121. Moulds: Mucorales

Callum and Alyssa are joined by Dr Dora Corzo-Leon to discuss all things Mucorales and Mucormycosis. Listen in to hear about:

• Taxonomy (and why it is confusing)
• Pathogenesis
• Risk factors 
• Clinical syndromes
• Diagnostics
• Treatment

Show notes for this episode here: https://idiots.notion.site/121-Moulds-Mucorales-c2343d4588ea4dd19c54de538fd7fe22?pvs=74 

Transcript

0:00

Hi everyone. Welcome to the Idiots Podcast. That's infectious disease insights of these specialists. I'm Callum, and that's Alyssa. And we're going to tell you everything you need to know about fungal infection. Soon, may the editing come to discontinue the Tazo sun one day when the S piece done, we'll take our leave and go.

Callum: 0:22

Hello,

Alyssa: 0:23

Hello.

Callum: 0:23

Melissa and Dora.

Alyssa: 0:25

Hi Callum.

Dora: 0:27

Heya,

Alyssa: 0:29

So today we're going to be continuing our moldy theme. So last time we talked about aspergillus and today we're going to be talking about mucorales molds. And we're really happy to be joined by Dr. Dora Coflo Leon, who is our guest speaker for this session. So Dora is an ID specialist who trained in the National Institute of Medical Sciences in Mexico. She has a PhD in molecular mycology from the University of Aberdeen and is currently a postdoctoral researcher at the MRC Mycology. And that's at the University of Exeter. And we're really excited to have Dora with us today because she's a mucormycosis. she has extensive experience of managing cases of mucormycosis, mainly from her clinical practice in Mexico. And her current research focuses on these fungi, Nekorali's fungi, and in particular, anti clinical drug resistance, and Nekorali's bacterial interactions during co infections. So welcome, Doreen. Thank you so much for joining us today.

Dora: 1:39

Thank you very much to both of you. for the invitation. It's a very exciting opportunity to share and talk about this that is one of my favorite topics in Mycology. So, thank you.

Callum: 1:53

Thank you. So we traditionally start episodes with a pun to get, or dear loyal listener smiling at the beginning of the episode, because we don't know what you're doing at the moment maybe you're having a very stressful day. So, Dora, you a curiosity or tidbit. to start

Dora: 2:09

Oh yeah, I think As we, I think we are going to start talking about bad about this fungi. I think I'll start with the nice part of them that I think are very, very important side of this group of, fungi. They are involved in many aspects of a, human life and environment. They are the main responsible for rotting and spoilage of food specifically fruits. And another interesting thing that not many people know is they have been used for centuries in food production, like in soy or beans fermentation. So you probably have heard about the making of tempeh. Some of these species are used for this fermented food, and it's actually considered a superfood. So, yeah, I think this is the side of fungi that sometimes we forget as clinicians. They're also being investigated in biotechnology. As their biotransformation can lead to the discovery of important compounds that can be potentially used in the future to make anti cancer therapies and also anti inflammatory compounds. So, they are very versatile as you can see, but obviously will be. I'm talking about the not very nice side of them.

Callum: 3:28

As a recent Tempe convert, I really intrigued to know that it's delicious. You haven't tried it. Go out and get some.

Dora: 3:35

Yeah, I need to say here that I have a colleague in Mexico. He's been off a company that it's dedicated to produce everything with fungi and uses these new corallis to do like a Mexican type of tempeh. So he's changing the beans. Instead of soya beans, they, he's using corn, I think. So it's like a Mexican version of the tempeh. And also he does leather made of mycelium as well. It's like a leather substitute material as well. So, yeah, they are very versatile.

Callum: 4:15

Maybe we should come back and do a whole series on

Dora: 4:17

Oh I, I can give you, yes, no, definitely. He also yeah, I can give you later his Instagram stuff. He uploads everything that they do. And it's really amazing to. To see how they transform this fungi to find more environmental friendly options for materials.

Callum: 4:38

I guess we'll just end the episode there on a really bright note

Dora: 4:41

Exactly. Yes. It's just, yes. Yeah. Yeah. I think that's a good idea. Yeah. I think that's a good idea. Yeah, for sure.

Callum: 4:54

So Dora, that's the nice side maybe we should move on to the less nice side. So over to you, Dora. to tell us about.

Dora: 5:06

I would say that this group of fungi that is actually quite big is It's a group that that contains more than 200 species. And they are multidrug resistant fungi usually neglected. And they are, I think we have them misconceived as rare. However, I think that they are not as rare as we think. It's maybe because we don't have enough and good tools to identify them. And the other important part, before I go to the populations that are at higher risk, is if you see the pipeline of new antifungals to be available in the future, none of them is active against mucrales. So there's also a neglected part of that. Of research in new antifungals that are forgetting this group of fungis. So these fungi are mostly affecting populations that are deeply immunosuppressed immunocompromised neutropenic individuals. With all those with transplant recipients and corticosteroid use the other important group. It's those with uncontrolled diabetes. And classically it has been said that in the group of uncontrolled diabetes, most of these infections will be in the area of orbital sinus and cerebral areas. And those with neutropenia and hematological diseases, transplant recipients, and corticosteroid use, most of them will have a pulmonary infection. So there's classically, it's been reported as two different phenotypes. In in this group of patients and the other recent because these are the 2 most important groups, but myocarditis can affect a wide range of patients. They are usually seen after outbreaks. They are usually associated to outbreaks after natural disasters. And it's one of the most frequent healthcare associated fungal diseases, mainly if they are in the skin, because they can contaminate bandages or linen or whatever. Also, after a trauma, these fungal infections can affect people after car accidents or also after war. And, but then there's a new risk factor or a new group at risk that we identified. After the COVID pandemic, that is the actual severe infection due to COVID, and that led to the major outbreak that a fungus can cause ever in India, where it's estimated that mycormycosis affected around 40, 000 people. in India.

Alyssa: 8:00

Yeah.

Dora: 8:01

Yeah. So I think that's in summary, I would say the importance and the relevance of this group of fungi to study and know about them.

Alyssa: 8:10

And the disease that they cause, so it's known as mucomycosis, and I would say it's probably the most acute, fulminant, fungal infection known. it progresses very rapidly. the hyphae invades the blood vessels. It causes angio invasion, leading to tissue necrosis and, and infarction. And I think you alluded earlier that diagnostics are limited, treatment options are limited, and as a result of that, it carries a really high mortality, doesn't it?

Dora: 8:40

That is correct. Yes. Despite treatment the mortality is around 50%. And one very key thing that I I need to like highlight several times during the course of this podcast, is that one of the main or the main therapy and what determines success is surgery. So yes, Seldomly, you will treat these infections only with antifungals and the rate of success if you look to the literature, will be determined by the patients that receive or didn't receive a surgical treatment. So, my professors the ones that I learned about for the first time about mycormycosis, they usually tend to say that with one hand you write and you prescribe the antifungal and with the other one at the same time you are prescribing the surgery and asking for the surgeon to be around. So, I guess that is a major factor. To help in improving the prognosis of these patients.

Callum: 9:46

You know, we're talking here about mucorales and mucormycosis. There's a really diverse group, as Dora has said, and we're just gonna run through some of the most common causes of species. There are others. And you know that we are talking about outbreaks. So I presume that when you see an outbreak, it's generally gonna be like, you know, you will see one organism causing the outbreak. Is that fair to say?

Dora: 10:09

Yeah. I think so.

Callum: 10:10

Okay. So I'll just start first and hopefully not butcher the pronunciation too much. So we've got Rhizophis actually, how would you say that?

Dora: 10:19

Rhizopus.

Alyssa: 10:20

Rhizopus, yeah, we've got Rhizopus eryizus, and Rhizopus microsporus.

Dora: 10:26

I will start by saying that rhizopus arises it used to be called Rhizopus oryzae, so most of the literature you will find the name Rhizopus oryzae but they mean a rhizus. And then, from the Rhizopus aereus, there's two varieties that people still are fighting about if they are a variety or they are a species. And you have Rhizopus aereus variety aereus, and Rhizopus aereus variety delamar.

Callum: 10:57

I'm glad you explained that because I didn't

Dora: 10:58

Yes, that's, I would say that's worldwide is the most important one. And the most frequently reported, at least. Okay,

Callum: 11:08

microsporus.

Alyssa: 11:11

Rhizomycopustulus? Pustulus? Pustulus. Pustulus eryizus.

Dora: 11:15

So the next one will be Kuningamela

Callum: 11:21

variobalis. Rhizophis.

Alyssa: 11:25

Sarcassinae vasiformis.

Dora: 11:28

Vertoletiae, Lichtimia, that used to be known as Absidia, Corimbifora and Lichtimia Ramosa,

Callum: 11:35

Mucor Cide and Mucor Osis.

Alyssa: 11:46

Syncephalastrum brassimosa.

Dora: 11:49

And Actinomuchor elegans. Yeah,

Alyssa: 11:53

And I think we've already talked a bit about you can see that Rhizopus arizes used to be arizy. Lichthymia corumbifera used to be a obsidia. So, nomenclature is quite tricky and confusing with these fungi. often referred to as black fungus. And that's a completely misnomer, because these fungi have clear hyphae. But they cause this invasive disease with black necrotic tissue, mainly affecting the skin and the sinuses. I think that's why it's named as black fungus.

Dora: 12:30

But they don't, they shouldn't be called like that because there's a whole group of fungi that are known as black fungi and these ones are not one of them. And if you are like, answering an exam, that might be confusing. And also in the clinic when you identify them, if you are expecting these fungi to be black.

Alyssa: 12:51

Yeah,

Dora: 12:52

they are not black, right? So it's always important to highlight that the reason why they call this group of fungi black fungus is because of the necrosis that they cause and they are visible in the face area. The other important thing that I mean it's it's something important to highlight mainly if you are reviewing all literature is that this mucormycosis used to be called Zygomycosis. But not anymore. It's also this came also with all the nomenclature And taxonomy changes. So they used to be called zygomycosis because the sexual structure for these fungi are zygospores. So that's why, that's how they used to call it like that. And also so many people will base the diagnosis on that morphology. But now after all the phylogenetic arrangements and changes in literature they are now calling it mucormycosis, and that's the name they go by now.

Alyssa: 14:01

that's also the fungal kingdom as well, isn't it, is mucormycota

Dora: 14:04

Exactly.

Alyssa: 14:06

zygomycota. think one thing that annoys me is that quite often people refer to the group of fungi that cause mucormycosis as mucor, but mucor is

Dora: 14:18

Yes,

Alyssa: 14:18

one of the many genera. So big list that we had that cause mucomycosis. So, so really it should be mucorales or mucoraceous moulds.

Dora: 14:30

correct. And I think that can lead to a misleading or wrong reporting when you find this fungi because they would put like mucor. So you don't know if that's mucor because they are looking, I don't know, like a histology finding. And they are calling mucor, instead of calling it mucormycosis, that it could be caused by mucor, or by rhizopus, or by lymphedema, and so on.

Callum: 15:00

So I guess that's what they are. You heard about why they're important in terms of. epidemiology story, you talked about this on a broader level. But how much mucor is there? Like we must know how many cases there are or what the instance rate is.

Dora: 15:17

where we actually don't know. yes there, there have been some attempts to do estimates. Of how frequent this infection is, and you can see some of it with in Gafi's page. But there's not like a like a surveillance program or anything. Everything is based on estimates and the, and the reason of this is because, as I said in the beginning, there's a lack of awareness. about the disease. A lack of standardized diagnostic tests, so you won't identify these infections. There's no surveillance programs, but what we do know is that there are certain countries that have heavy burden of this infection. Most of these countries are India and Pakistan that some estimates say that the frequency here is 70 times higher than in the rest of the world. This is also a Reflection of the lack of awareness and diagnostic tests, because there's a recent publication where after the introduction of PCR to diagnose mucormycosis in immunocompromised patients in France. the burden seems to be as high as it is in India. So, maybe we are just seeing the top of the iceberg in these two countries in India and Pakistan. And the other thing is the type of presentation and the type of patients that are affected. Because it's easier to spot. An infection that is in the face. I mean, it's evident, right? It's difficult to miss it, right? And these are the type of infections that are being seen in India for example, where most of the people affected there has diabetes. contrast, what they found in France is the infection in immunocompromised in the lung mostly. So. My point is the burden might be as high, but in different populations, in different presentation. And the reason why we thought the burden was different is because we didn't have a very sensitive tool to identify that infection because diagnosis in the lung is more difficult so, Yeah. Thanks to these molecular tools, we are learning more about mycormycosis, and because the preconceived conception of this infection is that it's a rare one, and maybe it's not the case. So this is new evidence and how the knowledge of this infection is progressing in the last years. So Yeah.

Alyssa: 18:04

Yeah. So like you said, maybe we're just seeing the tip of the iceberg with the cases are diagnosing.

Dora: 18:10

I think so.

Alyssa: 18:11

And I even though, we're probably not picking up anywhere near all the cases, the trend globally over the last 10 years has been a really steady rise in reports of these cases. And we'll come on to this in a minute in more detail, who gets this because we have a growing population that is immunocompromised and it's just been slightly rising.

Callum: 18:37

so you mentioned there the sort of sites of disease. So I guess what you tend, my understanding is that, that you. sort of rhino orbital cerebral pattern that you were describing that's more obvious than you can see is the most common, followed by pulmonary, but you can also cutaneous, like with many fungal pathogens, if there's direct inoculation or trauma, and finally you can get gastrointestinal, and with all of these, because the hyphae invades healthy tissue, you end up getting dissemination in about a quarter usually by contiguous spread. patients. So transplant, malignancy, prolonged neutropenia, purely controlled diabetes, prolonged steroid use, also desferioferoxyamine therapy. Why is that a risk factor?

Alyssa: 19:30

Does Ferrioxamine therapy? It's all to do with the iron availability, so fungus needs iron to grow. And interestingly, does Ferrioxamine is an iron chelator, isn't it? So I've never quite understood why it's a risk factor. But Dora, do you know?

Dora: 19:49

well, iron is a main component for mucormycosis pathogenesis and metabolism is the major source of micronutrient. Free serum iron is iron is a major source of micronutrients. So people with this chelating agents therapy or with overload of iron are classically considered a higher risk as well. So people that get a lot of transfusions and stuff. They are also considered at higher risk because iron metabolism plays a central role in, in mycromycosis pathogenesis. So this, the ferocidox some years ago went into clinical trial because they observed that the chelation with it will limit the access of iodine to rhizopus aureus. And this will cause the deprivation and fungal apoptosis in the fungus. And and then they it went to a clinical trial, if I remember it was with hematological patients. and they started to die more. That's why they stop it. Yes. Huh. So this is like one of those examples that not everything that, that, that works in vitro and even in mice models will actually work when you put them in clinical trials. So, that's why also this kind of chelating therapy is considered a risk factor because in a clinical trial it demonstrated that instead of helping was actually increasing the probability of having this infection and dying more of the infection.

Alyssa: 21:19

I think it's probably key to highlight in these risk factors that whilst we classically think of mucomycases affecting immunocompromised who have been previously very fit and who then sustain, huge trauma or extensive burns, are also at of me for my cases from cutaneous site. So it's important to think about that brief as well.

Callum: 21:43

Yeah, I guess the pathogenesis maybe differs between the immunocompetent and immunocompromised. so in your immunocompetent host, the spores are, brought up into your macrophages and neutrophils are phagocytized. So that, but, the role progressed in a slightly different way. But still cause disease, whereas if your immunity is impaired and then you get more direct invasion. So I guess you would expect to see a difference in the clinical presentation because of that.

Alyssa: 22:11

So the spores either inhaled or inoculated into skin abrasions, or I guess in case of gastrointestinal, they're ingested. And then in healthy, immunocompetent individuals, these spores, they're phagocytosed by macrophages and neutrophils, but in your, patients with defects of their cellular immunity, or I guess case of your immunocompetent patients who have burns, the immune system is overwhelmed by the burden of spores. the, then these spores are able to germinate and then develop hyphy that, that grow and invade the tissues.

Dora: 22:52

Yeah, so there's macrophages and neutrophils are main layers in the innate immune response to this fungi. So as you know, in diabetic individuals, the macrophage response, it can be affected by the glycosylated processes that all tissues in diabetic individuals. Are suffering so that's one of the factors that are associated to a less efficient response, but there's a receptor that is called GRP78 that it's highly expressed during hyperglycemia and ketoacidosis. So this GRP78 is the receptor in the host and it's highly expressed. And mucorallis have a virulence protein that loves that receptor. So, the expression of that receptor during hyperglycemia and ketoacidosis, allows the fungi to join and bind that receptor. And that has been demonstrated before that allows the invasion. So, yes, that's in the case of patients with diabetes. Most of the expression that occurs is In the zhinoorbital cerebral areas, but in the case of patients with hematological diseases, obviously not having neutrophils. It's a major thing to start with, but then you have all these factors we were talking about. That is the iron overload. But the other is that in these patients, there's a new protein that has been identified in the lungs that are overexpressed. And that are the receptors for mucorales in the lung. So, and that explains partly. The differences that you see in the presentations between patients with diabetes and patients with hematological malignancies. The fungus will not bind the area of the rhinoceno orbital cerebral areas, because the GRP78 probably is not enough expressed. But what is expressed is in the lung, so this integrin protein is highly expressed, so they will bind that area, and that's why the hypothesis is, that's why you see more lung infections in these patients. Yes.

Callum: 25:26

yeah. Thank you for explaining that. Do you think we've covered the clinical syndromes now? Or do we want to delve into that bit more detail?

Dora: 25:35

Maybe only saying that the one of the theories why patients with COVID associated mucormycosis are at higher risk of mucromycosis is one that, I mean, in COVID most patients that were at risk of having severe COVID was patients with diabetes. And and also then they receive high dose and in some areas, like an overuse of steroids. So, you have two of mucormycosis, but then there's also findings that this protein GRP78 was highly expressed in patients with severe COVID. And that some of the variants of SARS CoV 2 will use GRP 78 as a core receptor as well. So it seems like there's like a combination of factors that we know that will cause mucormycosis, but others that are also triggered and taken advantage of by the actual virus. So this is something that is still in study, and there's some nice work on patients with COVID and other interesting theories about how is it that these patients got more mucormycosis as well.

Callum: 26:48

So we've heard about the different clinical syndromes there, so rhino, orbital, cerebral, pulmonary, cutaneous, gastrointestinal, and I guess some others as well. So maybe we could go for each of those. briefly in turn what presentation that would you see and when should you be suspecting this? So I guess starting with our most common, the rhino orbital cerebral, we mentioned that you could get the sort of black SR. What other symptoms might people present with?

Dora: 27:13

Yeah. So, this is actually one of the reasons why I started to study mycomycosis because early in my ID career, these were the first big cases that I saw. So some of the symptoms that you are expected is ptosis. You will expect to have edema around the eye and the face. There is also proptosis, ophthalmoplegia, that's another important finding. You will have not all patients, but some of them can have necrosis and an ulcer in the palate. they go so fast, right? But not all of them will present with a necrotic ulcer and a hole, because you can't even see a hole in the palate. Sometimes it's just like some kind of bruise. Or if it's very early. like necrotic tissue inside the nose as well. So here the eNT. those that have the endoscope to go inside, that's a great tool to identify how advanced and how damaged and also take samples and also to follow up those patients. So, that's another thing that you can find. they can have sinusitis, obviously cellulitis as well in the face. Not um, frequently you will find them with thrombosis, like cerebral thrombosis how you call it the, I just know the name in Spanish. Trombosis del seno cavernoso?

Alyssa: 28:46

Venous sinus thrombosis.

Dora: 28:48

Yes. So that's a very frequent feature as well in these presentations.

Alyssa: 28:57

Pulmonary disease then, so you said that mainly occurs in your kind of, haematological malignancy stem cell transplant patients, neutropenic cohort. that fairly nonspecific in presentation or are there any hallmark features that might make you think, could this be me

Dora: 29:16

I mean, it classically has been said that the reverse halo sign is very typical of mucormycosis, but I think you'd need to take that as a pinch of salt, to be honest. It's not very common, and there are other infections that can cause the same, even aspergillosis can cause that feature. You can see nodules. You can see for example, in COVID, obviously, this is a different type of patients, but you will see non specific affection in the lung. We are currently running a a study in Mexico. In a respiratory hospital.'cause they are they started to see more cases after covid. Like more cases more and more. The, their statistics are like all the way up. But not all of patients have neutropenia or are classically immunocompromised, let's say. And they see a lot of cavities as well. Yes, so you can see cavities as well, and the other thing is like they are very destructive. So usually they are like one nodule or one, area of consolidation because you can also see consolidations with nodules. And then they will progress rapidly. So that progression as well, I think, should be a sign of alert for this.

Alyssa: 30:33

symptom wise, like a severe respiratory infection, I guess things like hemoptysis and pulmonary hemorrhage that's going to happen quite late in disease when you've got significant angio

Dora: 30:47

Yeah. I guess it depends on where the infection is And how close to the, bronchus and the trachea and everything is, right? If it is far away, probably will take longer

Callum: 31:00

and then for cutaneous disease, is that quite similar to the rhino orbital cerebro in terms of you get these necrotic lesions? And how does it progress? Is it spread outwards or is it, just, does it go like deep in a narrower area?

Dora: 31:14

Eh it also depends on the type of patient that you are you are seeing. For example, after trauma, it will be like an extensive destruction and necrosis. Right. And and usually they are also associated to to like bacterial infections, for example, a multi polymicrobial infection and not. You won't find them or probably you won't find them in the beginning. You will find them afterwards when the patient is not improving to your antibiotics and your treatment. And then you go and reassess the wound. You take a new sample, once the burden of bacteria probably decreased, you take a new sample and then you will see them. And the other important thing is, there are some reports that they will show that many of these post trauma infection, it's caused by apophisomiasis species. An apotheosomized species is difficult to grow in the lab, so you will probably struggle a little bit recovering it in the lab, so here is where also sending your sample to histology, to? cytology, and even molecular identification will save you a lot of delay in your diagnosis as well. Because it's probable that you won't recover it very easily in the lab.

Alyssa: 32:45

Yeah, I remember coming holiday in France and my Apophis Mices had grown and I

Dora: 32:53

yes, yeah, it they have like certain needs. They are not as Rhizopus or other type of species that grow in more regular media apophisomiasis might take more TLC, so that's one of the things that why you need to also send things to histology, even when it's a wound infection Yeah. send to histology, send to a cytology or like a direct microscopy. And also the the molecular identification, but in that's the trauma, but for example I also saw a couple of cases in patients with hematological malignancies, and the ones that I, I saw it's after a puncture. Okay.

Alyssa: 33:38

Ah, okay. Like a cannula.

Dora: 33:41

Yes, Yes. Like a permanent catheter there, and then the adhesives they are all the time Like a embracing the skin. And if there's a contamination, they can be a signs of infection and the way they start is as a bruise, but it's a bruise that one hour is like this size and then after 12 hours is this size. So, yes, Yeah. they yes, they are highly immunocompromised. There the time is like you need to really rush for the surgeon and for the antifungal. Yes. And they look like a bruise like a purple, that you could associate to the trauma.'cause they are neutropenic and you could probably associate the beginning to the trauma of the puncture. But that it start to grow. And then underneath feels hard and they start to grow. So that should be a red flag for you for this infection in this type of patient. So it's a different kind of like cutaneous and subcutaneous presentation.

Callum: 34:50

And then but ultimately gastrointestinal. I presume that's quite rare. I had not heard of that before. Is that something that you've seen and how does that present?

Dora: 34:59

Yeah, I am, I've personally haven't seen any, but I've been consulted for four cases, The first one was after a procedure. It was a patient after that had a severe COVID, so needed a a gastric tube to be fed. So during the endoscopic procedure, they inserted the gas, the gastric probe. And then that night, he started to bleed and bleed. And they had to go into the surgery and they found a 10 centimetre ulcer in the stomach. They had to take the whole stomach. And then they found that in less than 24 hours the spleen was affected. So they had to take the spleen out as well. It's very aggressive. And then they did, during the surgery, they did a direct microscopy. And they saw the wide ribbon like hyphae. And they started immediately the antifungal. So that's one, one of the cases that I was consulted. And then there's other three that seems to be associated to ingesting. Either forage food here in the UK is the only risk factor, or at least the only kind of like thing that like being walking in the woods and maybe foraging something in immunocompetent individuals. And the other is after eating these natural products that are like supplements. It was the only risk factor for this person. This was in the States. It was made of a plant, like dehydrated plant. And they tested the product and they found a fungus there. And they found it in the intestine of the patient. So, those are the cases that I've been consulted and said, but they can happen also in immunocompromised individuals, the literature is full of those. And here, as I said before, the high suspicion and every time I give a talk about myocromycosis I tell that for myocromycosis that is not in, in the zhinoorbital cerebral areas your threshold to suspect myochromatosis should be like really low. Because in all these areas in the lung and in, in gastrointestinal, you have like a list of 20 things before you think about mucormucosis so, and that's, I think, a major factor for non identifying this fungi, and then patients dying. So, I think, and then you could rule it out very simply by doing direct microscopy. Or at least being less scared if you don't see anything. Because it could you could not see it, right? But at least if you see it, you will start the antifungal straight away. So I think that's something that We need to be aware of. And the other thing, again, is that some of this fungi won't grow in the lab. The yield of culture for 50 percent. And you have to have a special protocol to improve the recovery in culture.

Alyssa: 38:31

could move on to lab diagnostics at this stage, couldn't we?

Callum: 38:34

Yeah, I actually I was going to mention in terms of other sites, we had a series of infected endocarditis from like themia, but I just wondered if that is that useful. Is that super rare and not really worth mentioning or is it?

Alyssa: 38:48

I think it's useful because it just shows that these pathogens can cause all sorts presentations. Yeah.

Dora: 38:55

Yeah. and the other thing is we have been receiving strains from Bristol, most of them are clinical. And the second most common isolate in the UK after Rhizopus is Lichthemia. So I think, Yeah. so I think it's important.

Callum: 39:09

Yeah, Yeah, certainly with Lythemia I wasn't really involved but there was a series of cases associated with cardiac surgery and valve replacement. And, obviously we've talked about how difficult it is to diagnose and treat and that was very challenging. post surgery. And they're in the environment. So if we're putting prosthetic material in, then, that it just highlights the importance of infection prevention, control, and ventilation. fungal spores are in the environment, but certain things make them more, more common. So my understanding is like building works one of the big risk factors. So. if you're working environment where there's building work going on nearby or a major disruption to the environment that might lead to more fungal spores, then, before that works, making sure that there's adequate precautions being taken. And, the ventilation systems are in adequate working order and have been maintained So, we're not an infection prevention control. Podcast but feel remiss not to mention the importance of that and preventing these diseases and in that sort of setting where you're putting prosthetic material in so guess that's the clinical syndromes and I've just learned so much there. So thank you, Dora for that sort of masterclass. We've touched a little bit on diagnostics. So I guess when we've talked with other fungal things, we think about microscopy, culture, histopathology, molecular, and, biomarkers. So I guess we'll just talk about those first. we've had an episode on fungal diagnostics in general. So what, about beta D glucans, very useful test in many fungal diseases about, as a sort of rule rule out test, What about that for mucoralis, something that's helpful.

Dora: 40:38

I will leave Alisa to take the lead on this.

Alyssa: 40:41

So ptca is not helpful for diagnosing the. So they lack B2G glucam in their fungal cell wall. so it's just really not useful at all. And this kind of adds to why diagnosing this disease is so challenging. Currently there's no antigen biomarker in clinical use for their detection. Dora and I have been very privileged to work together, with a novel monoclonal antibody. That, that binds to these different Nekorali species and looking at whether the antigen that it binds to could have potential as an antigen biomarker. So that's really exciting potential feature development for Mucroales diagnostics, but Currently, there is not an antigen biomarker that's in clinical use to detect them.

Callum: 41:30

We'll fingers crossed because we definitely need something.

Alyssa: 41:33

And some, some of the fungal pathogens grow in blood cultures, mucorales do not. So again, you're not going to be expecting to grow or see mucorales in blood cultures. And I think Dora's already mentioned quite a few times the role of direct microscopy. And I think this is really important for that early diagnosis. of the sample getting to the lab. like Dora said, sometimes these fungi don't grow well. So in some cases it can be more sensitive than culture. You might see hyphae that are characteristic of Nucorales, but then you don't grow anything because They're quite fragile. They can be easily disrupted during the culture process or something like apophisomysis is really challenging to grow. One of the downsides, direct microscopy it's not, it's not hugely sensitive, so it's not a out. If it's, if it's negative, but if you do see fungi there, then it's hugely helpful. And fungal brighteners such as calor white, which binds to chitin in the fungal cell wall can be used to enhance the sensitivity of direct microscopy. So it literally up the fungal element. And mucroales have really characteristic features under the microscope. So Dora mentioned this earlier that they have these broad kind of ribbon like hyphae. And you can differentiate them from Aspergillus because they have many scepter. Sometimes they don't have any scepter. They don't have as many branches. And when they do branch, they branch at a right angle. So if you see that, it's quite typical, and that means that you can get in early with your mucorales directed therapy.

Callum: 43:50

That was microscopy. So you're saying that the culture is very difficult and we've talked a lot of mycology diagnostics can be really challenging. So in the UK, certainly this would, if you were suspecting this and you were able to culture something, you would send it to the mycology reference lab. And there's some images in the show notes which show some classical features. I think that would, certainly be something that you would look up. But there's certain sort of classical features. And then the other modality that might be using particularly from theater samples is histopathology. as Dora said earlier on, making sure that when you get biopsies of tissue they're going for the different areas. I think one challenge of histopathology is certainly locally that it's quite a long wait for these results. you see those sort of ribbon like hyphae the same sort of appearances. But there's also this sort of angioinvasion tissue necrosis patterns and inflammatory response. But there's no specific stains, I believe, for histopathology, so they're just using their paths and crocus stains.

Alyssa: 44:48

And I think that's another, just come in again, that's another area where Dora in particular has looking at using the TG11 monoclonal antibody.

Callum: 44:56

Oh, right. Okay.

Alyssa: 44:57

has shown that it can specifically detect Mucorales species. And that was in a mouse model. So,

Callum: 45:06

an immunofluorescence, you know,

Alyssa: 45:07

yes,

Callum: 45:09

okay, yeah, cause I know there's a lot of research in that and but molecular pathology and how that might, might be a huge tool. So another sort of thing, but you've mentioned already Dora, molecular and the role of PCR. So maybe we could just complete diagnostics by talking about that and is that something that's available now to clinical teams? And how is it being used, in practice

Dora: 45:28

yeah, The the molecular diagnostics by PCR has been used and there've been. different targets to be amplified by PCR that have been used in many research contexts. Most of them have resulted in being very useful in diagnosis, mainly in, in for screening and do a screening program for a pathological patient with high risk of of mucormycosis. They do the screening every three, four days. And then when they identify the star, the treatment, and they have reported that this strategy could identify mycormycosis even one week to ten days before the culture and histology. And even four days. before the halo sign or reverse halo sign appear. And this is very important because with this time is precious. And the earlier you started the treatment, the best a prognosis you will get, and also the surgery that you will need, it will be less destructive, potentially, and less affecting the function. So for standardizing these tests, there's a fungal PCR initiative that is working on having a standardized protocol. to use PCR in hospitals, so you can have a standardized method to be implemented in your hospital. There are also commercial assays but I'm not entirely sure if those are in the UK available though, but there, are definitely commercial assays available that, have also been used and I think there's a lot of literature now using them and that have demonstrated being very useful as well.

Callum: 47:22

Yeah, there's a link in the show notes that this is put about Mucor genius, which I think is one of the commercial ones. Can I ask a question? And, I guess with molecular techniques, my experience in a more bacteriology realm has been that sometimes with molecular methods, we're picking up stuff and it's, it can be a bit difficult to interpret that clinically, is it relevant or is it not? With the sort of fungal pathogens often being environmental and widely spread. so for example, what I'm talking about bacteria, so Legionella species PCR we find that we do often get low level positives and samples, suspected to do with contamination at some point along, one of the reagents that's being used PCR. Is that something that we need worry about with fungal, molecular diagnostics and or is that not something that we tend to see?

Dora: 48:13

Well, that's a very good question.

Callum: 48:16

Sorry, just bringing that on you, that

Dora: 48:17

it's more I mean, the way to to to formulate the response. First thing You can't do the PCRs in in plasma or serum, which it's difficult to say that. if you identify the fungus there, it will be a contaminant. And most of these screening programs they do the screening using cell free samples that is basically serum and plasma. So that reduces The probability

Alyssa: 48:50

so it's not all in from the lab

Dora: 48:52

yes. yeah, And then I mean, there's obviously a potential to get a contamination in the lab of the regions and whatever. Right. But then I think it's more probably a colonization. Right that you could be, like, not differentiating from the disease you could probably have a positive without the actual disease. Right. So, that part, it's not very well known. We are, in this study we're conducting in Mexico we have identified some features of colonized patients. Usually, they don't have a sign of mucorales disease. So you can find, for example, even in histology, this ribbon like hyphae, But there's no destruction around it, and the patients are actually well, and usually they have another explanation for their pathology. And then the other thing is, if your PCR is positive, and the PCR goes with A range of other features, right? The clinical presentation, the actual presence of disease, right? So if all that is in line your diagnosis will potentially be true, right? I haven't seen a paper specifically studying this Because, in people's head, I think, the colonization, if you find a positive PCR, is like really low. It's more probable that would be the cause of the infection. In a sterile site, Please assume. And then the other thing is you see these definitions of proven invasive infection, probable invasive infection, and possible that they consider these risk factors and microbiological evidence and everything. So, if you have the disease, and if you have the positive PCR. in a sterile site, it's more probable that's a real thing. If it is in a non sterile site, you might or might not, depending on how sick or how compatible the infection is with the patient. Right?

Callum: 51:08

What I was thinking when you were explaining that, I was thinking about, your case, who you're doing the test on, your pre test probability, and, we talk about sensitivity and specificity, but it's far more important to think about, the sort of positive, negative, predictive values and likelihood ratios. So, okay, so we've done the diagnostics, I think it is, but there's lots more we could talk about because it's obviously very challenging. So, I guess probably we talking about Susceptibility testing and resistance, solar. They go hand in hand, don't they? So we've mentioned before that, the extremely high mortality, 50 percent or even up to 90 percent disseminated disease and how we're so reliant getting that early diagnosis prompt appropriate treatment. So let's say we're in a situation where, we've suspected it, we've got the immune compromised, so they've got a compatible clinical syndrome. You what would you go in with first treatment wise and what specific considerations do we need to have in mind for that?

Dora: 52:02

Well, I mean, considering that you have the diagnosis, right, you will start usually with amphothericin B, that's the keystone of antifungals and then surgery, or at the same time, Basically. Surgery.

Callum: 52:21

Do surgery, so I guess when we think about malignancy, we're thinking about clear margins and so on. because obviously we're talking about, like rhinoceros, So, these are really sensitive areas, disfigurement

Dora: 52:32

Yes.

Callum: 52:32

the important structures. How do they know? Do they just do it to like macroscopic appearances or any other way that you would, they would be guided about how much to, to debride. Okay. Okay.

Dora: 52:44

Yeah, it could be microscopically, but there's some recommendations it's more like an expert recommendation, actually, but to treat this as you do with cancer patients, to do a margin evaluation microscopically. And if you don't see hyphae, then you are probably in the safe side. Now, most of these infections will be diagnosed. When you are doing the surgery because you think that this is another infection or another disease. And then if you do the cytology during the surgery, and you see the ribbons, then you should have a good communication between the person that is doing the direct examination with the surgeon team And say, This piece, we need to know the margin of it just to make sure, because otherwise what it usually happens is That patients go out and then you are on the antifungals and then the patient need to go to surgery again in a couple of days because they will be. The infection will progress. So that's something that people usually recommend doing.

Callum: 53:59

That needs a huge support from your laboratory as well, because I'm thinking locally, like, how we would deliver that, a lot of orgies in the UK are hub and spoke model. Now, they're not that close and they may not have 24 7 covers. So there was a survey shared at FIS last year in 2024 where they were looking at what was available. And yeah, having someone there to do microscopy. So how do you know, sense that are managing this more of is that, and you would need a trained, not all, even in microbiology, not all our biomedical scientists are that comfortable with looking for hyphae and mycology diagnostics. That sounds like a real challenge to deliver

Alyssa: 54:36

I think that is a real challenge. And think our mycology reference labs are excellent. they're excellent at, processing fungal serology, and branched in tests. culturing fungi, performing identification, antifungal susceptibility testing. But what worries me is, do we have adequate diagnostics at local laboratory level? And this is something I've started looking into in Exeter and, really the answer is no, we don't. And, we're trying to get funding now to get, a fluorescence microscope so that do direct microscopy on these samples. I'm thinking about, the sort of out of hours provision. I in respects to bacterial necrotizing fasciitis cases where, we have one of our colleagues, Dr. Marina Morgan is incredibly knowledgeable and passionate about cases. And she will come in the middle of the night and go to theater take those samples take them to the lab and form the grand saying. if it's clear, she'll call the surgeons. and she'll go back and get samples. So, sometimes you are reliant on somebody who has a true of passion to go above and to facilitate

Dora: 56:01

Yeah. it's like really engagement and involvement and passion for it. If you don't have the resource. The other thing is if you got the diagnosis afterwards, after the surgery, if you don't know what the patient has. And it's identified after the surgery. You know that patient probably will need to be in the surgery room again. So maybe get prepared like knowing that the patient's going in get prepared to receive the sample and analyze it or asking for help to do that. And the other thing is because this is not only for mycorrhiz. I think it's for many infections where. A direct microscopy could make the difference in diagnosis in terms of not delaying the diagnosis. So learning and having strategies like Alisa is implemented in Exeter. It's very important to to improve the local capacities of diagnosis and And provide faster diagnosis and treatment for patients. I will encourage people to try to do more of these microscopy strategies.

Callum: 57:13

And just to circle back, antifungals very briefly. So I guess the treatment is high dose amphotericin B. And when we talk about that, we talk about usually weight based dosing. So I think high dose is generally greater than five milligrams per kilogram, but you can go all the way up to even 10 milligrams per And I think some of the options and the guidelines are, considering adding in azole. So, or you might add in a kind of candon and then thinking later on about, an azole as step down therapy and. Usually the treatment is, 68 weeks, but it's until his clinical resolution. that's my very basic understanding of it is that about right? what sort of things you would starting dose? I don't really like guidelines where it says, here's a range of doses. what's your take that Dora, in terms of the antifungal side of things? Do we have evidence for that? I suspect that we don't have great evidence

Dora: 58:13

Not much. No. No. And to be, to be honest, I think here I will leave Elisa to go for it.'cause back. home we don't have liposomal amphotericin B so we used to treat these patients with Deoxy, coate, amphotericin B. And there's some evidence that it's, it doesn't seem to be. like a difference in terms of if you use one or 1. 5 milligrams per kilo in deoxycholate. So, yeah, in terms of liposomal, I, my, my experience is more limited. And then the other thing, what is important though, is that if you are going to use these high doses, even when liposomal AMFO has less probability of having adverse events, Obviously, the more you increase the doses, the more careful and keeping an eye on the patient you need to be in terms of having adverse events even with liposomal formulations of AMFO.

Callum: 59:13

I feel that a lot in, when something's really severe, you want to give the best treatment you can. And you end up thinking the higher dose of antibiotic, the more likely I will to kill it. If you take a holistic approach and think, two things I think about with high doses is one, drug toxicity and that can have a significant impact on the patient, might even lead to, severe outcome. And, that might lead to death on its own. But also if you give such, so I always think about this with PCP, we give huge doses of cotrimoxazole, end up with toxicity, which ends up needing to go on to a second or third line therapy. But then you think would we have been better getting a lower dose of our best therapy? and then we don't have to go to, line therapies because it talks to the renal impairment of amphotericin. So, yeah, I think, we just don't know, do we? But I think this is just left with a question. We're not really giving an answer here to the listeners.

Alyssa: 1:00:06

Yeah. And I think there is a, there is a huge degree of uncertainty because we don't have any large randomized controlled trials looking at. at different treatment regimens. And, like you said the initial dose that you go for, I guess has to be weighed up against the burden of infection, how sick the patient is their existing renal function. so lots of to, to take consideration. Yeah, and those were the the guidelines that I used. With regard to the treatment options were the ECMM global

Dora: 1:00:43

Mm hmm.

Alyssa: 1:00:44

From 2021. Which are really worth the read, they're a really nice comprehensive set of guidelines. And I don't think they give much more clarity on, which dose to go for Yeah, so I think it's still an area of uncertainty.

Dora: 1:00:59

Yeah.

Callum: 1:01:00

I guess. I know the in terms of treatment, we talked about diabetes and, I think that the far bit in the guidelines is about the book. Correction optimization of those underlying predisposing conditions. So, we had a lot of focus of that during the COVID pandemic locally in people that were using steroids. I'm conscious of how long we've had to talk about this how much we've had to talk about this I think maybe the final thing to talk about is antimicrobial susceptibility testing Testing, I guess the summary answer there pretty limited. on how to do that

Alyssa: 1:01:30

so Mucor, Doris said that, that they are quite resistant to antifungals, so they're intrinsically resistant to Fluconazole, intraconazole, voriconazole echinocandins, and Flucytosine. So considering we don't have many antifungals anyway, treatment is very limited. So we're left with amphotericin B, which is first line. then posaconazole and izavuconazole also have, activity against the majority of eukaryotes and species. So these be either used as an oral step down agent. Or they might be used as a salvage therapy, saying somebody who has a reaction to lifosomal amphotericin B. So I know we did have case of pulmonary mucomycosis who we did treat with posaconazole. Primarily because it, it was an incidental finding, actually. They had a nodule, it was biopsied. Had classical mucorales hyphae on um, hology. And they did very on And I think that there is mileage in, getting species specific, identification, Professor Borman at the Mycology in Bristol has published profiles, anti, in vitro antifungal drug resistance profiles for all of the clinically relevant members of mucorales. So if you can get that early speciation, you have a good idea about distributions of drugs. That species that you can use to, to guide your anti fungal drug choice usually in discussion with the mycology reference lab whilst you're waiting for those susceptibilities to come back.

Dora: 1:03:18

Yeah. but I think in mycology, we're still not at the point, at least in molds where we can make a clear recommendation on how to guide or practice based on MICs. I mean, to start with, we don't identify these infections. And then seldomly, we know what's the. genera that is causing, because if you don't recover it in the, in culture, and then you don't do molecular PCR identification, then you don't know which genera you are having there, right? So, that's why the treatment is very generic. So, I think that's something that in the field that we study in micromycosis, we need to move to, start identifying species. And then knowing more about the susceptibility pattern and start investigating how much will like impact on using the mics in the in, in guiding the therapy. But at this point, I don't think we are in a position to make a recommendation to guide or practice in mics because that part is still unknown,

Alyssa: 1:04:21

Yeah.

Dora: 1:04:21

think.

Alyssa: 1:04:22

Because we don't have clinical breakpoints and any

Callum: 1:04:32

so, dear listener, we are, I'm blown away by the, just the amount of content there. That certainly I've learned a lot because this isn't something I'm very familiar with. so thank you. Thank you both for the contributing there. Just to summarize what we've talked about. So we have gone for our mucorales the different fungal molds that cause mucormycosis. We talked through the different genera that are contained within that and clarified some of the taxonomy and nomenclature around that. So we heard about the epidemiology of these conditions. In particular, the patients that are at higher risk namely the immunecompromised states, the high blood sugars and iron high states. and really some detailed information about the pathogenesis, the clinical syndromes of rhino-orbital, cerebral disease, pulmonary cutaneous, gastrointestinal on some others. And then we've talked about lab diagnostics, which centers on the sort of direct microscopy as our main tool supported by things like culture and molecular techniques along with histopathology. And finally, we talked there about the treatment, which is based on amphotericin B with potential for posiconazolase of a conazole and surgical debridement and A little bit about antifungal susceptibility, testing and resistance mechanisms. So, just to close, I guess we could point on the show notes, there's some more resources that you might want to look at. what sort of things would you suggest Alyssa and Dora that people, if they wanted to read more and solidify their learning, where could you go?

Alyssa: 1:06:09

really recommend is the WHO fungal priority pathogen list that was published in 2022. We talked about that in our overview of mycology. And it gives a really nice concise overview of mucorales and really highlights the main knowledge gaps, of which there are many. But I think it's nice to focus. Future research and think and thoughts about where to go with mucorales

Dora: 1:06:39

yeah, there are some resources that I guess you are you have shared in other podcast episodes. There's some online resources. Like, Dr.. Fungus, and then the LIFE one as well where you can find interesting resources I think Gaffey has as well some of them, and I don't remember if LIFE or Gaffey have a microscopy course, I think.

Alyssa: 1:07:05

It's I think it's life, it's microfungi. net. But if you want to find more details about that we covered that in the fungal diagnostics session.

Dora: 1:07:16

Yeah, perfect.

Alyssa: 1:07:17

Also the educational webpage on the BSMM website.

Dora: 1:07:22

Exactly. You can find it there.

Callum: 1:07:26

one final thing, I guess, if Dora, if I had been practicing my Spanish more which is now, how do you say not good, muy bueno, or

Alyssa: 1:07:35

That's very good.

Callum: 1:07:41

If I was a Spanish language speaker and I wanted to listen to a podcast about infectious disease, is there somewhere that we could direct our listeners?

Dora: 1:07:51

Oh yeah it's actually a non Spanish name, actually, which is easy to find. It's called The Mycoloyeast, with a Y. Yes so, and I would say 90 percent of the content is in Spanish, so for those that want to practice their Spanish or are Spanish speakers the content is in Spanish, and we have some talks including Mucormycosis and and some resources for diagnosis and For other interesting topics in my ecology there that we hope to update in 2025 as well. So, yeah, that's another resource. If you are interested.

Callum: 1:08:41

That's great. I I would need to do a lot of Spanish practice sure that there's so much on there that's really useful. And I guess all that remains is to say thank you so much for giving us your time and expertise. And thanks on behalf of our listeners for sharing, sharing that with us. And thanks as always to Alyssa for putting together the show notes and your expertise as well.

Dora: 1:09:02

Yeah, thank you.

Alyssa: 1:09:03

you. It's a pleasure. Thank you so much,

Dora: 1:09:07

Bye. Thank you.

1:09:08

This Mycology series has been supported by the British Society for Medical Mycology. The BSMM aim to bring together clinicians and academic researchers in the field of medical mycology. For more information, you can find the link to their website in our show notes on notion. Please consider joining up to become a member.