of antibodies where there was a high total mold count in the indoor environment that’s an interesting but not unexpected observation and um aspergillosis diagnosed in two patients who presented with increased levels of
Antibodies so this is the conclusion of the paper antibody detection levels of antibody associated with chronic lung alteration functional alteration and or domestic mold exposure okay finally then what about monitoring and I’m not going to encroach upon anybody else’s time but um these are available on Amazon now 100 Euros or
More there are many many of them but just note here that we haven’t looked into this mold and vocs um I don’t know if anybody has any experience of these but because of this highlighting of this tragedy in Rochdale two and a half years ago now there’s
Quite a lot of research money in the UK for investigating Maori houses so we have in collaboration with some inventors and and housing surveyors we are looking at the the use of real-time monitoring so here we have again the Maori bathroom now this is a device that could be planted put inside
Um house where there are damp problems or there are vulnerable patients and so a constant stream of air over a whole series of slides and using um photography the development of these colonies is monitored in real time say over 48 hours and then that image is sent to an AI artificial intelligence
Type of station and the colony is identified based on on Photo recognition of the colony so we also have been working on AI recognition of fungal colonies a lot of people are doing this but this is just a prototype it’s going to be installed in a number of problem
Houses in and around the UK that sea where we get from go from this but as I say there is grant money now for this type of investigation so that’s me and and fast literature of course I found these these two reviews here this one in particular this covers more than just
Allergic Disease and aspergillosis and COPD covers all aspects of human exposure to fungus so um thank you very much for your attention I’m very happy to take any questions thanks [Applause] yeah so thank you very much Malcolm you have a real huge experience I don’t know
Why you are all Specialists on malls in Manchester is it a umid weather climates this is where we come to Ren because it’s always sunny here could I ask you please to send that one slide with the government you should yes you mean in terms of the the going
The committee is going forward yes of course please it’s it’s on it’s in the public it’s the main but I can send you that slide of course there’s a big document associated with that summary so yes some question well thank you again for the talk and I’m really glad you talked about the
Case I was just asking before yeah but for what I recall also from this case so this was even a private housing situation no no no okay so but I mean housing it’s the same in France you have you have owner occupied dwellings houses flats whatever mansion houses a few and
Then you have what we call social housing yeah yeah so there are two categories of social housing the social housing which is organized by housing associations so they are if you like private landlords and then you have and then you have um vast fast areas of social housing
Which are if you like the landlord is the council city council but then you have a third group of landlords so these are if you buy a house big house in in wherever you come from in France and you subdivide it into Flats it’s a Victorian
House you don’t want to spend too much money so that’s a private landlord and they’re the ones who are so resistant to improving the property they ignore all the complaints hence this government directive now you’ve got to get your act together because we’re going to come after you if you don’t
Um so we have a an organization called environmental health officers and they can actually declare a house unsuitable for habitation based on what they see or the landlord has to fix things and if they don’t then the consequences are pretty severe if it is good no I’m glad
You mentioned that because those were my two concerns I wanted to raise so I’ve been in the Netherlands now for two years although I moved there and then left and came back and I remember when I arrived the two first houses I had were really important conditions and I feel
Like nowadays that we’re we are moving so much we are migrating so much we are not buying certainly something in a place we are not sure we will stay in science we move a lot so how can we I mean I fell at the time who should I
Complain to about the situation that’s why I was so interested to know if there’s regulatory bodies checking the housing conditions um and especially in in cities like the one I am now that buildings are from the 60s still and another concern just said if I can quickly mention this will be
Also yeah okay no but this is another concern because you mentioned the AI and I feel like now everyone is doing AI nowadays and is it really necessary for this issue to have well I mean I’m just thinking in that particular situation I mean um they have the alternative is that the
The image of this developing Colony let’s say it’s clavasporian is is then viewed by a human being to say yes this looks like Gladys Warren based on the topography and the color of that Colony uh or you make a slide press but this this machine will send that image to an
Uh photo recognition library and we have over 50 000 images now of both developing mole colonies and the microscope preparation so it gives you an answer I mean this is very very familiarologists with these automated track systems like the WASP boy cholesterol system it’s all photo recognition and there’s the answer on
The screen so okay thank you very much we have to to move to the next you will ask your question after no problem so now I’m really happy to welcome Marina Richardson so right now is also working at Manchester and she will talk on a very particular clinical situation cystic
Fibrosis and you are really expert on the field of mold of course so thank you very much okay thank you so much for inviting and and yes um quite the challenge to uh talk to this audience about CF and and fungal infections because so many of you are involved
Um but I’m I I decided I will take more of a sort of clinical approach practical approach the reality check how we do and how we think about things um having both clinical and laboratory background myself my disclosure sorry I failed to use the template but it says it all
Um and uh none of this is relevant for the topic of the talk so first starting so not everybody is necessarily completely aware of what’s what cystic fibrosis is a couple of facts about it and facts that are relevant for for what we’re talking about today so yes it’s called it’s a genetic
Disease um inherited disease caused by mutations of the CF TR Gene so cystic fibrosis transmembrane regulated Gene and the mutation leads to dehydration of fluids in the body so that that is in the respiratory tract GI tract everything becomes sticky and gooey it’s alive saliva is thick and sticky
Everything is is concentrated in a way and that leads to mucous plugging in the lungs um blockage of fluid secretion inflammation and tissue damage and fibrosis we also know that recurrent pulmonary exacerbations that are mainly caused in childhood by bacterial pathogens first normally starting with staph aureus as as little kids and then
Later with pseudomonas um they are the predominant feature for many patients chronic colonization with candida and aspergillus has been linked with accessibations and um and more sort of Rapid decline of lung function um and patients colonized with aspergillus have an increased risk for particularly for abpa so abpa is the
Disease in fungal disease typically seen in in CF and the exacerbations are treated with um oral and IV antibiotics many of them are sort of on suppression uh suppression with oral antibiotics and then they come into hospital or via outpatient or Pet Care sort of pulses of IV and antibiotics
I think the other thing that is really important to in this context to to just to know that there are sort of completely novel disease modifying treatments now in clinical practice so we have the cftr modu modulator therapies which basically partially or quite fully return the normal function
And that stops the sticky gooey excretions so they become more or less normal so the question with CF and fungi is is always what is colonization and what’s it infection and as Malcolm’s presentation showed clearly that we have um we have to separate that immune response um
Uh and having antibodies does not equal to disease humans have evolved in in rainforests and in and lived in mud Huts through the evolution of humankind and our immune system and everything has been developed so well that we respond to infection and every resp and and some
Of the response is just a reflection of Health so if if antibodies don’t automatically mean infection finding fungus doesn’t mean infection how do you tell the two apart and it becomes even more difficult like Malcolm mentioned NGS the more sensitive methods are used the more fungi you find what on
Earth does it mean and that is a big problem clinically and there’s a massive risk for over treatment and a complete loss of antifungal stewardship and the reality is that the clinical evidence or benefit of antifungal treatment is very very limited in CF patients Beyond apba so APPA yes we know
It will and it’s needed and it works uh but um actual clinical trial showing that somebody is colonized with fungus X you treat that and the fungus goes away does the patient get better that that evidence is very shaky clinical trials are very limited the other thing is to remember is that
CF itself the mutation itself doesn’t affect your immune system sort of directly that that badly it’s it’s really historically the the old-fashioned treatment steroids and other things um so the question is that what is looking forward what’s going to happen so we have these novel treatments they will reduce the need for
Antibiotics they will reduce the need for steroids and other immunosuppressants Biologicals have been are being used and then later of course for lung transplant so what is it going to look like in the future is another question so is the fungus an innocent bystander is the question
Just couple of facts these been measured already we know that a lot of species couple of them only um are associated for human disease this is evolving little bit with more and more research and NGS and other methodologies but I think that’s sort of the bigger picture the important thing that has been
Mentioned was topic of yesterday was about you know what is how do we resist fungal infections it’s not really about us it’s mostly about the weakness of fungi and that um the poor dermatolerance of most fungal species is the the thing now how that is relevant
In CF setting is that many of them uh require uh um uh nebulizers and and um uh saline nebulization just illusion that sticky gooey stuff to moisturize it so that they can cough things up so they use a lot of nebulizers um and the the systems are often cool
And the nebulizers they can become dirty and colonized so they are inhaling who knows what and um so those fungi so often the sort of oversimplified scenario is that you get a sputum sample from a patient you throw it in the laboratory at 25 37 and 45. if
It doesn’t grow in 37 and 45 it’s an environmental contaminant can’t cause human disease can’t live in human body temperature um now with this setting it probably can colonize that nebulizer so don’t that’s not as straightforward as we thought this is from the casa de Val review this
This uh picture but it’s just a reminder that our ability to resist fungal diseases has these two main pillars that’s the new side of the things and then the endothermy and I’m not going to cover the endothermia anymore because I think that’s that’s what needs to be
Said about it really uh other than keep your eyes and ears open What’s Happening new pathogens may be popping up but the immune side yeah we’re doing all sorts of things and and this is a wonderful picture of how it all works yeah uh important thing every part of
The innate and adaptive immunity and all the soluble components of both systems and all the cellular components of both systems ranging from complement to innate cells of macrophages and neutrophils and da da da to your BNT cells to your antibodies the whole cycle the whole immune system is is dealing with it
But this is the curve that Malcolm already briefly showed but it’s really key to uh think what the fungus can cause in humans so yeah any any in the if you’re an average person with normal immune system having a few spores in your Airways you’re in the middle there
And the curves shows that you are that’s colonization there’s no damage and the weaker you become your immunity is defected the more damage you start seeing because the fungus is able to invade tissues important things the stronger isn’t the better we always think something a bit a
Little bit more detergent in the washing liquid it’ll be good no optimal is good because the stronger you start seeing damage again but that is now caused by the immune system stupid thing doing too much so the range of diseases is there and as I said
Already the main disease in CF is in the hypersensitivity side they are hyper reactive and that’s their type sort of um what is typical however the important thing is that CF patients represent the whole spectrum of that in different conditions um stages of their life so they are at
Risk of invasive aspergillosis if they develop a hematological malignancy if if they need a lung transplant they are then pushed to the left and so and if we manage to control their their immunological the hypersensitivity the burden of disease with the new wonderful drugs they are maybe in the center
So the whole Spectrum so there are two components that the sort of physical physical sticky gooey long side but then there is the immune Spectrum which is independent different genes and the whole spectrum of humanity is there so this is a I like it pretty good um uh review recently from Journal of
Fungi actually yeah this year uh summarizing the uh most common allergic fungal diseases and the only place where CF is mentioned is with APPA allergic bronchopulmonary aspergillosis so that’s just to say yeah that’s recent review and that is based on a number of papers I just picked up couple of them
Um and this is this is now from uh 2014 so a couple of years back definitely and they showed that uh 15 of CF respiratory specimens were positive for moles and um that was in contrast to two percent of non-cf respiratory specimens and um and 32 percent of CF patients had one or
More cultures positive for molds uh in contrast to less than three percent in non-safe patients yeah so they are more colonized full stop nobody’s arguing about that the interesting part of the paper is this list of species and you can see that there is only a couple of them a
Couple of lines there where you have bigger numbers with three you the last row can’t see anything of the detail but you can see that it’s only once and twos single digits here but there are a couple of them with um hundreds of of um cases and those are as particular species
So aspergillus vermigators and flavus and when you drop them together um uh with with the type aspergillus vermicators and aspergillus non-fumigators scheduling and then what they considered was contaminant the only group that gives you p-value is the aspergillus one so okay definitely CF aspergillus is important
And this is an older review already but similarly summarizing all the literature up to the point saying that yes as particular’s colonization is bad news basically in CF and um and then what do you do with antifungals is unclear so going back to this table now diagnosis how do you do the diagnosis
The next next call the one of the columns there just so that the back row can see it basically says you have your Immunology that you need to do microbiology and your Radiology and in Immunology it’s ige mainly but IGG as well and specific ige and GE for aspergillus
Or other molds if you suspect other moles but mainly aspergillus has said and then total IG So What proportion it is of that eosinophilia is is a typical finding microbiology you do culture and you do your aspergillus PCR and Radiology yeah you do that anyway but mainly to rule out other diseases
CPA and and such um so we looked at this we looked at because um we have um one of the country’s biggest CF centers um the Pediatric CF Center and then the adult CF Center in Manchester um and uh we had decided we did a lot of work up
On this uh sputum samples from these patients to identify exophylla to identify all sorts of moles and other things the question was what’s the point of it does it relate to anything and we looked at particularly at exophylla because that was one of the main projects that we were working on
And we wanted to see that how did it go pre and post when they first were identified with exophylla how did it how did the lung function evolve and you can see from here that the midline is where the identification the day Zero was the first exophylla found
There’s no real difference left and right the patients go their own way we even looked at then that many of them were treated for exophylla and they may have had co-colonization with aspergillus or other things so they got causes of mold active antifungals that made no difference
And we looked at all sorts of parameters all clinical things and and so forth and really the the um the number of p-values is very limited in in um you know some link to see if mediated diabetes don’t know what that means but the conclusion of our paper was that
Despite the frequent isolation of exophylla um it was not associated with any clinical progression of the disease it didn’t make difference left right or Center at the same time it’s really interesting Paul Vive and others from 9 Megan coordinated this Dutch study published in medical mycology same year and they found that
That’s um basically the summary of the paper was that this study suggests a schedosporium and exophila are probably no major pathogens and made no difference and this was a nationwide study and these are the the kind of being at the clinical um interface yes the lab can do wonderful we can
Isolate all sorts we can do NGS what does it mean and stay away from treatment unless you have any evidence that it works at the same time we did an analysis this was just triggered so we analyzed um the beta D glucan levels in our CF patients because we’ve got a gut feeling
That when they got sick and we did beat the d-glucans as per the protocols they were always positive always positive so we wanted to analyze and we have a tissue bank for for CF patients because we are a center so we went to the tissue bank and we collected patients uh CF
Patients um um a set of them pre and post and during six samples per patient uh with excess patients without exacerbations and looked at their blood beta the glucan levels and I’m sure we all know what petery glucan is but it’s basically a pan fungal um sugar I call it always fungal dandruff
Been shed all over the place when it when it’s around and yeah disappears by it’s cleared by neutrophils taken the liver sugars are dealt with in the liver and that’s basically their life cycle so we noticed that there were some patients who had very high level of Betadine glucan antigenemia and it was
Persistently you can see on the top graft there that they they had over many many months that’s a year follow-up they were all the time over 300. um or two hundred hundreds clearly positive others were all the time negative and when we plotted them um against various things age was a thing
That came up as a p-value and it’s interesting that when you’re younger the spectrum is is wider but when you get older it’s low our interpretation of that was that it actually probably reflects the basically the natural um a lot of patience through death and the survivors cannot have continuous anthogenemia
So beta di glue gun is highly monogenic and causes systemic inflammation and if you have systemic inflammation for 30 40 50 years I mean you you end up with problems so the older patients can’t have that um either they cope better with bdg and get rid of it or they die away and
So yes that’s interesting it’s not to be ignored funky yeah but not just an innocent bystander probably but um yeah but what do we know about treatment Cochrane they haven’t bothered anymore they did this in 2014 uh maybe due to be done again but they’re they’re basically they didn’t find any systematic
Randomized controlled trials for treatment of APPA in CF and they basically say we don’t know we could do better we could do those clinical trials but nobody’s doing just to mention about fungal biofilms broadly so um they develop on any surfaces that are either non-renewing non-shedding like lines and catheters and whatevers or
Nebulizers spaces or they develop in cavities preformed cavities or sinuses now they are innately resistant to treatment and but the key thing in the CF setting is that they are very rarely in human body single species and it’s not even single a couple of species but mixed Kingdom
Biofilm so you have bacterial fungal biofilms and the the thinking is that they are protection against each other and Immunity and antibiotics or antifungals so should you treat for both to get rid of them otherwise if you treat for the Mixed biofilm only with antibiotics it ends up with an empty
With a fungal skin not penetrating anymore and you have persistent bacterial infection or the other way around so we looked into this as well already a couple of years back now and as the title said says there interestingly the intravenous antibiotics reduce the presence of aspergillus and that just reflects that they are
Very symbiotic they hang on to each other they hold hands and when the other one goes the other one goes with it it’s so the assumptions we make and extrapolations we make from other settings have to be made very carefully it’s a different disease entity and um this is um uh
Uh an interesting study from you you’ve a local study from here and um uh some Rising hold on um very clearly that you get a lot of espadillas in these patients so the the environmental exposure is there so we are back to basic yes espadrilles is important and
Um and that maybe we should do some surveys how surveys and this leads to this final part of my work which is um about Norman’s paper on from Manchester about our CF units or Ps Ward is our CF unit and um it’s very old-fashioned part of the building where they have a wonderful
Courtyard with trees and other things inside there and the safe patients can get fresh air they open the doors and so much about ventilation and filtration so that’s that’s the PS word and because of some construction work and other reasons um we did weekly um so air sampling there for about a
Year 14 months and all the sampling data has been put together into this paper for that of a year period and not going into detail of any of this but um I think the findings are and then yes they were combined with the Met Office data so the UK
Um uh basically weather forecast data and wind speeds temperatures rain no rain everything was put together into magical statistical analysis the findings are less surprising so aspergillus fumigator spores were more abundant during summer months yeah we have leaves we have plants excellent High counts were driven by higher temperatures and lower wind speeds
Okay not surprising but indoor counts directly correlated with outdoor counts well obviously so if you open windows and doors um but the link of indoor counts with exacerbations of APPA is is so well established so this comes to the point that what instructions do we give these patients
Do we tell them not to do composting not to go outside keep windows close and and so forth and the final reminder although we are very clear now what what diseases are there definitely the story isn’t all written and clear so get keep keep your eyes and ears open with with
Climate change new things may may be coming our way so summarizing it’s important to remember that CEF patients are not one group they they represent the broad spectrum of immunological responses ranging from deficient to hyper reactive the main fungal pathogen as we know at the moment and probably stands is aspergillus for megatus
And APPA is the most common disease but getting to the point and the point of this afternoon is that of course we know that prevention is better than cure but controlling exposure is better than prophylaxis how do we control the exposure and that requires better understanding of the environmental exposure sources so
That we can Implement better non-medical non-drug based approaches to keep these patients safer so at the same time yes CF management is evolving the tolerances evolving that’s all messing up the picture so just uh sort of reminder that yeah let’s keep our eyes and ears open that’s me thank you [Applause]
Thank you so much for enough for This brilliant presentation so we have time perhaps for one question but once again we will we can talk on this topic this afternoon so Joanna thanks Rina that was really insightful um my question is very sort of philosophical
Um with the with the Advent of the cftr corrector drugs being approved um how do you see the landscape of fungal diseases changing it requires monitoring who knows but the simplified thinking is that there is nothing wrong with the immune system of these patients other than if we give
Them steroids and Biologicals and whatevers but essentially there’s nothing wrong and if we can get rid of the sticky mucus by fixing the problem they’re just like anybody else but I don’t know with the correcting drugs yeah so the um the regulated drugs they actually they’re amazing when you
Look at these patients they are amazing yeah I think we will have results in in a few months or a few years because uh everybody’s monitoring that and it’s really impressive so it is yes yeah okay so thank you very much and now I’m very happy to welcome Francoise she’s
Professor of medicine in in Paris in Paris Crete uh and she’s the the president of the French society for medical mycology so Francois is going to talk on bacterial and viral infection during cystic fibrosis and respiratory diseases thank you very much um good morning everybody here and in
Online I would like to thank Jean-Pierre my friend for giving me the opportunity to share with you some data about bacterial and viral and and the fungal of course Associated respiratory fungal infection and the question is can we are you before dangerous lesions of best friend forever and thank you Dina for your
Introduction about biofilms this is my disclosure let’s start with a few definitions the respiratory tract infections as number one as you say of consultation worldwide uh indeed the respiratory tract infections refers to a range of infection um on the upper respiratory tract and the lower respiratory respiratory tract
And of course including viruses of bacteria and fungi and sometimes together um we have to describe distinguish co-infections and super and fictions indeed the co-infections are concomitant and function while super infections are sequence sequential infections by two different pathogens it’s very important to this to this and then to distinguish sorry that
And the co-infections are associated with the severity of respiratory respiratory diseases and sometimes lead to Shear resistance through biofilms and of course is very important to quantify the risk of co-infections or super infections to address the prophylactic treatment for the for the patients and you can see here that the
Influenza virus and Sask of two virus infect respirate all the respiratory as of course Asperger’s formulatus uh the partners of the the poly infection in the respiratory disease as of course all the microorganisms and the uh cell epithelial cells tractor and uh uh at the bronchial level and
Alveolar level and I propose in the first part of my talk to um analyze with you the the interaction between microorganisms and epithelial cells and in the second part the interaction between microorganisms especially in in biofilms and in fact the coup pathogenesis of co-infections is complex and involves dynamic
Interplate between the ostisman these differences and the virulence of the microbes um I choose to to um to speak about um of co-infections to to analyze the the weight of bacterial and fungal coin fixtures in flu and we know that most of estimated 20 to 60 million death from
Is due to from bacterial super infection rather than from Direct effects of the virus we know also that one in four patients admitted in intensive care unit with severe influenza in fiction presented bacterial or viralco infection and we know that the frequency of bacterial and fungal co-infection are highly variable
From 2 percent to 65 percent the Civil bacterial pneumonia is following influenza with a Streptococcus pneumonia a staphylococcus hemophilus and friends are recognized and are known to increase disease severity and mortality and of course with the recent literature about in the current fiction between influenza and aspergillosis we know that
Influenza virus is an independent risk factor for aspergillus asperger’s yes in immunocompetent and immunocompromised host and we know that about 20 percent of influenza infected patients are diagnosed with aspergillus um we have a lot of listen from the literature with uh covid-19 of course there are a lot of Articles of
Um from the subject and I choose a meta-analysis published in 2021 about 118 studies with ICU and non-icu patients in this um meta-analysis the prevalence of bacterial co-infiction um after covid-19 is about eight percent and the co-efficients are higher amongst non-icu patients for the very well coin fiction after or
Concomitant with covid-19 the rate of this coin fiction is about 10 percent and the most virus implicated are influencer virus and respiratory Sensational virus and for the fungal co-infections the rate of this uh this is co-infections are about eight percent and aspergillus is the most frequent
Um fungi implicated but you can see uh on this figure that the percentage are different between coin fictions and super infections now we will see together the physiological physiological mechanism for vowel and fiction and we know um in the in the flu that the virus binds to um
Alpha 2 6 salic acid receptor coupled to glycoproteins such as measin in respect in epidural cells and it leads the decrease my calorie mucosiliary equivalence we have to loss of cilia and you can see in the in the figure on the on the on the right uh that there is a decrease of
Cilia beating frequency dyskinesia of uh cilia and we have two dysfunction of tight junctions and cytoskeleton frequently observed after a viral infection and the impact of integrity and functionality on respiratory tract contributes to create to creating favorable favorable environment for secondary bacterial and or fungal infection
Um for the very well and bacterial and fiction after the viral and fiction the window of susceptibility to bacterial co-infection is approximately um to seven two days after influenza various and fiction and we have a lot of consequence after the secondary bacterial infection after viral infection with a lot of production of
Cytokines different between bacteria and virus and it leads a cellular inflammation a fibrosis and apoptosis and necrosis of epithelial cells for the viral and fungal infection especially for aspergillus fumigators we have an alteration of respiratory tract after the viral infection as you can see here in the figure after
Um after infection of 24 hours and the epithelium damage can alter of course the respiratory epithelium display of transplant protein and it’s it’s leading and exposing apical sites receptor such as anti-green Alpha One Alpha 5 and a beta1 and this exposition of apical site leads the
Fungal at the runs in the tech tracking tree after that the the major epithelium facilitates the addition of fungal cornidia and of course subsequent Invasion and in ICU patients the tissue repairs takes longer and the basement membrane of the epithelial cell is more exposed as you can see here favoring super infection particularly
Particularly with aspergillus of course close to the physiological mechanism the antiviral immune response also plays a role in mixed in fiction and you have here um the the lot of alteration of the human eye Moon response after bacterial and we will infection with a decrease of the numbers and functions of innate and
Adaptative cells after the viral and fungi viral and fungal infection at the alveolar level we have a phagocity phagocytosis fungal killings and cytokine production in pair the recruitment of neutrophilia is also affected by the by both infections and the loss of neutrophils compromises cytokine production the cytokine storm which is very different
Between the the virus and the net mediated fungal killing of course we have to release of fabinus material which obstructs the the small Airways at the end uh the very the virus destroy the normal site and it leads a decreased oxygen and carbon dioxide diffusion capacities and the the environment
Changes with the epoxic environment we we change the property of aspergillus virulence and we have a biofilm formation and in the in the biofilm the macroscopic for morphotype and the architecture of aspergillus regatus are modified because um influenced by oxygen tension oh of course all these interaction between
Epithelial cells and viral bacterial and fungal infection have a consequences on diagnosis treatment and prevention of the poly infection in respiratory disease it’s because in recent year a lot of publication talk about different management and guidelines on diagnosis and treatment of this poly infection let’s go back on my figure with the
Partners of the poly poly infection in respiratory disease and now I will focus on interaction between microorganisms and in the literature there are few data about biofilm With viral and fiction at my in my knowledge I I don’t know but there are a lot of few data about a biofin between
Filamentous fungi and bacteria and I will take two examples to to illustrate this talk uh the as you can as well as as you say the the biofilmoid is a change of life for bacteria and uh fungi which um change from planktonic and individual to uh fixed and uh life in communities
And the biofilm is a structured physical and metabolic community of microorganisms unbade in an extra cellular as you can see in green on the figure which protect from external aggression and which is in contact with biotic or abiotic surface it’s very important to this to to distinguish the bacterial biofim and
Fungal biofilm because the the thickness of the biofilm fungal biofemme for for the former filamentous fungi are is very important and it’s uh it’s not the same thing um with the bacterial biofilm um the bio film have a lot of properties with localized gradients gradients of oxygen where the oxygen decrease
According to the deepness of the of the biofilm we have a gradient of nutrients a gradient of pH gradients of Quorum something or so in biofilm we have a lot of social interaction with the core version of competition between microorganisms and of course the problem of biofilm is the ability to
Survive after antenegrobial exposure this properties include extracellular Matrix that form physical basis of the biofilm architecture and stability of the biofilmer the the biofilm is characters I I said before by heterogeneity of gradients um no I would like to to give you two examples the the specific interaction
Between the microorganisms and the first one is between aspergillus femigators and stenotophonous meltophilia which is a gram-negative bacteria saprophytic bacteria opportunistic bacteria which which is found in cystic fibrosis in COPD in immunocompromise with aspergillus femigators and in my lab we analyze the the the cooperation between the Asperger’s filming
In mixed biofilm and the wind direction with a human nasal epithelial cells which are primary cells for the physiological relevance uh yes in this video you can see the the formation of fungal biofilm with and without bacteria and uh on the left we we can see uh the mixed biofilm between with Asperger’s
Responseophilia and you you can see the slowing down of the fungi fungus in presence in bacteria and the typical morphology of aspergillus fugitis in presence with bacteria this phenomena is called antibiosis is known between staphylococcus pseudomonization with Asperger’s fumigatus and it’s a molecular communication between this microorganisms
Known in mixed biofilm which alter one or two um this in terms of development morphology or growth for one or two partners and here the the antibiotics of standard of minus maltophilia on Aspergers is probably due to um in part uh to secondary metabolites as Nicola papon said yesterday but yeah
Of course it’s uh this metabolism secondary metabolites are secreted by stenotrophone as meltophilia we we have developed a lot of characteristic in our biofilm between aspergillus and stenotrophomonas maltophilia and you can see from the left to the to the to the right direct interaction between stenotrophonesophilia and aspergillus fumigators
Binds to aspergillus fumigators you can see in a Range the bacteria and in green uh the the filamentous fungi we we know that aspergillus is inhibited by the stereotrophonesophilia with a specific morphology of the filamentous fungi when we we observe the extracellular Matrix which signs the mixed biofilmer we observe that the the
Increase of thickness yes of Asperger’s cell wall when the the bacteria is present and the the last figure the last picture is on epithelial cells and we suppose that the galactosabino galactan polysaccharide of Aspergers is the link between the bacteria and the fungi this year we analyzed to the the
Frequency and the quality of the oh sorry of the Cilia we beat them sorry for the video and we we note that when aspergillus and female formigators and symptoms are together um on in biofilm on the epithelial cells we we know the reductions of frequency and quality of the ciliary bits
And without no additional effect when we had stenotrophomonas maltophilia the same interaction I’ve I’ve been note by um how colleague especially between first part A cooperation between both microorganisms which facilitate the pseudomonas and Asperger’s co-colonization when they are at distance in the epithelial cells and after that it seems to have a
Competition between both microorganisms with when when two microorganisms have colonized epithelial cell with a negative interaction with uh via different secondary metabolites secreted by the other microorganisms in fact in respiratory diseases in in on epidural cells the microbial Community is dynamic very complex to explore with a lot of intra and inter-species and
Intercedental dialogues as you say a dinner there is a phenomena the different phenomena of co-occurrence of coexclusion between microorganisms and we know that the iron regulation and redox potential play a k role in these interactions for the perspective we have a lot of interaction to explore between the inter or intra-kingdom
Species in respiratory diseases and for example we analyze thanks to the metagenomic analysis the fungal ball due to aspergillus figures in 30 patients and we found a specific interaction with a biofilm with hemophilus influenza we don’t know why exactly but we found thanks to the beta genomic this interest specific interaction between hemophilus
And aspergillus and we know we can discover a lot of interactions of course we have also a regulation of disease exacerbation by the gut and the long axis and we know that some forms of chronic lung disease exacerbation such as in cystic fibrosis in COPD in asthma that maybe due to translocation or
Expansion of microorganisms content from the gut and direct aggression to the learn could be could also exacerbate diseases via the effect on the on the lung or gut microbiota conclude finale the interaction between microorganisms in respiratory diseases it is a dangerous lesion or best friend forever that is a question I think it’s
Difficult to to answer because the interaction between these microorganisms are very complex and very complex too with host cells and I think that nature of different pathogens microbial and cellular contexts sequence and timing of fanfiction are essential parameters to to answer these questions I think finally that these microorganisms are more Frenemies
Than enemies or friends as described in 2019 by how colleagues dark I will thank you for your attention [Applause] thank you very much Francois it is really important to to have a a wide panel of microorganism in in the in mind because of course there are many competition cooperation Etc so it was
Really really interesting so do you have one question yes what is your opinion in terms of vaccination in terms in respect to to combined infections a were practically could you say if someone was vaccinated that they didn’t have it or or or or there’s no influence
I’m not I’m I’m in a routine so I I can’t I cannot Trace answer this question but I’m vaccinated of course I think for the moment we we describe all the the the interaction between all these microorganisms for the moment we don’t know which implication which which implicated which implications are um um
In the in in the in the context in the medical context I I we don’t know we we have to explore all this interaction to to answer from gut feeling if you have to deal with one bag that’s bad enough but then if you have to deal with two
And more and more so so I think my feeling is I have no data to to show I don’t think there are data maybe there are and I don’t know it would be worse yeah it’s yeah just a sort of an other side of vaccinations is always that we know
That over vaccination can push people to hyper reaction and and overreaction so not that much of a problem with viral antiviral vaccines but when we start talking about anti-fungal vaccines or antibacterial vaccines pneumococcal vaccination um yeah how do do we actually know what we’re doing the
There is no no real vaccine no for the moment yes but there are some uh some teams that are working on aspergillus vaccine so okay so thank you very much for attending and now we have time for lunch and we will come back at 2 pm please thank you foreign