106. Fungal diagnostics part 2: biomarkers, serology and molecular

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 biomarkers such as beta-D-Glucan and galactomannan, as well as serology and molecular techniques.

Notes for this episode can be found: here.

Transcript

Callum: 0:00

Hello, Alyssa.

Alyssa: 0:01

Callum, and hello again to, Professor Malcolm Richardson, who is joining us in this second, part of our fungal diagnostics episode. Thank you so much for coming to speak to us today.

Malcolm: 0:13

thank you again for inviting me to reflect and give my own views on fungal diagnostics, something I've been intimately involved with for many years, far too many years to really think about.

Callum: 0:26

So on the last episode, we went over, and if you haven't listened to that already, then what are you doing? Go back and listen to that one. we talked about, direct microscopy, histopathology, cytology, culture, and then a little bit about identification using morphology and things like MALDI TOF. And so in this episode, we're going to be continuing and the things that we're hoping to talk about are fungal antigen detection and serology, and then molecular diagnostics, including a section on various point of care testing, And this is a start of overview episodes. And as we go forward in this mycology series, we'll be delving in more detail on some of these aspects when we talk about the specific pathogens. So, Alyssa, do you want to take us,

Alyssa: 1:12

Yeah, so I think we're going to start about talking about, fungal antigen detection. And I think the main ones that will be, and trainees will be familiar with in clinical practice, is B2C Gleason. And Galactomannan. So perhaps we could start, talking about what these, antigen tests are and how they work and how they can be used in clinical practice. So, Malcolm, can you tell us a bit about the B 2D Glucan assay?

Malcolm: 1:39

Yeah, so the concept of, detecting cell wall biomarkers, including, Beta 1, 3D, Glucan, has obviously, has been around for many years. In fact, when I was a postdoc in Birmingham, we published the first, paper on the detection of aspergillus circulating antigens in 1976, a long time ago, and since then of course this has developed into the Galactomannion test, which we'll talk about in a minute, but then more recently we've had the recognition of a pan fungal biomarker, let's call it Glucan, it's, or BD, Glucan, let's call it that, as a circulating, biomarker cell wall component. Remembering, of course, that the cell walls of, fungal pathogens are pretty complex, multi layered, entities, and glucagon being a major component of the fungal cell wall, both molds and yeast. So this has been recognized over many, many years, originally by some Japanese, researchers. as a potential biomarker for, a number of fungal pathogens. So I think the main emphasis has been, for the diagnosis of systemic invasive, candida infections, but of course it can be applied to a number of other pathogens with the exclusion of, tryptococcus neoformans, and the agents of mucormycosis. they do contain, those agents do contain glucan in their cell walls, but not to any great extent, and so the whole idea of testing for Glucan has not really been applied to those two diseases. So yes, it has a very varied Glucan test and it's available now in many formats, ELISA type, formats or kinetic assays. perhaps the one that most people are familiar with is the Associates of Cape Cod, fungital test that's been around for 20, 30 years now. And that's the one that we use, I think, primarily in the UK, though there are other, formats available. Fujifilm, for example, is another one. So for a long, long time, these tests were thought to be expensive, and they were thought to be difficult to perform. they're very easy to perform, but of course the expense was because you really need to batch a number of samples to make the test cost effective. But now we have formats that we can use just a single sample. So, the usage, and this is how we use it in Manchester. We use it for antifungal stewardship. The best usage is because it's very high negative. predictive value. It's very good for excluding a fungal infection. but of course it can also be used as a confirmatory test to support blood culture positivity and other markers for the diagnosis of Candida. So that's how we use this, particular test.

Alyssa: 4:29

Great, thank you. So, I think it's, my understanding then is that. it's generally got very good sensitivity, but somewhat lacks specificity, both with regard to, it doesn't specifically detect a particular fungal pathogen, but also it's, false positives are fairly, fairly frequent. So it's mainly it's negative predicted value that, that is useful in ruling out invasive fungal infection with the exception of cryptococcus and mycorrhizae. So that's, an excellent point about its use, in antifungal stewardship. And what clinical samples can be used to perform this test?

Malcolm: 5:08

I think the most appropriate sample is serum. It has been used for, for respiratory secretions, but not particularly useful. And, some some more recent evidence has shown it can be used, quite usefully in CSF. so we, we do see levels of glucan in CSF. Yeah, I think the one thing we have to remember is this is a very ubiquitous molecule and the risk of environmental contamination or just carriage of glucan in the sinuses, elsewhere can actually obviously influence the positivity or negativity of this particular test. So I think that's very important to bear in mind. And as you've mentioned, There are a number of well recognized interfering agents that cause false positives. So I think from a laboratory point of view, I think it's absolutely crucial to understand that the glucan test really has to be carried out in a glucan free environment. So we're talking about all equipment is glucan free and ideally in a cabinet in a separate room for actually performing this test. Otherwise, you're not really going to be too sure about whether or not you've got a true positive or not. So I think that's an important point to make here.

Callum: 6:16

here. And you're talking there about this sort of laboratory aspect of this investigation and the potential for false positives. I've heard lots of people talk about the the clinical characteristics of the patients and the risk of false positives there. So some examples being, certain antimicrobials that patients are receiving and so on. But I seem to hear like different lists of things to worry about from different people, different sources. And sometimes people say that this antibiotic is risk. And other times it's said that actually the manufacturing process has changed and it's no longer such a risk. And what are your thoughts on that? Is that something that is there a definitive list or is it just something that we always to be mindful of?

Malcolm: 7:03

I think it's something you have to be mindful of. It's an evolving list, so some things have dropped off. I think the concern about, Exhibients and, components of antibiotics, I think that has disappeared now. I think we do, Take, note of the fact in terms of, there are all sorts of dressings that can contribute to, false positivity, there are various, food items that have a lot of glucan in them, especially if patients who using various sort of staple products, like beans and, and cereal crops and all that sort of thing, they contain a lot of glucan. So these can contribute to a circulating level of glucan in the blood. So I think it's just this, yeah, there is a list, but it's constantly changing. And I think really the product inserts of the various kits, it should make this very clear. And I think the companies are quite responsible in this sort of way that they do actually update their product inserts to accommodate any changes in our understanding of false positive results.

Callum: 8:10

And, the other thing you mentioned there, Malcolm, was about CSF. So, sometimes locally we've had complex cases where the patient has some sort of, CNS infection and fungal infection. causes have been in the differential. So you saying that if we performed a CSF would that have the same role as it does in Serum of having a high negative predictive value?

Malcolm: 8:37

I wouldn't really want to comment on that. I'm just really basing that comment on one recent paper I've seen. So I think the experience of using this test to either corroborate or independently, diagnose, a, A cerebral sort of fungal infection. I think it's too early to say. I think why we say, that this particular test is not useful for cryptococcus, of course, we have the crag test there, but it's thought that, of course, the glucan that's released from the cell wall of cryptococcus. can't really escape through the capsule of the organism. as you know, it's a very, dense, wide, thick, capsule. So that's why I think, I'm one of the theories put forward why it's not useful for cryptococcus. Whether it's, the Grucan test is useful for cerebrospiridylosis, for example, I think it's too early to say. I wouldn't really want to comment too much on that. Not having any sort of, Personal experience here in the Manchester center.

Callum: 9:36

That makes sense. Thanks.

Alyssa: 9:37

And then often, what constitutes a positive test? I think often I've seen the cut off of 80, is it, pg per ml, is the cut off for a positive test. But does that vary depending on the different commercial assay that's used, and is a higher reading more indicative of invasive fungal infection than a lower reading, and is there value from having consecutive positive readings?

Malcolm: 10:08

Yeah, I think all of those points are good ones. yes, I think the ACC test 80 is given as the positive negative threshold. You do have this. indeterminate zone below that. these thresholds, these cuttles have been based on many years of work and how precise they are. I'm not too sure about that, but I think yes, the higher the value, picograms per mil value, then I think the more indicative. of the fungal burden. And I think if you have persisting levels of glucagon, and so that does obviously, suggest quite frequent testing. So in the past, of course, the problem has been, that this test really needs to be done within, the day of the sample being taken. So that, until recently, that has never been the case because a lot of labs would send away and, horror stories of nothing to do with the lab, but in terms of all the logistics, two, three days or a week even to get an answer back for that particular request. You that is not satisfactory. These tests should be used, In the same hospital where the patient is. And I think that's going to change. that's my wish. Especially if, you can now test single samples. that's, I think has been a major change in our whole approach to this particular test. And, and of course, we're going to perhaps talk about this a bit later on. There's quite a lot of work. on the development of a point of care test for glucan, but I can foresee all sorts of problems there with this contamination issue, environmental contamination. So how will we get around that? And of course, what we haven't talked about really is the use of the, the glucan test for, pneumocystis infection. It's quite useful in that setting, perhaps to confirm a positive PCR test. That's how we, we use the glucan, test as well. So it's interesting that particular organism does have glucan, which is expressed from the cell wall.

Callum: 12:04

And, I guess it's a test that, can be useful in monitoring and that negative predictive value serially in patients that are high risk of fungal disease. One question I've had before is whether. Once you've had a raised one and made a diagnosis of invasive fungal disease. Once you've had a positive B2D glucan, is this a test that is, is there any value in Monitoring it, do you see it come down with treatment or is it something that just persists so long that there's no real value in monitoring it?

Malcolm: 12:40

Good point, good point. Yes, I I think if you do get a positive is to do a repeat test and see whether that can be repeated. Yes, one of the issues with this particular molecule is it does hang around for quite a long time. So whether or not it can be useful for monitoring treatment is another issue. Our problem is. we very rarely get repeat tests, and even though you might chase them up, this is a lab which gets, gets samples from all over the north of England and parts of Scotland as well, and also, to the south of the, northwest down to Nottingham and places like that, it's very difficult to track individual patients. It would be very nice to follow on, individual patients where you get a positive result. Life's not really like that, you, the lab releases a result and that's probably the last you're going to hear about it. Though of course increasingly, people do make contact to get some further explanation of what can that test mean, but obviously, in relation to all the other parameters and things that they have, information they have about that particular patient.

Alyssa: 13:42

There's a trial going on at the moment, looking at the utility of, of B2D Glucan for screening for fungal infection in haematology patients. So it's an NIHR funded study called Biodrive AFS. trial. So it's biomarker driven antifungal stewardship in acute leukemia. so it's a phase three open label randomized control trial, 404 patients, AML. and there'll be randomized one to one to control arm, which is standard of care and intervention, which is a biomarker straight diagnostic. Um, and then they're going to look at cost effectiveness and survival impact

Callum: 14:30

I put a link to that

Alyssa: 14:30

Yes.

Callum: 14:32

It's the biomed central, protocol, link. so I guess that's something to keep an eye out for, that was published in 2024, the protocol. So I guess a while till we get the results, but.

Malcolm: 14:43

Yeah.

Callum: 14:43

will be useful in looking at this in the future. So if you're listening to this podcast episode several years in the future, maybe I'll have already read the paper so maybe we could move on to talking about collect Manon next. The other big, big G for talking about the two big G's here.

Malcolm: 15:01

As I mentioned very briefly, this whole concept of circulating antigens for the diagnosis of invasive aspergillosis has been around for a long, a long time. So the early work we did in Birmingham was, animal models and then extending that to human samples. and then the whole idea became refined when the circulating antigen was characterized. as Galactomandana. That was some work carried out by Jean Paul Lachy and others, Institute Pasteur, but also some people at the CDC in Atlanta. So this is now sort of 80s. And then since, and then of course it was initially developed by Sanofi. but to get FDA registration for use in the United States, basically the trials and the studies, evaluations had to be repeated with, samples from American patients. So then, I'm not sure exactly when Bio Rad, picked this up, but since then, of course, it's been available through, Bio Rad, but there are lots of other companies that make, but I think the one that we use. In the UK is still the BIRAD. test for aspergillus galactomannan. So this is again, even though we call it the, or it's labeled as the battellia aspergillus galactomannan test, it is in many ways a pan fungal test. Again, fungal pathogens have galactomannan in their cell walls, and even if you Take, quite a high inoculum of candida, put it into a test tube, leave it, for a few hours in water, you'll detect gala, gala in that, supernatant. But still it's used primarily for, agents of hyalohyphomycosis. So that includes obviously aspergillus. It can be used for fusarium infections. We don't see too many of those in the uk. So it's, its main usage is for diagnosing, pulmonary and invasive aspergillosis. And, its utility is, in serum samples, but also it's very useful in, respiratory secretions, like BAL. We've done a bit of work looking at levels of galactomanna in sputum, but the threshold there is very difficult to establish for obvious reasons with a sample like sputum. So that's really a bit about the historical background of this particular test.

Alyssa: 17:22

yeah so Galactomannan, so as you said, it's a component in fungal cell wall, particularly. so I guess it's particularly prevalent or it's released more readily from certain fungi, such as. aspergillus and infusarian, but I guess the main role, is used for, diagnosing invasive and pulmonary aspergillosis. so it can be performed on the main sample types, serum and BAL, How does its sensitivity and specificity compare to beta-D-Glucan, for example?

Malcolm: 17:52

I think, quite, superior. I think it's, sensitivity is pretty, pretty high. but the specificity, as I've just said, really is not as, is not too great, but I think in the setting, in our setting in the UK, where in the sorts of patient groups that we do this test on, where the expectation is that these may have. Aspergillosis, as opposed to anything else. I think then, I think you can be reasonably confident if you have a positive test. This indicates, a true infection. Of course, with the caveat, again, of all the interfering factors that can give you a positive result with this particular test. And again, I'm referring to diet as one thing to bear in mind here, but also a lot of other interfering factors, which may give you a false positive result.

Alyssa: 18:39

I guess the utility of galactomannan, you need to consider your patient group as well, because it's, the levels of galactomannan detected in serum can be, affected by whether or not patients neutropenic, so non neutropenic patients can get false negatives in the, or it's less sensitive isn't it, serum galactomannan, and similarly, patients receiving mold active, antifungal prophylaxis, the serum sensitivities reduce. So I think, you just need to be mindful about its sensitivity, particularly in those groups. And then I think another important thing to say is that it's an ELISA, so an enzyme linked, immunoassay. And the result is given as an optical density. and the cutoff varies for whether that's a serum or a BAL. So I think the cutoff for a positive is 0. 5, in serum, 1 to 2 in BAL. And Malcolm, is this, is the trend in optical density? Is that a useful thing that can be used to monitor treatment response?

Malcolm: 19:46

I think generally speaking, you're right, I think if you do sequential sampling and you get a persisting strong signal from the ELISA test for a galactomannan, I think that gives you. some indication that basically the antifungal treatment is not clearing that infection there's a focus somewhere there's a sanctuary site or a focus somewhere where the antifungal drug is not being effective so I think if it's used like that I think it can be that can be very useful and of course we are aware of the fact that if you do get a high positive on a single sample that again indicates further investigations and so on and possibly even instigating treatment but also again with the understanding of false positives. It's again a ubiquitous molecule like glucan. It's found in various dietary food products, dietary products, and so on. paints, varnishes, pretty common in the environment. So again, I think it's just a case of good laboratory practice, I think, to make sure that, you're trying to minimize any possibility of, um, contamination. And of course, there's that wonderful story, isn't there, that came out, The case report came out in the New England Journal of Medicine, a lady who, had a transplant recipient had extremely high levels of galactomannan and they just could not really work out. And based on that, the patient was given voriconazole, but it turned out, I think it was particularly hot in this hospital. She was, sucking ice, little ice pots, many of them per day. And of course, as a stabilizing compound in ice lollies, galactomannan was there, and that was the reason why. So all those little stories, I think, are quite, quite interesting. just be aware of that.

Callum: 21:34

So Galactomannan, or Mannan, is used as a stabilizing

Malcolm: 21:38

Yeah.

Callum: 21:39

powder in ice lollies,

Malcolm: 21:41

But I think probably a lot of other things as well. Yeah, so just, just bear that in mind.

Callum: 21:46

yeah, okay, I did not know that. I feel like I'm learning lots of things about the importance of fungi in there. Everyday life and the industry as well, so another little top fact there for a pub quiz.

Alyssa: 21:57

And for pulmonary aspergillosis, BAL really is the superior sample to serum, is that right?

Malcolm: 22:05

Yes, I think it is. During COVID with, COVID associated pulmonary aspergiosis, I think that was really shown quite conclusively that the BAL sample was far superior to serum in picking up CAPA, in those patients. If you believe in Kappa to the extent that some centers were reporting, And so you did get some high positives in BALs. but of course that then, raises the whole question, what are you picking up? Are you picking up, heavy, heavy colonization? And we did discuss this a bit last time, or true tissue invasion by aspergillus. And again, I don't think we're really sure about that. So again, I think that does reflect perhaps sequential sampling and Quite a few algorithms now that are appearing quite recently, suggest that we, we should do sequential sampling for galactomannan. The paper that came out in CID, it's one of these accepted manuscripts. Before it was a diagnostic algorithm in patients with invasive pulmonary aspergillosis. So this is primarily from workers in Graz, in Austria. So that's really worth, I think, having a look at. I just found it this morning. It appeared on Twitter. That's my usual source of information, as I said last time. So that's worth having a look at. And there's a very nice graphic at the end of that paper,

Callum: 23:27

so, I guess those are the two big G's of fungal antigen detecting, detection. Glucan and galactamannan. there are, as you mentioned, there are other, biomarkers of fungal disease. So maybe we could talk about Serology and I know before you said that people often say serology when they don't mean serology. So apologies if I

Malcolm: 23:49

Sorry,

Callum: 23:50

up.

Malcolm: 23:51

but when I see that word I then, of course a lot of people mean they encompass biomarker detection under the heading of serology, but as you probably are aware, I don't need to tell you this. Strictly speaking, it's a reflection of the host response so yeah, talking about, so your colleague Ian Page and myself, we wrote a review a few years ago about Asperger's antibody detection, Crove Artists, that was the title of the paper. It's probably five years old now, but I think it really did try to review all the literature that we knew of. starting off with the so called precipitin test, which is still actually used quite extensively, especially in France. They really have a thing about using the precipitin test for more sort of chronic forms of aspergillosis. it's something that we don't, do now, as I said, we've moved on to the lateral flow device for IgG and IgM. And of course, along the way, people have looked at, of asperger's antibodies in the setting of IPA, using ELISA, for example. So there is a literature there, but I don't think anybody has really embraced the idea of antibody detection. even with more sort of sensitive methods for diagnosing, invasive aspergillus. And then we have the whole thing about, immunohemagglutination using red blood cells. that have been sensitized with aspergillus antigens. This goes back many years, but there are today commercial tests for this type of approach. But I think by and large, serology, aspergillus serology is used in the setting of chronic hormonal disease caused by aspergillus.

Callum: 25:35

And I guess the serology available for some other fungal things is just specific ones that are obviously a reference lab test for things like and paracoccidioides and

Malcolm: 25:47

Absolutely. You're absolutely right. Yes, these tests are used in combination with perhaps congruent fixation for blastomycosis for, as you say, for valley fever. capsule mycosis and so on and so on. the number of companies make these. I think the ones we use in the UK are made by IMMI, the American fungal diagnostic company these are available, not in every center, of course. We don't, run these tests. We usually send them either to Leeds, or to various labs in London.

Callum: 26:19

interesting. So we've talked about antigen detection and serology., Is it worth maybe just about point of care testing? So let's say you had mentioned this before. what point of care testing are available for fungal pathogens and what's the benefit of that? That

Malcolm: 26:36

Increasingly then we're reading about, we're hearing about point of care tests for a variety of fungal infections. Of course, we've been using the, crag test for cryptococcal meningitis for a long time now. And that really is the test of choice. We've had point of care tests for a while now for aspergillus, for Clactamannan, and that's the other one that really does spring to mind in terms of antigen detection, but also point of care tests for other antigens, other diseases. like histoplasma, histoplasmosis. it's a very good question. Where would you use, in the UK setting where, a number of labs now have access to collapsive anion testing, or the turnaround time of the reference labs that they're using is pretty good, where would you use a point of care test? And I think that's the big issue. So as I said, we use this point of care test for aspergillus antibody. So if we get a positive for aspergillus IgG, we would then move on to testing that sample with the immunocap to give us a numerical value. And you have quite a variety of antigen types and you're thinking of all the allergens that aspergillus expresses. Where would you use, where would we use an aspergillus, Glaxoannan point of care? we don't. I don't know whether anybody else has any experience of this. But, can't really see much use of it. the idea is, of course, is to perhaps, is to do the test once a BAL has been taken and again if you get a positive, so it's a rapid screening test, get a positive and then move on to a more sort of quantitative test like an ELISA for galactomannan. So I don't really see in our sort of settings how these tests can be used. Yeah, there was development also, let's not forget of, point of care tests, which have not really gained much traction for candida infections. And now I'm talking about not a point of care test for a molecule like mannan or glucan, but for the antibody response to enolase. Now enolase is an interesting biomarker. It's an enzyme. part of the respiratory pathway of yeast. And in 1980s, there was a point of care test for candida enolase that was published in CID by Thomas Walsh and colleagues. That all disappeared because the company, Beckman Dickerson, did not see this as a worthwhile commercial. products. So they pulled the test. But more recently, there are people now looking at the antibody response to a marker like nase. And also people are looking at the antibody response to glucan. So, we are talking about what's available at the moment, but I still think it's worth just mentioning for the future or else is hopefully going to change the availability of these tests.

Callum: 29:31

Yeah, and hopefully we'll see that move, as you say, to having it more available, closer to the patients so that they can, we can get those results

Malcolm: 29:38

Yeah.

Callum: 29:38

that's really where it's useful, isn't it? It's part of our antifungal stewardship.

Alyssa: 29:42

so fungi are difficult to diagnose. I think with, but fungal infection can be really challenging to diagnose. I think we've already, talked in detail about that. and it feels like one of the area where huge progress is being made, is in the area of molecular diagnostics for fungal infection. It's an area that I don't know a huge amount about personally, and I think, aside from using PCR to detect, pneumocystis on respiratory samples, I don't think we use much, I don't think we use any other local testing, but it feels like this is an area that's. ever expanding, and has really exciting future for fungal diagnostics. so I was hoping we could, yeah, talk a bit more about that.

Malcolm: 30:29

Again, being somebody who's interested in the history of medical mycology, I think the first, molecular tests for aspergillosis was published in 1990. So that goes back quite a long way by, Jonathan Cohen and colleagues in London. so since then we've had this enormous, explosion in the development of all sorts of test formats for circulating, DNA in BALs, serum. So many different platforms, and these are very nicely, reviewed in great detail in, the review I sent you after last time, the evolving landscape of fungal diagnosis, current and emerging microbiological approaches. So, it's really worth, having a look at that. There's a lot of very useful, information there. So you've got various platforms, various kits. I think there are 13 Asperger's PCR kits for, available. we in Manchester, we use a particular one made by a company called Elitech and we've got a good understanding now of the thresholds, the cutoffs. And again, this is in our setting, this is mainly in the area of chronic pulmonary aspergillosis because they're the patients that we see in the National Aspergillosis Center. but obviously other people have a lot of experience. So Louis White in Cardiff, has written extensively based on their experience. on the various approaches where molecular testing can be used. I think it's an obvious development over the years. Let's look in terms of, sensitivity and these tests can be made very specific depending on how you design your tests. And I think as time goes on now we're reading, very recently and hearing presentations on, plasma cell free, DNA PCR for both aspergillosis and mucomycosis. And there are quite a few talks about this at the recent ISHAM fungal diagnostics course in the US, in early December. so We're now seeing this type of approach being used. But of course, it comes down to Standards and protocols. So isham has had a fungal working group a working group on PCR the PCR initiative that's been running for a few years now, trying to optimize the type of the right part sort of sample, whole blood, serum, plasma, and so on. And of course, the various platforms.

Alyssa: 33:00

And is that one of the major challenges with molecular diagnostics that a lot of these assays, vary, the target varies, and therefore standardizing the assays can be challenging. Some of these assays are developed in house, whereas some are commercially available assays.

Malcolm: 33:18

I think it's very difficult to compare one center with another or one, one particular test kit with another. I think it's virtually impossible to make any sort of comparison. firm conclusions about molecular testing. I think the concept is very good, because of its sensitivity, but I think in terms of coming up with a standard, I think that's going to be very difficult, even though the, ISHAM initiative, the fungal PCR group, are attempting to do that. And of course, it's quite interesting, isn't it, when you think about the various international guidelines that we've had. It has taken a long time for the, the Infectious Diseases Society of America guidelines to adopt. Fungal PCR as a diagnostic test, for all sorts of reasons, I think now having been and talked to a lot of, American clinicians and labs, it's lack of availability. Now, why is that? Because, very few labs, considering the size of the country and the patient populations, very few labs do full, mycology testing. Even a big centre, for example, like Duke University Medical Centre, it's sent out. taking one or two weeks to get an answer back and so on. So I think there's all that going on in the background

Alyssa: 34:38

And what's your view on, the risk of false positives with, molecular diagnostic approaches? Because I know that they're very sensitive, but I guess that they might be, there might be risk of false positives from colonization or sample contamination.

Malcolm: 34:56

yeah, and I think also the environment where the test is being done, and we talked about that in relation to DrugA, and again just thinking about our setup, we've got, a suite of molecular labs, where the environment is controlled extremely rigidly, and to the extent that we do weekly air sampling in those labs to make sure there is no contamination from the ventilation or from other sources. So all of those things, yeah, can impact, on, and also, of course, from how a sample is taken, and also, transport. It's difficult to believe that contamination could actually arise during those steps. before the sample is received in the laboratory, but I think it's just, again, it comes back to good laboratory practice, I think, and I'd like to think that the UKAS inspections that we all have to undergo actually delve into that to make sure that laboratories are performing these tests in the right sort of way.

Callum: 35:52

And I guess an area of molecular practice in both bacterial and fungal is the sort of, ribosomal PCR. So I guess what gets termed as the 18S PCR, although I've heard that's not really technically the correct name for the test that's, that's performed at the moment at Great Ormond Street. in London. What's the sort of role for that? Because I certainly locally with that's being utilized alongside things like the 16s PCR and mycobacterial PCR, and are very difficult to diagnose cases, particularly CNS infections, where we've got like an immune compromised patient who has, there's a really wide differential and we're a bit stuck. so yeah, is it, called 18s. And what is that looking at? And is that something that we should be doing more of?

Malcolm: 36:40

Yeah, as far as I'm aware, and just as a provider, I am not really a qualified molecular mycologist. Perhaps I'm the wrong person to really answer that, what exactly is happening. But I think 18S, as far as I'm aware, is still a valid term, but I'm not absolutely too sure to give you a good definitive answer, for that.

Callum: 37:01

Yeah, I it's looking at the one of the subunits of the fungal, ribosome. And because it's highly conserved, it allows you to speciate down. And there's something called the internal transcribed

Malcolm: 37:16

ITS yes, sure. Yeah, Yeah,

Callum: 37:19

right. And using a combination of those, you can. You can hopefully different, the good thing about that is that unlike most of our PCR tests, because you're amplifying up all the genetic material and then sequencing, you are able to detect things without having to know what you're looking for, because I think that's often the problem with molecular is, you do your specific panel of the pathogens that you suspect, but, it's always the thing that you not suspect, suspecting that you're going to miss isn't it because you won't test for it and unlike culture we're just not going to find it unless you do that specific

Malcolm: 37:53

In Indeed. And of course beyond the ITS, there are other domains as well, which have been used quite extensively. But, I think you. You're absolutely right, there's always that risk of missing something. I think it's going to be very difficult to incorporate all of these targets into one test. You know, we do panfungal PCR on tissue and we find that is, that usually does accord quite nicely with culture and with histopathology. But yeah, I think there's still a lot, to be gained here, a lot of work to be done. Hopefully these various working groups can come up with a definitive answer, but it's taking a long time.

Callum: 38:28

Yeah that's a lot of sciences taking a long time isn't it hurry up and give us all these answers So many questions, particularly in mycology, it seems.

Alyssa: 38:37

Did we talk about ITS sequencing?

Malcolm: 38:41

Yeah, we've been using ITS. sequencing for identification for a long time, but since we've, a few years ago got our mold it off, it's not approached that we particularly use now, but there has, obviously has had a lot of potential for identifying, difficult to identify, fungal pathogens.

Alyssa: 38:59

So it stands for internal transcribed spacer. and is that, is that a relatively conserved area within DNA?

Malcolm: 39:08

It's not particularly well conserved, so that's why there have been other domains, other sequences which have been developed, because it's not particularly well conserved. ITS1 and ITS2.

Alyssa: 39:19

And I guess if you've got a novel fungal pathogen that you don't already have, data for on the multitask database that you're not able to identify, then that's when you'd reach for your ID.

Malcolm: 39:32

absolutely, yeah, so that's what we would do if it's a particularly, if it's an organism that has not appeared on, the commercial databases, or we have not added It to our database from previous work, then we would revert back to, the ITS 1 ITS 2 approach.

Callum: 39:50

okay. That makes sense.

Alyssa: 39:54

great. I just wanted to ask what are the most, useful clinical samples for fungal PCR? Obviously, blood is one of them. Tissue samples. I think we've even sent paraffin embedded, sometimes you don't get a tissue sample to microbiology, so you don't, you're not then able to perform, fungal culture or bacterial culture. So I know we've sent paraffin embedded tissue before for, fungal PCR if, elements have been seen on. on histology or whatever.

Malcolm: 40:23

Yeah. So we, that's what we get in the lot. we get a lot of scrolls from, or even the whole block. Sent to us from labs around the north, from the pathology department. So we find that very useful. So we do a pan fungal PCR on those and of course we also, yeah, as you said, we view that against, the histopathology slides. So that's something I do a lot of. Now, almost like a self taught histopathologist in a way. So it's nice to be able to match what with what the panfungal PCR, turns up. Yes, because quite often there's no cultural work done. And, so that's the sort of material that we have to work with.

Alyssa: 41:00

and beyond blood and tissue, I guess other sterile fluids.

Malcolm: 41:05

Yeah, sure. Absolutely.

Alyssa: 41:07

And then obviously we test in, respiratory samples and VAL. Yeah.

Malcolm: 41:11

Thing we haven't talked about, you may want to think about a complete session. is NGS. Next generation sequencing, I'm aware of all the developments and I read a lot, but I don't have any personal experience. And of course, the interpretation of the data that comes out is tricky. The BSMM is formulating, is forming a NGS group, that's with David Moyes and others, including, Rena Richardson from Manchester, and a fair number of other people. But, that's an interesting development. But the interpretation, as far as I'm aware, and having looked at some of the data, is very difficult. how do you interpret Malazesia in a BAL, for example, for a patient? What does that really mean? And you're going to find Candida in all respiratory samples. So I think it's how that data is used is a crucial thing. But I think we just have to wait and see. And as far as I'm aware, very few people in the UK are using NGS from a diagnostic point of view. I think it's a research based at the moment. I think we've touched upon most of the things which I think are where we are currently with diagnostics. And again, I would urge people listening to this podcast to perhaps access that, review article in the Journal of Fungi. It's a very comprehensive review on everything that we talked about and more. and if you have a burning interest in fungal infections as a result of these conversations, we have the Mycology 25 fungal update meeting in London in March, 14th, 15th of March at the QE, conference centre, and that's, I think, the 21st meeting. It's a nice gathering of people and it really has expanded. I think the last year was over 200 people. It's, and it's really good for trainees as well. It's pitched that type of an audience.

Callum: 43:03

And is that every year that

Malcolm: 43:04

Yeah. Every year. Yeah.

Callum: 43:05

So even if you're listening and you've missed it this year or you're too late to get the study leave, then you

Malcolm: 43:10

Oh, don't!

Callum: 43:11

go apply for next year.

Malcolm: 43:13

if you can get study leave, our hospital, I'm not involved now, but our hospital, I think, has put a block on all study

Alyssa: 43:19

Oh God.

Malcolm: 43:20

applications. And you're expected to fill in your CPD return, aren't you? Of course, get your number of points per year.

Callum: 43:28

Don't need to learn anything. Or do that in your own time.

Malcolm: 43:31

You should know it all by now.

Callum: 43:34

Great. As always, you can find the articles and guidelines mentioned in the show notes, which are linked in the episode description and your podcast player of choice. And just to summarize what we've talked about there in the second part of our fungal diagnostics episode. So we've talked through antigen detection, Specifically, B2D, Glucan, and Galactomannan, and the benefits and pitfalls of both of those tests. We've talked about serological tests, so the host response against the pathogen, including some things like aspergillus antibodies. So aspergillus precipitans as an example. And then we've talked about point of care testing and the utilization of that. And then we talked about molecular diagnostics. So, PCR for specific targets and also pan fungal 18s PCR and ITS sequencing. and As we go on through our mycology series, we'll be talking about some more of these in more detail. So I guess all that's left to say is thanks so much for coming on again and for sharing your wisdom and impressive amount of experience in this. This field. And it was really interesting to hear a bit of the history about how we got to where we are and maybe where we're going next as well.

Malcolm: 44:55

Thank you very much. Pleasure.