105. Fungal Diagnostics part 1: Microscopy, Culture & Identification techniques

Callum and Alyssa are joined by Professor Malcolm Richardson to talk about all things fungal diagnostics.

In this, part 1 of 2 on diagnostics we discuss the use of microscopy, histopathology and culture in diagnosing fungal infections.

Notes for this episode can be found: here.

Transcript

Alyssa: 0:05

Hi, Callum. Hi, Malcolm.

Malcolm: 0:07

Hi. good to see you again. Hello.

Alyssa: 0:09

Hi. So today we're doing an episode on fungal diagnostics and we are super excited to be joined by our guest speaker, Professor Malcolm Richardson. so Professor Richardson is a consultant clinical scientist in medical mycology. and an honorary professor of medical mycology at the University of Manchester. He's currently the president of the British Society of Medical Mycology and leads the ISHAM Fungal Diagnostics Working Group. His research and clinical interests have focused on the pathogenicity, diagnosis, and epidemiology of superficial and systemic fungal infections. So we're absolutely delighted, to have, Malcolm here with us today talking about, fungal diagnostics., So fungal diagnostics, this episode we've decided to put second in our series. why was that Callum?

Callum: 1:02

Well, often when I'm not certain about stuff, one of my hobbies is role playing games, and I often roll a dice, or sorry, a die, when I'm uncertain. So you might say that, um, I was die agnostic? Diagnostic? About what to do.

Alyssa: 1:23

Diagnostic. Okay.

Malcolm: 1:25

Yeah, that's, that's being well crafted. Yes.

Callum: 1:29

was, that, I guess as we go through this series, we're going to be talking about quite, common and uncommon fungal diseases. And, maybe the diagnostics are less familiar to people so we thought it would be good to start off just giving a bit of an underpinning about how we diagnose fungal infections. So as we go forward, every time we mentioned these tests. We don't have to then explain them, so hopefully if you've listened, listen to this episode before you go on to the next ones, when we're talking about the different diagnostics, you'll have a better understanding of, what they are and how they're used and, the, potential pitfalls or benefits of the, so hopefully this is building the knowledge, as we go up our pyramid of learning,

Alyssa: 2:11

I was hoping we could start off by talking about the value of direct microscopy. in diagnosing fungal infection. I always think that fungi look absolutely beautiful down the microscope, and that there's so much that we can,, learn from such a simple test. so Malcolm, in what sort of specimens, and what sort of circumstances is direct microscopy looking for fungal elements, valuable?

Malcolm: 2:36

Well, first of all, I'd like to say, of course, I'm an advocate of microscopy, and in fact, I've just finished a whole series of 21 lectures for the BSMM's Masters in Medical Mycology module on microscopy and histopathology. So, yeah, I've been familiar with direct microscopy actually from, the beginning. a PhD student in Leeds. I was actually called to the dermatology clinic to look at nail clippings from cases of supposed onychomycosis. So that's where my interest in direct microscopy has really come from. And of course, yes, Fungi are large structures. They have unique morphologies. So in many cases, they are recognizable by their structure and of course, the way in which they interact with the host immune system and so on. So the types of specimens. It's broad. So from, yes, from skin scrapings, hair bulbs through to, nail clippings. So, the Manchester lab deals with about 8, 000 nail clippings a year. That's quite a workload, that's looking for dematophyte, elements, candida pseudohyphae in, nail specimens. and more unusually, where you have cases of pituitary vesicular, the appearance of malazea in skin scrapings. And of course, occasionally you can come up with an immediate diagnosis. thinking of a mold like, a non scopolareopsis that's instantly recognizable by direct

Callum: 4:05

Yeah.

Malcolm: 4:06

So it has a lot of, it has a lot of value. And then of course, the other very useful sample are, sputum and BALs. And that takes a little more thought and processing of the sample. But again, that can be a very valuable technique and can in many cases give you an immediate diagnosis, but not forgetting, of course, the appearance of a filamentous mold in a BAL doesn't necessarily tell you it's aspergillus. There are many molds, 13, 14, even more molds much rarer infections that do appear very similar to aspergillus in respiratory secretions. So, they're the sort of normal specimens, and of course there are a whole variety of other specimens. There can be smears, there can be punch biopsies, and so on and so on, and especially if you are faced with a possible endemic mycosis, a patient who has returned from an area of the United States, for example, where there's a possibility based on the clinical presentation and the travel history of coccidioidomycosis or blastomycosis. Again, the ret microscopy of, superficial lesions, which you often see in those patients, the ones I've seen anyway, that can be very valuable Again, because of the unique structure and the size of that structure, an instant diagnosis. So it's a very valuable technique. But unfortunately, I think the numbers of people who are trained to do that is few and far between. There was a recent note in the British Journal of Dermatology, a survey of dermatologists, how many of those dermatologists in training has actually done the potassium hydroxide preparation of a nail specimen, and the figure was zero. So obviously it's not part of any practical clinical training for that speciality, but of course in the ID micro world I would like to think that, during, trainees training that they would have the opportunity to see Aspergillus spout or, or in a BAL. And of course, you can enhance the presence of sparse, fundamental elements using various enhancers. So, chalcol 4 white, blank 4 white is a typical example there, but you need access to a fluorescence microscope. That's not always available, but again, extremely valuable, because there are so many artifacts, whether it's an artifact in a nail specimen, or a or in a sputum sample. they can be confused with, with fungal structures. So if anybody's listening to this and they want to learn more, they need to sign up for the BSMM Masters in Medical Mycology that the University of Exeter is providing for us, a whole module on microscopy.

Alyssa: 6:45

Fantastic. And also I've included in the show notes, if doing a whole masters isn't for you, then, it's worth checking out microfungi. net, which is a free fungal microscopy course. It's designed to teach identification of fungi by direct microscopy and histology in a range of tissues.

Malcolm: 7:05

Yes, the whole team in Manchester has been involved in assembling, and creating that site. So I think over about now over a thousand images. So that's, I don't know whether you've looked at it, it starts off with all the basics, staining and, the principles of microscopy right through to advanced imaging. histopathology. Thank you for promoting that and it's in a number of different languages as well. So I, because of my interests and my connections with a new lab in China, we've been trying to get Chinese, medics to, to have a look at that. And they're pretty good at microscopy, but they're not aware of that site because of the lack of promotion. So thanks for mentioning that

Callum: 7:42

I wish I'd known about that when I was sitting my part two fellowship exams because I hadn't seen it before. the majority of this is going to be done by, or, biomedical scientists and laboratory these days. But as a clinician, one, it's useful to have an understanding of how it is useful and to, Bye. May well be in an exam near you and you know the question of a clinical case and there's a microscopy.

Malcolm: 8:04

like me, you. be sent slides, histopath slides for review. So, the week doesn't go by in the Manchester lab, where when, where we're sent, whatever, often from the lungs, but elsewhere as well, and some quite tricky cases. so we're looking, we're exploring the use of AI as a possible way of identifying fungal elements in stained tissue sections. And of course, and there's also, a wonderful new book called the, Histopathologic, Diagnosis of Systemic Fungal Infections by Henrik Jensen, who is a professor of pathology in Copenhagen. That's available now on Amazon or it's as an ebook. So, if your unit is going to be, involved in all things fungal diagnostics, then that's, I think these are very useful resources if somebody's going to be trained up. because as you probably are aware, there are very few pathologists who have an interest in fungal pathology and histopathology,

Callum: 9:05

and you mentioned there, Malcolm, the basics and so on. I guess I, I'm lucky to have a sort of my ecology lab locally and was able to spend some time with our specialist biomedical scientists recently, which was honestly just really helpful. and one of the things that struck me was, obviously there's this wide range of specimens and if we think about, you've got a person with a clinical you suspect a fungal infection and you're asking for, Microscopy. What's the sort of journey that sample goes through? Because there's a sort of, preparation of the sample and then how do you, how do we, when we say microscopy, what is, what are we actually doing? And then there's a little bit about how these stains are helping us. I think that might. How people get their head rounds, you know what we're talking about later in the series

Malcolm: 9:45

Very, very good question. So once that specimen arrives from wherever, could be your own hospital or from another hospital, that is a big issue in terms of turnaround time. So yeah, let's just go through the stains. First of all, that you would use possibly for direct microscopy of onychomycosis, that is purely potassium hydroxide. You can add Parker ink to that to enhance the fungal elements. And as I said, you can use brancophore, calcophore, white quite effectively. Then if we talk about sputum, for example, we do a lot of sputum microscopy because we have an adult cystic fibrosis unit and we do get sputum samples from those sorts of patients and others. We tend to, we don't tend to process that sputum, which we try and do microscopy directly on the need. specimen, a portion of that. And again, that can just be potassium hydroxide with an enhancer added. And that really is a useful tool. Some laboratories, some manuals procedures will advocate digesting the, sputum and then centrifuging that possibly to improve the sensitivity of the technique. The same with BALs that can be looked at directly with an enhancer like Carcophore White. Again you can actually centrifuge that type of specimen because it's a larger volume and you may want to, considering the dilution effect, you may want to centrifuge that. Then of course we have Cerebral Spinal Fluid, the familiar India Ink. technique. Again, that takes a little bit of expertise. there are host cells sometimes that can be, can be confused with budding cells of Cryptococcus, but normally because of the feature of that, encapsulated budding organism and the presence of intercellular organelles, with experience, you can actually identify that. Then we come on to Any sort of biopsy that, can be sectioned to give you a thick section that can be stained directly with potassium hydroxide or chalcophore white. And then you move into the whole area of histopathology and there are a whole range of stains there. So there's, the methenamine silver. Gamori grocot stain, specific fungal stain, which will stain the cell wall of, fungal hyphae, for example. there's periodic acid shift is a very useful stain. So usually, the two are done together if there's a fungal infection suspected. And then you have more specific stains for the melanin, the in the cell walls of demetiacious fungi, like the, Fasun Montana stain. That's not always available in everybody's, pathology department. And then you can, use the Southgate's mucicarmine stain for, again, for Cryptococcus. So that really is it in terms of the specific stains that we do use for fungi.

Callum: 12:47

I recently was looking at aspergillus on the microscope and the, we mentioned fluorescent microscopy there briefly, and it was amazing to see. It was very, it was quite hard to find on the initial samples just under drip microscopy. And then we did the fluorescence and it was like, wow. So, I guess that's a bit of a step forward in improving the sort of sensitivity of that test. Yeah, how does carcofluoride work, actually work? Because we know that it enhances the sensitivity of diuretic microscopy. what is it doing?

Malcolm: 13:14

Yeah, it's binding to the chitin of the cell wall, which is exposed on on the cell wall, but also usefully, it does stain up the cross walls, the septi of a filament of fungus, like Asperger. So that's the basic mechanism, obviously is a lot more chemistry, behind, behind that. But that's how it works. It's similar to what. whether it's still the case, but it used to be added to biological, laundry detergents, that sort of thing. To make white shirts even whiter, but I'm not too sure whether that's still used, for that purpose.

Callum: 13:49

strange, world of mycology.

Malcolm: 13:50

something I've been passionate about now for 52 years. I started my PhD to work on Canada, University of Leeds, in the early seventies. So yeah, I've tried to be an evangelist and an advocate of all things fungus, going along.

Callum: 14:08

the Mycology series will hopefully help with that

Alyssa: 14:13

I found that I'm increasingly becoming a big fan of direct microscopy. And I think, we're very good at using it for wet preps on, genital swabs to look for, evidence of VVC or using it in skin scrapings and nail clippings for looking for denatified infection. But I think there's a real role for using it in tissue and respiratory samples as well for diagnosing invasive fungal infection. And I think what you mentioned earlier, Malcolm, was that the huge benefit that direct microscopy has over, cultural history path is that rapidity of results and getting, that was it within hours of a sample arriving in the lab, getting that clue that, hey, this could be. could be fungal infection if we've seen, fungal elements in, in, in tissue or BAL sample.

Malcolm: 15:05

So I think what we're talking about, of course, is a training course. I think we have not had a specific training course on microscopy for a very long time. I mean, sometimes these one day courses are, tagged on to the beginning or the end of a international conference. So the upcoming conference in Brazil in May, that's going to have a one day, a one day training course, but that's a long way to travel for a training course. we could do something similar in the UK, and I think there are enough people who could actually, provide that. There is a core expertise in the UK, in our society, who, I think could do something quite useful there, but it's a very good It's a very good point to, try and encourage people to do more and more microscopy. And of course,, in terms of laboratory accreditation, of course, that's something which would have to be, validated and people would have to sign, be signed off for competency in that type of technique. So whether long term, whether AI is going to Help us overcome this, lack of trained people. that's wait and see. I was in the United States, last week for a couple of days. And the, the following morning before traveling back to the UK, we had a tour around the NIH, laboratories in Bethesda and, There they were showing us, it's a combined mycology and, parasitology laboratory. There they were showing us some very nice image recognition software that's been developed by an American AI company for looking at cysts and worms and so on. in faecal specimens. It was very impressive, but then they have not, um, produced a fungal one yet, but that will be really worth having. We, the laboratory in China has developed an AI system for direct microscopy of, of nail specimens, about 50, 000 images in a image bank there. this is to avoid, of course, this very tedious direct microscopy that we do with. potassium hydroxide and microscopy, you can have these scanners that take 10 slides and they're read very quickly and then matched against the library of images. So yeah, hopefully that becomes a reality,

Callum: 17:11

so, Lysia's mentioned the benefits, was just that rapid results, the, provisional identification, I guess some of the other benefits, maybe aiding differentiation of infection from colonization. So if you've got, Candida hyphae in a non cell sample, it indicates infection rather than colonization. and then, for, nail and skin scraping is that some of these dermatophytes are very hard to grow. So, actually the gold standard is a microscopy. If we talk about the pitfalls, so you mentioned about, this is difficult. I'm always struck when I'm in the mycology lab, they're showing me that in explaining it every time I find it quite challenging to remember, which one has branching high, hyphen, exactly how to differentiate them is, it requires a skilled operator, or maybe AI will answer that. And what are the sort of other pitfalls that you might, why is, why can't we just rely on microscopy alone,

Malcolm: 18:05

um, you may not see anything. That's the first pitfall. you just may not because you can't do microscopy on the whole sample. And we may come on to culture in a minute where we have been developing a method to enhance the yield of fungal elements from samples. But, it really comes down to that. And of course, the other pitfall, especially with, onyomycosis is artifacts. I think that can really trip a lot of people up. In terms of the basic morphology of filamentous fungi in respiratory samples, for example, you've got the basic differentiation between aspergillus hyphae, the branching pattern, presence of septi, you can actually identify almost down to a species complex level aspergillus terrius as adventitious sporulation on the side of the hyphae but you don't see many cases so it's difficult to build up that sort of experience. from Mucroales for example, I think they are pretty, and they are pretty recognizable. But, again, going back to this book I've mentioned, if you're interested in histopathology, the author does have pictures of look alikes of confusing images, which you might think are fungal hyphae, but they're not. And it's quite interesting, the, there were two pathologists, presenting At this isham diagnostic course in Bethesda last week, and one of them, showed a lot of images and there was a quiz attached to each image with the differential and, a lot of us got them wrong because ultimately because based on culture and other techniques. A definitive diagnosis was made because he showed some very tricky slides to read. So they're all, that's a pitfall. It's really, and what does that come down to? I think that just comes down to experience. And we're not seeing a lot of these infections of any of these infections, but your point about candela is interesting because again, we've been having this debate and I'm sure you have as well. And we also talked about this last week in Bethesda. is, there's such a thing as candida pneumonia. So if you see candida in a prep or a BAL or a sputum sample, is that colonization? Is it such a thing as candida pneumonia? And you probably know years ago BVC was thought to be, if you see candida hy on a wet prep. Of course that means there's infection, but that's not the case. So yeah, all of those issues, I think, just have to be borne in mind,

Alyssa: 20:31

Ah, so I thought that, yeah, the presence of candida hyphae was actually Enabled you like in a BBC sample enabled you to differentiate colonization from, infection, but it's that not necessarily.

Malcolm: 20:45

and that's not necessarily the case. So if you look at the BASH guidelines, the British Association for, Sexual Health, that's made very clear there that there is this, confusion between true infection and colonization. And I think the same thing comes into either colonization or infection with aspergillosis. How do you tell the difference? I mean, these spores will germinate in any sort of environment, not necessarily causing infection.

Alyssa: 21:16

Okay.

Malcolm: 21:17

that's another aspect to microscopy. Can you say anything about? And of course, if you're looking at tissue sections, I think the presence of a host response is pretty indicative of an infection. If you've elicited, if the fungus has elicited a host response, I think that goes some way to saying, yeah, this is invasion. This is tissue necrosis and, and a host response.

Callum: 21:40

I guess just to summarize, microscopy, so we've talked through there, the different types of clinical specimens and situations in which you'll be doing microscopy, some of the common stains and the use of potassium hydroxide, which I understand is that sort of breaks down the tissue to allow you to do the microscopy. and then the benefits, rapid results, provisional identification, potential differentiation of colonization, from an, infection, although, maybe that's not the case. And, you might grow, might identify things that don't grow, and then pitfalls, Sensitivity is an issue, required experienced operator, and then, you might need special equipment. For example, the stain or, doing fluorescence, then you need a fluorescence microscope with the right light bulb, which is quite expensive, apparently very expensive, and filters, and then we talked briefly about histopathology and cytology, the GMS and PAS stains, which is a sort of definitive diagnosis., So maybe we could move on to culture next.

Alyssa: 22:40

So my understanding is that generally, we refer back a lot to the UK SMIs. about when we should be performing fungal culture. It's rare that we would automatically put up a, SABC plate for,, a specimen routinely. But if we're, suspecting that a patient's at risk of fungal infection or fungal infection is expected, then we will try to, inoculate the sample on a media that supports fungal growth, to try and isolate the fungus.

Malcolm: 23:10

Yes. Okay. So if we can start talking about culture, I think my, advice is if there's enough sample is to always culture, cause you are amplifying the fungal elements, the spores, the hyphae, the blastoconidia that are in that specimen. So which could be Mr. Microscope, as we've already said. How do you culture? Well, fungi, pathogenic fungi in general, they're not particularly demanding in terms of what they need to grow on. Most will grow on a sub earth plate. And of course, you can manipulate that medium to cut out any overgrowing organisms. If you are processing,, superficial, samples. Then you have the whole question of volume. So that's something that we have been publishing on a bit recently in so called high volume culture. Instead of processing the, the BAL, for example, and, and then plating out a small volume of that, concentrated sample, we would plate maybe five plates. for a BAL, and that has been shown to be, certainly enhanced, sensitivity, somewhat similar to, sputum PCR. And, so that's pretty effective, but you can't always do that, and that takes up a lot of place. It depends on the throughput of samples. Then, of course, you've got the whole question of temperature. if you're interested in what's going on in somebody's chest, you can obviously incubate plates at a whole range of temperatures. And then once you've got something growing, you can then increase the temperature even more to try and differentiate the taxa. I'm thinking of mucorales now, some grow at 50, some grow at 55, and so on. So there are many aspects to, culture. How often should you look at the plates? And of course, I'm talking all the time from the mycology lab in, in Manchester, which of course is a focus lab. So, we have time to do this sort of thing, but in a much, much bigger,, general microbiology lab, there may not be the time to do all of this or the resources. And of course, we're all looking at cost savings at the moment. So yeah, some general points about culture.

Callum: 25:14

I guess, as Alyssa mentioned at the beginning, it's about that pre test probability in a way so that the UK SMIs outline, specific syndromes where, say you've got a bone marrow or CSF, when, when do they recommend doing it? I guess it should be as per the guidelines in the protocol. But also, if you've got a patient with an unknown clinical syndromes, they're a risk of fungal infection, or that's the differential, then ensuring the laboratory that you're working with know that that's in the differential. And, certainly look, we have, if you label something as specifically for the mycology lab on our electronic system, then it goes straight to that specialist part of the lab, and they will do the mycology stuff. Okay. Whereas if you're just sending, say, a routine sputum or another sample, unless you tell the lab that you're worried about a fungal infection and which fungal infection, then, it may not actually get anything. and whilst we might grow some fungi, like candida on a routine sort of blood agar plate, we might not grow it. so if you don't look for it, you'll never find it.

Malcolm: 26:15

Pose you a question then, a case that came through this morning actually from from the lung transplant team in Manchester And so here we have a lung transplant patient. And from a BAL, we've isolated one colony of scopolareopsis and five colonies of penicillium. So the question was, how do we report this out? This is the lab asking us now, how do we put that out? Do we do sensitivities? What's the significance of that? I immediately found quite a few case reports of scopolariopsis, not just brevicordis, but the other species of scop and quite a few case reports, in lung transplant patients. I don't know anything more about the patient. that's one thing. And then the whole aspect, if you isolate penicillium from a BAL, is that contamination? should you get contamination of a BAL with penicillium? I mean, we find masses of penicillium in sputum and so on. So there's all that going on. The interpretation, I think, is very difficult.

Callum: 27:13

I guess we spoke in the fungal overview that fungi are, omnipresent in the environment and it's definitely something to think about with culture where, if you're growing something it doesn't necessarily mean that it is either a pathogen, so it could be a colonizer, or it's actually from the patient, aspergillus, or other fungi, can be real problematic, laboratory contaminants because of the way that they grow. Once they set up and spores are in that area, that room, then, in terms of the agar, if we're suspecting fungal pathogens, we mentioned SAB and SABC, SABARO, what are the different types of agar that, that can be used and what's the sort of benefits that we use for each one?

Malcolm: 27:52

there are multiple, agar you can use. So modifications of Saborode. we do a lot of environmental sampling using volumetric air samplers. So we use malt agar there, which is very useful. So most environmental malts Aspergillus, Penicillium, Calasporium, and so on. they grow on malt. most fungi within the UK setting, will grow on Sabarodes. But of course, I'm sure your electronic ordering system is the same as ours. We, we use, epic Greater Manchester. Whoever is ordering the test has to put in the travel history of that patient. So that immediately alerts you to the possibility of other molds, which might grow on a patient. Saburo's plate. So that's the sort of primary isolation. And then of course you have all those techniques for converting those, dimorphic pathogens into a different morphological phase. just a little hint here. in terms of Cryptococcus you've just mentioned, their malt extract agar is very useful because if the organism is producing a lot of mucilaginous capsule, if you hold the plate up vertically, that growth will slide down the surface of the culture plate because of that palosecular capsule. So malt in particular, if the suggestion is that the patient may have cryptomeningitis or whatever, that, that is a little useful. trick to remember. But I think, yeah, by and large, most molds will grow in terms of primary isolation on Saburo's.

Callum: 29:21

I always wondered what that stood for, and I don't know why I never looked it up. There's maybe too much stump, but I didn't realize it was named after a Raymond Saburo in 1892.

Malcolm: 29:30

in many ways, he was the founder of, medical mycology. So if you go to the Institut Pasteur in Paris, a lovely place, it has a museum there and a lot of sort of artifacts and memorabilia from that particular time. Yeah, there's been a whole string of very well known, French, Psychologists, they're very active. And, the whole psychology setup in France is somewhat, different, from we have in the uk it's well supported and they have many units. So I don't know if you remember, we did publish a survey and it's even gonna be repeated quite soon, of what is available in the UK in terms of fungal diagnostics. And the paper came out in the Journal of Infection, and the title went something like this, that basically, Medical Mycology in the UK, colon, results of a national survey, and the chief editor at that time, he changed the subtitle to, Evidence of Suboptimal Practice. So let's see whether the new survey, which is being conducted at the moment, actually, highlights that something, some things have changed, but I'm not sure. I looked at a copy of my PhD thesis the other day, University of Leeds, and the first sentence was, we need, to see whether or not we could differentiate between antibody responses to candida yeast cells compared with fungal candida hyphae. First sentence was we need better diagnostic methods. That's a long time ago. And, so here we are talking about fungal diagnostics.

Alyssa: 31:04

I think it's worth bringing up chromogenica, you guys, such as, something like the chroma Canada plus, because I think that's, another commonly used agar in, local, laboratory, level.

Malcolm: 31:19

I'd see, we see that as the subculture goes onto that. you can use ChromAgar as a primary isolation media and that really does. speed things up a bit. But yes, so chromogenic agar with expertise can differentiate between a few of the, the major pathogens, but also, yes, as you said, in terms of candida auris, there's quite a lot of interest now in using, the enhanced chroma agar for, pretty rapid, diagnosis and avoiding, sequencing, of that particular organism. So yeah, there are lots of, there are lots of, media like that for secondary, for identification basically, not for primary, but for secondary, subculture and identification. In the, in the environmental mold world, there are media like Rose Bengal and Vegetable Juice, V8 Juice, and so on and so on, to select out particular groups

Alyssa: 32:13

and we touched briefly earlier on the different incubation conditions. So, generally, air is the atmosphere. So generally air is used to, incubate, SAVC for most of, most fungal pathogens. Would you use aerobic conditions for, if you're trying to isolate

Malcolm: 32:28

We know just simply 30, 30 degrees for the required length of time, just in a standard, standard incubator. If you want to, in a research setting, if you want to actually produce masses of rubrum, for example, you can introduce carbon dioxide into an incubator chamber because, there's quite a high concentration of CO2 on the skin's surface. So that's trying to mimic the environment. But that's for producing arthroconidia for, for research. So many years ago, Rodhay and I, and we developed this type of method and published on that, but that's not routine diagnostics.

Alyssa: 33:08

And then what temperature wise? incubation is usually done at between 35 and 37

Malcolm: 33:15

if you're Cultivating respiratory samples, yeah, between 35 37 that would be standard practice.

Alyssa: 33:23

and when would you use the lower temperature range? So looking in the, UK SMIs, sometimes it will say to use, a lower range, 26 to 30 degrees, or 28 to 30

Malcolm: 33:33

for whatever reason, if you're interested in fungal allergy and you're interested How much penicillin there is in somebody's lungs, then, you would obviously have to use a lower temperature than very few penicillin species. So we have a particular interest in penicillium, allergy, because we do see quite a lot of that in terms of severe asthma with fungal sensitization. If there was an indication that that might be the, uh, the clinical, scenario presentation, we would use a lower temperature. So yeah, to modify the nail samples, that, that's 30 degrees, but anything respiratory, we would incubate at a a higher temperature.

Alyssa: 34:10

okay,

Callum: 34:10

And then I guess for the dimorphic fungi, which, obviously mostly ACDB, category three or four pathogens. So, shouldn't be doing in the normal lab, but, if you are looking for them, you're doing The whole idea is that you do the two different temperatures and then because they grow as a mold and a yeast at different temperatures. That's, useful. Even the temperature you incubate at is part of the diagnostics, isn't it?

Malcolm: 34:35

I think it's very irresponsible especially if you are, sent a sputum sample with no travel history, no history at all, barely, or you are the lab is sent a, a fluffy mold on a plate. I got involved many years ago in a potential co mycosis, outbreak because, I was still working in Helsinki and Finland at that time. A lot of, fins, went and competed in a model aircraft flying competition in California, Southern California, and they, and there were a lot of contestants from the UK as well. They were warned about the hazards of going into that area in terms of coi. But, somebody came back to a smaller DGH hospital in Finland, produced a, they had a sort of a mild flu like illness, produced a sputum sample, and the mould from that sample was sent to the main fungal lab in Helsinki with no information whatsoever. Yeah, that was very irresponsible. of course, once it was identified as cocci, of course, you can just imagine, all the lab staff wanted prophylaxis and the serology was positive. So those things do happen, but hopefully not very frequently.

Alyssa: 35:45

yeah.

Callum: 35:47

Culture duration is maybe the final point to make. So I guess, most Canada, what we're seeing on clinical samples will be growing within, 24 to 48 hours, things like cryptococcus. you need to, extend the culture to serve 7 to 14 days and then. Some of the more unusual molds or endemic dimorphics might take up to three to six weeks to grow. So, I guess just think about which pathogen you're looking for, and then that will guide how long to incubate for,

Alyssa: 36:15

So, Malcolm, say we've grown a yeast or a mold on a plate in the lab. What's, what's our first sort of approach to identifying what this, organism is?

Malcolm: 36:26

first of all, obviously, sort of visual assessment. expertise you can tell the difference between albicans candida nebrata and one or two just by the topography of the, colonies. so the first approach would be would be through a germ tube test. done. And again, that takes a little bit of training and expertise. I'm not sure if you've spent much time looking at germ tube, tests, but again, there can be confusing structures.,

Callum: 36:50

Can we just very briefly explain a germ tube test is because I think some people might not

Malcolm: 36:56

Yeah. So this is, this goes back into the. 1950s, some mycologists in the U. S. found that if you took candida cells and put them into a rich protonaceous type of medium, and that can be serum, that can be, egg white or albumin, then, candida albicans and candida dubonensis will start to germinate. So that is the germ tube test. So that's a test tube, small test tube with that medium in it, lightly inoculated, not over inoculated, and then incubated at 37 degrees. And after three hours or two to three hours, the guidance differs a bit. You will see a germ tube, basically a filament, that's originating from that, from individual candida cells.

Callum: 37:44

certainly locally we use germ tubes for candida first stage identification because it's cheap, it's quick and we're going to talk about MALDI-TOF later on, but like our multitask overwhelmed. it's a great test to quickly say, is this germ tube positive or negative candidate? And that actually is really helpful. First step, isn't it? I guess we're talking about yeast first before we can get to that, in the fungal overview, we talked about yeast versus mold. you're seeing yeast release these single colonies. So say we've, I've done a test. we've said it's, germ tube negative, for example. So it's not Candida albicans or double nexus or something. How else might we identify

Malcolm: 38:17

well, traditional ways, biochemical tests and these oxanagraphic methods like the ID32 gallery of sugars and so on. That's something that we still use. and of course we do, as you said, we will come onto the Molditoff, which again, the match that I've had for about four years now. But yeah, we would use a variety of, of biochemical tests and come up with a profile. And I think that's a pretty, that's probably used a lot in laboratories still.

Callum: 38:41

plate appearances, about the various yeasts that Alyssa's put in the show notes. But I think in the interest of time, we can jump past that. So maybe I personally think that molds are more tricky to identify than yeasts. I don't know if that's a fair statement. So maybe we should spend some time talking about how do we identify if you've got to say something that's growing on mold. So it's a colony and it's got that sort of typical appearance.

Malcolm: 39:04

So basically you've got the various criteria. You've got the, color, you've got any particular zonation segmentation, you've got the rate of growth. that's a very useful, um, criteria. And again, I've always, said, basically what are the most likely Yeasts or moulds, are you going to isolate from that particular specimen type? You're not going to get a mould, you need to bother about too much from a urine sample, but you're going to get a variety of moulds, and so the list is longer, from a respiratory secretion. So you can begin to narrow down. But then when it comes to molds, yes, you've got, you've got many species of Aspergillus. And so one that we see a lot of now in environmental samples is Aspergillus calidastus or calidustus. we need to identify that if people are being exposed to that. We've got all the familiar terrius and flavus aspergillus nidulans. We see that quite a bit in our respiratory samples from the National Aspergillosis Center clinics. You've got aspergillus niger, so they're in many cases recognizable the culture colony, the color of that colony. Then of course it's microscopy. It's recognizing the various shapes and sizes of the fruiting body and the arrangement of the spores. That becomes quite tricky, I think, in terms of these, cryptic species of Aspergillosus. Some people think they probably can identify these species. Then you've got other models like Skelosporum. I think that's quite easily identified. You've got Fusarium species, Oxysporum complex, and so on. Solani complex. easily identified, but you know, there are always going to be exceptions. And we find, sputum samples from our patients who've been on azoles for years and years, you, you get all sorts of albino colored, aspergillus fumigatus colonies. There are always those tricky ones, which, do need a lot of time and before the moldy we would sequence identify them because we need to know what they are and of course there are some wonderful identification manuals if you can afford it have 300 euros for the two volume identification of clinical fungal pathogens That type of book, there are quite a few new books, coming out, and I think the, UK HSA Identification Manual, edition three is, is being, is being written at the moment. Yeah.

Alyssa: 41:34

yeah, fantastic. We have a copy of that in our mycology lab. So, we're always referring to that and looking to seeing if we can make a provisional identification based on looking at, like you said, the colour of the colony on the front or reverse, the colony morphology. How quickly has it grown? We had a mucorallis this week that was, pushing the lid up off the agar plate after a couple of days. and then looking at those microscopic features. And some of them are, really quite characteristic like just something like differentiating a mucorallis from an aspergillus, even to a fairly untrained eye. you're able to recognize the broad, ribbon like hyphae that have few septa and the sacs of spores, as opposed to the, the finer, hyphae of aspergillus with the, conidia. So I think it's, yeah, it's definitely even for, the non expert, the great book to refer to when you're getting these, growing in the lab.

Malcolm: 42:29

And I think the proficiency schemes that we have available are very useful to the NEAS scheme I think that's a tremendous training opportunity for people, but also. there's a very nice, it's obviously online, fungal identification, proficiency scheme from South Africa, fungi of the week. And that's sometimes quite challenging. So they present you with a very brief, vignette of the clinical background and then, A picture of the colony or the plate, and then a picture of the, microscopy. And some of those are very challenging, stuff we just never, never see in the uk. I think it just reflects that part of the world. But you can get CB, D points of that as well. And so that's another very useful scheme that we, get all the staff to have a look at in, our lab in Manchester, yeah.

Callum: 43:14

One of the things I struggled with a lot when I was doing a little bit of mycology in the lab was all the mycology terms. So we've talked about things like, Canidia, I think we could have a whole

Alyssa: 43:25

Highlights. Naturally.

Callum: 43:26

fungal, basic biology and. I guess we can't really do that. It's no time. but, there is an appendix from the identification of pathogenic fungi, which is the one that we were mentioning earlier on. I just basically open it up every time. I'm like, what does it mean again when it says K spore, anamorphic, presidium, it's like speaking a different language. but, yeah, one of these training

Malcolm: 43:50

it

Callum: 43:50

would cover that, but

Malcolm: 43:51

there's one, one book, which we use a lot. It's been around for many years. It's in its seventh edition now, is Lauren's, manual of identification. So this is, it's a wonderful book. It's not just, identification of fungal cultures. It has a big section now on, on molecular techniques and also on, histopathology. So. It's, um, the author's quite some new authors, but anyway it's called Larone. It's available on Amazon as well as an e book, but that is a very useful book. It's got line diagrams of the basic fungal structures, the phyllis and metruli and so on, but also it has a lot of color images as well of the fungal cultures.

Callum: 44:33

Yeah, you're, you, if you wanted to learn more, there's lots of places to go, but I guess For now, those are the main things to think about in identification, macroscopic and microscopic appearances. So maybe now we should talk about the Malditoff, which, you know, we, Jame and I did an episode very early on., on episode 25, we just have a little bit of a deep dive in what Malditof is and so you can go to that if you've not heard of it, but, to the loyal listener that's listened to every episode, how do we use the Malditof in the context of fungal identification, because it seems to be a bit of a game changer for some things.

Malcolm: 45:08

Yes, so if you can afford the MALDI-ToF, we bought one four years ago, 200, 000, I think, capital purchase it has actually revolutionized the identification of unusual, molds, so there are a number of companies who make MALDI-ToF, so getting a result within a few minutes It comes with a database of a lot of the pathogenic fungi, you can obviously build up your own database and add that to, the standard database, both for yeast and molds. So yeah, it has a very high capital cost, of course. It, but it's very cheap to run. It does give you, in many cases, a definitive identification. So extremely, extremely useful. So I think it's a good example of automation. My one worry is, of course, it's going to reduce the level of expertise using these classical techniques that we've just been talking about. Because people say, well, why do I need to learn how to do this stuff? And the other, with the microscope, with culture identification, We just put it onto the, onto the MALDI-ToF. it doesn't really require any previous knowledge of the organism that you're dealing with. It's simply onto the machine and a name is generated.

Alyssa: 46:19

Yeah. And I guess my understanding, so, locally we use the MALDI-ToF to identify our yeasts. So my understanding is that, the commercial databases, represents yeast pathogens very well, and it's very rapid and reliable for, identifying yeasts from the colony growth. but I wasn't sure about how, how useful it was at this stage for molds. And if, it's only used in more specialist laboratories where they have developed an in house database to sit alongside that commercial

Malcolm: 46:51

Yeah. So, yeah, we've been developing, our own database for these environmental molds because that's a big part of our, laboratory, operation. So that's our own database, but I think, back to my favorite organism at the moment, Aspergillus, Callidustus or Callidoustus, the standard, database that the machine came with. It picked that up. Whether it'll pick up aspergillus lentilus and a lot of these other, unusual cryptic species, I'm not too sure about that. And the other thing which I think is going forward, I think how it can be used for antifungal susceptibility testing, but that might come into a separate session that you're contemplating running, but that can be quite useful. We haven't gone down that route because we do optical pyrosequencing to look at, mutations in aspergillus, azole mutations in particular. But it can be used for that purpose as well.

Callum: 47:43

So yeah, MALDI-ToF, amazing if you have it, but if it breaks, then, uh oh. And I think that actually could be an exam question, You know, what do you do if Maldipoff breaks? other than panic. so yeah.

Malcolm: 47:54

The advice is to buy two.

Callum: 47:56

but yes, yeah. That's even more of an investment. But

Alyssa: 48:00

you've just got, spare 4,000

Malcolm: 48:02

Yeah, yeah. Buy one and get one free. Haha. I don't think that's not in the interest of the companies, I don't think.

Callum: 48:10

so just to summarize what we've talked about there. So we've talked about culture for fungal pathogens and thinking about doing that in patients that are at uk standards or microbiological investigation the smis Which are linked to in the show notes, and, the, the utility of culture, being additive to that of microscopy. and we talked about the types of culture media. So mainly talking about saex drills, malt extract, agar, and also some chromogenic. Agars talked about the incubation conditions of the temperature. which to incubate them and how long, and the important point about, things that are hazard pathogens like the dimorphics are, and notified to the lab. Now we moved on to talk about the identification. So we've talked about the morphology and how to identify these using for yeast germ tube tests, multi TOF. and how they appear in the plate, and then for molds, we spoke about macroscopic appearance on the plate, the rate of growth, the appearance, the color, and some microscopic appearance about different aspects of the fungi, and there's more links to some excellent resources about how to get better at that, if that's something you're interested to, and then we touched on the moldy top, but there's so much more to talk about, isn't there,

Alyssa: 49:25

There's probably not mushroom left in this episode.

Callum: 49:29

Oh,

Alyssa: 49:30

But in a future episode, it would be wonderful to cover, fungal antigen detection and non culture based methods of diagnosis. So fungal antigen biomarker detection, fungal serology, which is detecting a patient's immune response to fungal pathogens. and there's been huge progress in molecular diagnostics, for fungi. so I think these will be amazing things to, to come back and explore.

Malcolm: 50:01

So you mentioned show notes. I've got a wonderful review in front of me here, which covers absolutely everything. It's called the evolving landscape of fungal diagnostics, current and emerging microbiological approaches. It covers absolutely everything from. What we've talked about right through molecular point of care tests, exhale, breath analysis, and so on. I can send you that paper

Alyssa: 50:25

that'd be fantastic.

Callum: 50:26

That's great. so I guess we've summed up and we've set you up for the next episode where we'll be talking about the rest of fungal diagnostics. As always, there's so much to talk about and it's obviously a very important topic. And so again, thanks as always to Alyssa for putting all the hard work in and preparing the show notes and we'll make sure to add in those references and papers that were discussed. During the show, if you, if there's anything that we talk about that, you want some more information on, you can email us at idiotspodcasting at gmail. com. and just a huge, thank you, um to giving up your time and, lending us your expertise, Malcolm on this, important subject. I'm looking forward to talking to you soon, about more of the same.

Malcolm: 51:06

Thank you very much. Thanks for inviting me.

Alyssa: 51:08

Thank you very much, Malcolm. It's been great to hear your expertise in this.