113. The yeasts: Cryptococcus part 1, overview and diagnosis

In this episode we are uncovering the most cryptic of cocci, Cryptococcus!

Alyssa and Callum are joined by Jeremy Day to learn all things neo(formans) about this important pathogenic yeast. In this episode we discuss the epidemiology, clinical presentations and diagnosis of Cryptococcus.

To further encapsulate your learning, be sure to listen to part 2 next week!

Show notes for this episode here: https://idiots.notion.site/113-114-Yeasts-Cryptococcus-14fd0e7ed6a2478cb5734f1e21f49029?source=copy_link

Transcript

0:00

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

Alyssa: 0:27

Hi Callum.

Callum: 0:29

Hello, Alyssa and Jeremy,

Jeremy: 0:32

great to be here.

Alyssa: 0:34

So we're delighted today to be joined by Dr. Jeremy Day. Jeremy Day is a consultant in infectious diseases at the Royal Devon and Exeter Hospital and Principal Research Physician at PROTAS, which is an organization that focuses on designing and delivering clinical trials with global health impact. So Jeremy was a professor at Oxford University, and led the CNS and HIV infections research group in Vietnam, where his research primarily focused on clinical trials for cryptococcal meningitis. and he has extensive experience of treating this disease. So we're really delighted to have, Jeremy joining us today to talk about cryptococcus.

Jeremy: 1:15

Thanks very much, Alyssa. Great to be here.

Alyssa: 1:18

So, shall we start off just talking about what Cryptococcus is?

Jeremy: 1:24

Yeah, it's a good place to start. So, it's a yeast, and there's actually about 50 different species in the genus, but only really, A couple of those cause problems for humans and cryptococcus is distinguished by having a capsule, which is an important factor that helps it to cause disease in humans and other animals. Why it has a capsule is, Not entirely clear in the environment. It's found in places like, nitrogen rich sources like, bird guano or back guano. it's also found in rotting wood and in soil, and it's probable that it's capsules evolved to help it evade, predation by free living. Amoeba in the and other microorganisms that live in the soil. And so we talk about having what we call bystander pathogenesis. So it's got these qualities that enable it to live and survive in the sort of harsh outdoor environment. But those accidentally give a couple of the species, the ability to cause disease. in humans and other mammalian, hosts, and it was first really described in, at the end of the, 19th century, and the first case of human disease was in, I think it was in 1894, and it was actually a woman who presented with, a swelling of her tibia, and she had an osteosarcoma, but she was infected with, this, mysterious caucus which, can to be known as, cryptococcus and it's, yeah, and of course it's really become famous globally, due to the HIV epidemic, and the vast majority of patients who are affected by cryptococcus around the world are immunosuppressed and the most immunosuppression is HIV infection.

Alyssa: 3:11

Yeah, that's really interesting. So I think there must have been like a huge surge of cases and real recognition of this disease. Following, patients developing HIV. but it's interesting to know that, cases have been picked up for years and years, prior to that.

Jeremy: 3:27

Yeah, and disease can manifest in lots of different ways. So, of course, the 1st case that where it was identified was a sort of osteomyelitis. But, the syndrome, it's famous for causing, of course, in is in HIV patients is meningitis. But all. Parts of the body are infected and that varies a little bit with the underlying lineage that's causing disease. So skin infections, well reported in Western Europe, particularly in France, pulmonary disease is common, particularly due to cryptococcus gatti infection. But brain infection is the most frequent presenting syndrome.

Alyssa: 4:02

you mentioned that there's kind of two main species of Cryptococcus that cause disease in humans. So can we just talk through which those are and how, and, how they differ?

Jeremy: 4:14

Yeah. So there's a number of ways that you can type Cryptococcus. And one of the first ways that it, that the species was typed, from the species causing disease in humans was using antiserum, raced in rabbits and that divided, the organism into four main serotypes, A, B, C, and D. And then there was also a hybrid serotype that would spot up between A and D sometimes. And, serotypes A and D, have become to be known as the Cryptococcus neoformans species. And there's probably two species there, Cryptococcus neoformans and, which is serotype A, previously called Cryptococcus neoformans variety Grebeii. And then serotype D, which used to be called Cryptococcus neoformans is now, and it's now really known as Cryptococcus D. neoformans. And then the serotypes B and C organisms, are known generally as Cryptococcus gatti, and they have been subdivided into probable other species, but you always have a bit of a debate about these things when you get a group of people fascinated by Cryptococcus together. The most important things to distinguish are Cryptococcus neoformans, deneoformans versus the Cryptococcus gatti species complex. Because they affect patients slightly differently. So, neoformans, deneoformans are almost entirely associated, with immunosuppressed patients, although there is a little caveat to that. And Cryptococcus gatti is a much rarer cause of human disease, but does seem to occur in apparently immunocompetent patients. can cause meningitis, but also causes pulmonary, more frequently causes pulmonary disease or patients present with pulmonary disease.

Alyssa: 5:52

Okay. And also, one of the, thinking about the taxonomy of Cryptococcus, one of the, notable things is that it's in the Basidiomycota kind of group of fungi, whereas most of our human pathogens are in, in the Acidomycota group. so it's a different beast to lots of the other human fungal pathogens.

Jeremy: 6:12

Yeah. So one of the few, one of those funny fungal pathogens that doesn't have a positive galactomannan test or BDG test Yeah.

Callum: 6:21

so Jeremy, obviously you've had a career where you've been working and seeing this clinically in other parts of the world. So just in terms of the epidemiology, I think we mentioned earlier on that it's found worldwide, but where are we seeing the majority of cases? You mentioned a couple of the different sort of clinical presentations being different in different parts of the world. And I guess that correlates quite well with the sort of HIV seroprevalence,

Jeremy: 6:45

yeah. Yeah. No, that's absolutely right. So disease is, largely driven by the epidemiology of immunosuppression around the world. So where there's lots of HIV, there's lots of cryptococcal disease, although, As ever, the tropics and subtropics are rich areas for biodiversity and for wildlife. And, I think that also plays in the higher prevalence of cryptococcal, disease in those areas. So lots of immunosuppression there, but also just a richer environment for microbes anyway, and there's more cryptococcus there. So, cryptococcus neoformans and, that has a serotype A, has a pan global distribution. there are a number of, different, lineages. We did a lot of whole genome sequencing. We sequenced about 800 isolates from our clinical trials around the world. And, there are multiple lineages and those lineages are represented in most countries, but there's usually a dominant lineage, in particular places. serotype D is largely confined to Western Europe, and then Cryptococcus gatti, is, associated largely with, tropical and subtropical reasons. So Papua New Guinea, Northern Australia, Africa. I mean, it has a global distribution again, South America. There has been a recent, well, it feels recent to me, an outbreak since, 2000 of gaffety disease in, the Pacific Northwest of the United States and Western Canada. and Gatti is an organism that is associated particularly with trees and in the tropics, particularly with eucalyptus trees. In the Pacific Northwest, it seems to be associated with sort of temperate rainforest environments. And as I mentioned before, Gatti is a disease that is more likely to cause an organism that's more likely to cause disease in immunocompetent. patients. It does, it can occur in HIV patients, but even in the tropics where there's high HIV prevalence rates, the vast majority of disease is due to neophormones. And I think that probably represents my speculation is that represents just a far greater prevalence of neophormones in the environment than GATI. And one interesting thing we found in Vietnam was that we were seeing disease in immunocompetent patients. not many, but. maybe 20 year in our hospital when we were seeing about, one to 200 cases, of meningitis a year in our HIV patients. And that disease in, apparently immunocompetent patients is largely driven by a single lineage within the serotype A, clade within Cryptococcus neoformans. why it can do that. We don't know. We did some experiments. We could show it had some differences in its virulence in some cell line models of infection that we had, but that's still a bit of a mystery really why that lineage can seem to cause disease in apparently immunocompetent patients. But vast majority of patients, as we said before, are immunosuppressed and usually with HIV.

Callum: 9:47

That's really interesting, I hadn't been aware that it could infect The immunocompetent person. So I guess, something to be aware of. Although, as you say, that's rare.

Jeremy: 9:55

So it's rare. I think it's something to think about if you have a patient presenting from the Far East or Vietnam, who has a clinical syndrome, a bit like TB meningitis, who's HIV negative, you should think about crypto

Callum: 10:08

right. Because, well, we'll come on to the diagnostics about when we might find it. Because I guess, sometimes we categorize patients into immunocompetent and, immune compromised, and then, build a differential from there, but just keeping an open mind about fungal pathogens like Cryptococcus. can I ask just a question? So for Cryptococcus gatii, as a more junior, Medic and doing the DTMH and learning about this, I just felt it was such a fascinating thing. to me, infectious diseases is all about going and asking patients really esoteric questions, which, everyone finds completely, befuddling. when you're asking like, have you recently stood under any eucalyptus trees? so the general populace seems like a mad question, but obviously, we know, and the your listener now knows as well, why we're asking that. What's the sort of exposure? I guess if you're living in an endemic area and exposed to these pathogens, but if you're, say, a traveler and how much exposure do you need? Is it quite easy to pick up? I don't know if that's an answerable

Jeremy: 11:12

So I think the vast majority of us would have an antibody response to cryptococcus near foremans globally. We're all exposed to us to it. It's around us and, it's a bit like TB, we think disease is most commonly recrudescence of latent infection, and the vast majority of people never have any problems whatsoever. In terms of GATI, what do you have to do to get it? I expect, I think it's probably rarer in the environment than near foremans, but I expect most people have been in endemic areas would have been exposed. So I don't know. I'm speculating. I don't think there's usually a clear exposure history, in terms of patients who are affected. And you mentioned right eucalyptus trees. Which is, associated with, cryptococcus gatti, and koala bears do get cryptococcal disease, along with many other animals, but, of course they live in the eucalyptus trees and they get cryptococcus I don't think it's generally because the late, because the, Incubation period is so long and disease is often related to recrudescence, at least that's the dogma. I don't think it's very easy to show, epidemiological relationships between exposure and disease, but don't let that stop you trying.

Callum: 12:34

So, so I won't ask that question anymore in my routine clarking histories.

Jeremy: 12:39

well I don't know, that's what makes my job fun, isn't it? I think you should ask me. Intrusive questions as you can.

Alyssa: 12:47

Yeah, I think this brings us on nicely to pathogenesis, so thinking that, Cryptococcus is an environmental yeast, and I guess it's infection often occurs through either inhalation or predominantly through inhalation of aerosolized and either desiccated yeasts or fungal spores from the environment. Is that fair to say

Jeremy: 13:08

that's what I believe to be this sort of infection. I don't think we really know whether it's spores or desiccated yeast or

Alyssa: 13:14

Yeah. Mm-hmm

Jeremy: 13:17

plump fluid filled yeast. But yeah, I mean, there have been cases associated with kinds of exposures, haven't there? So there was, in fact, the cases in Scotland associated with, pigeon droppings in an air venting. Was that in Glasgow?

Callum: 13:32

yeah, I guess the infection control aspects, I think that the example was, building error and pigeons getting trapped in the wall and then ventilation, allowing that to circulate to immunocompromised patients. so yeah, I guess that's the, thinking about it from travel history and that bird exposure and, I was working in another hospital and we did have certain patients reporting hearing birds flapping in the walls. And it was just an odd thing for people to say and I think it was dismissed by a lot of people. And so To, you know, move out all the wards and decant all the patients so that they could go and take all the dead pigeons out the walls, I guess hospital design is important, isn't it?

Jeremy: 14:09

I think those two cases were different species as well, weren't they? Well, there were different lineages, so

Callum: 14:15

Um, the menu's only published on it. Maybe I'll be able to find

Jeremy: 14:17

interesting. Yeah, look that up. But it's just, it. was just, yeah. I thought that was fascinating. Yeah. So, yeah. So to get back to pathogenesis, we mentioned a little bit before that this thing is not, it's not a primary pathogen. It's got what we. in the field., So it's got these features that enable it to, survive in the environment, but also to opportunistically, cause disease in humans. I think that's true for gatti as well. I don't think gatti is not a primary pathogen either, so a key amongst those factors that enable it to do that are the capsule, which protects it from being destroyed by macrophages in the immune system and, crypto, plays that game that various pathogens take, play like salmonella typhi for example, it gets taken up into macrophages and it then is quite comfortable within there and it can, and it can reproduce within the phagosomes, within the macrophages. And then there's this thing called vomocytosis, where the macrophage then expels the, its phagosome full of crypto back into the surrounding environment within the host. so that's one key virulence factor. Another key virulence factor is the ability to grow at 37 degrees and crypto actually grows better at 30 degrees. so those are two key virulence factors. what else are important virulence factors? The ability to make melanin. Melanin, has a sort of antioxidant property and probably present, protects it from, The kind of free radicals and, things that, our immune system sticks into phagosomes to try and kill pathogens. why does it have melanin? We think probably it's used in the environment as it protects it from, sunlight. And of course you can see that, melanin production if you grow it in the lab on Niger seed agar, bird seed agar, you get these dark brown, colonies and you can grow it on, Other agar that stimulates melanin production.

Alyssa: 16:09

nice picture actually on the Adelaide ology of it. Grain on bird. Bird seed go and you see the nice brown colonies and the white co compared to the white colonies of a Canada.

Jeremy: 16:19

Yeah. Yeah. Yeah. So it's a nice little test to do in the lab. So I think we've mentioned three virulence factors there that are important. There are more, and, There are more that we don't know about, and that would be something that would be fascinating to work on in the future. The other thing that we found in our work in Vietnam was that this, lineage, VM1. 5 lineage for the, which is a subgroup serotype A lineage that caused, The vast majority of disease in our apparently immunocompetent patients and have caused about 35 percent of disease in our HIV patients. they have variable virulence when we tested them in the lab. And the strains that came out of our HIV negative patients were more virulent in the lab than the strains that came out of HIV patients. But they didn't express those virulence factors I've mentioned differently. But they behave very differently in the Galerio model of, infection. And we could get those virulent strains to teach non virulent strains, or less virulent strains from the same lineage to be more virulent in the lab. And, they could then pass on that virulence to other strains, just to this one, um, uh, strain. one lineage. And so that appeared to be being driven by some kind of soluble factor. And we think that those factors were contained within extracellular vesicles that the cells were producing. So really interesting, but a lot more work to be done on that.

Callum: 17:48

Also quite worrying.

Jeremy: 17:50

I don't think, I don't think it's worrying because,

Callum: 17:53

Okay, I think we're just good.

Jeremy: 17:57

when you look at the, phylogeny, these different lineages have separated from each other tens of thousands of years ago, but we're not seeing global epidemics of crypto. So I don't think it's a recent thing. I think it's just a, it's still a rare cause of disease.

Callum: 18:14

Okay, you've reassured me.

Alyssa: 18:17

interesting

Callum: 18:17

Obviously still concerned about it, but yeah, glad it's not gonna be our next epidemic. yet.

Alyssa: 18:22

it's interesting how, features of cryptococcus that help it survive in the environment Also. our virulence, features, like the capsule and the melanization. yeah, that's really interesting to think about how that improves their survival and resilience in the environment, but also that, that translates to their virulence and the

Jeremy: 18:43

Yeah, I think that's your starting point is, as you said, how these things have evolved in the environment and they're accidentally, conferring pathogenicity, the ability to cause disease. Yeah.

Callum: 18:58

So people often say that white blood cells are, I don't know if this is true or not, but that the theory is that our white blood cells were initially amoeba and over time, or are very similar to amoeba. So I guess it makes sense that if they're, as you said earlier on, Jeremy, trying to resist. free living amoeba that would confer some resistance to our host amoeba, response.

Jeremy: 19:19

nice parallel there with what's happening with macrophages and potentially what's happening with amoeba and People were going back to look at the environment. A lot of work done by Arturo Casadevall and his colleagues in New York when he was at Einstein. Going and looking in, in the Pigeon Guano in New York and finding these amoebae and finding Crypto there and seeing how they interacted and thinking, Oh, that's a bit like what we see in human disease. So yeah, It's a lovely story, a lovely bit of biology. I think as, ID micro people were. we love nature, don't we? we love these biological stories.

Callum: 19:53

it helps you remember, doesn't it? so that's pathogenesis. So just moving into the sort of clinical, so we've touched on this as we've gone along, but repetition is always good for helping you remember. we touched briefly on what type of clinical syndromes it covers and the risk factors, we've already talked about the HIV, but particularly where your CD4 count is less than a hundred, although correct me if that's outdated, long term corticoids, post transplant, so, you hematological within the first, So a couple of months, malignancy and sarcoid. is that about right?

Jeremy: 20:25

HIV is the most important, risk for cryptococcal disease. And as you said, it's when you're pretty profoundly immunosuppressed, your CD4 count is. Less than 100. Here we're talking about Cryptococcus neoformans or GATI, as we've mentioned, it can occur in apparently immunocompetent, people. the other thing just to think about, and that we don't know an enormous amount about, but is these new, biological agents that we're using these days, we think about TP and so on, and the question is there an increased risk with crypto? And I think there, there is with the, Tyrosine kinase inhibitors with, Jack inhibitors with many of them. Cryptococcus disease has been described, although those patients are often on steroids as well. so I don't think it's entirely clear, exactly what the increased conferred risk is, but it's biologically plausible and disease has been seen and well reported in patients receiving those, treatments too. Otherwise, you've got it, solid organ transplants, hematological malignancy. occasionally other cancers use of steroids. And then the big one, HIV.

Callum: 21:29

and then the clinical syndrome, so, meningitis. Pneumonia cutaneous and other is how we've categorized it so it might be worth if we maybe could focus on meningitis first because that's the most common and maybe in particular how that maybe differs or maybe doesn't differ from other causes of meningitis. We've, recently spoken to Fiona McGill. so Jame and I had the pleasure of her talking to us about meningitis in general. So I would certainly recommend going back and listening to that. These are UK context on bacteria and viral meningitis, and we didn't really touch on fungal. So, interesting to hear how cryptococcus differs.

Jeremy: 22:05

Yeah, so we say meningitis, but we really mean meningoencephalitis. So the organ is obviously within the meningeal space, but it's throughout the brain as well. And, the syndrome that we describe as meningitis of course, is headache, neck stiffness, fever, plus or minus photophobia. And really the symptoms that predominate in, in HIV patients are headache, perhaps with fever, neck stiffness is uncommon, it can occur, but I think that's a reflection of the poor, inflammatory response and the disordered inflammatory response in HIV patients. And then of course confusion. So, in our trials in Vietnam. And across Africa, about two thirds of patients would have, confusion. Almost everyone would have a headache. Fever was fairly common, but neck stiffness was not a prominent feature. So then in terms of what else happens in, HIV associated disease, another, key, finding is elevated intracranial pressure, and we see that in about two thirds of patients. So we would measure the, opening pressure when we do our lumbar punctures and, we had opening pressures of over 140 centimeters of CSF. So we would be stacking our manometers to try and

Alyssa: 23:15

Wow.

Jeremy: 23:16

measure it within our trials. But again, about two thirds of patients would have elevated, opening pressures at diagnosis. And then as you treat the patients, you might develop elevated pressure. And I'm sure we'll come onto that later as we get onto the therapeutics. The other thing that's quite common in cryptococcal meningitis, is, visual symptoms. So some form of, visual loss from blurred vision through to blindness and in our HIV patients about 5 percent of patients would have or be left with total visual loss in one or more eyes. In HIV uninfected patients with cryptococcal meningitis, they have a slightly more florid presentation. Often the history is much longer because the disease has a more insidious onset, perhaps because their immune system is better able to manage things. So, I think the longest duration of symptoms we had reported for a patient in an HIV negative. who is HIV negative with crypto was over a year of symptoms that we could potentially have attributed to cryptococcus meningitis, like chronic low grade persistent headache and intermittent fevers. So cryptococcus gatti, as well as this, occasionally in, serotype A disease called cryptococcus neoformans can cause disease in immunocompetent patients. a more florid, a more typically, meningitis type picture with neck stiffness, but again, a very insidious onset. So TB meningitis right in the differential diagnosis. Again, in these patients are more florid immune response. So higher cell counts in the CSF, essentially indistinguishable from TB meningitis until you start doing the microbiological testing, low glucose. just to put this out there, if I could, as a sort of brain infection specialist, if there's only one test you could do for me on a CSF, it would be the glucose. Okay. And I did with a paired blood glucose, and that's more useful to me that if I could only have one test when I'm in the middle of nowhere with a patient in front of me, that's the test I want to have, because that's going to help me make treatment decisions, I think. So the other thing that's a bit different clinically about GATI meningitis and meningitis in the immunocompetent is this higher rates of visual loss. So visual loss, very common in Seaton, one of your colleagues from Scotland's work back, back in the day from Papua New Guinea, back in the late 90s showed high rates of visual loss in patients with GATI infection. And like similarly in our HIV negative patients in Vietnam who were 20 percent of diseases due to GATI and the rest is due to neophormans, we had rates of visual loss, a total visual loss in one or more eyes of more than 20 percent in survivors and a third of them were dying from disease. it's devastating for the vision. Moving on, pulmonary disease is well described globally from Cryptococcus gatti and also from Cryptococcus neoformans, presents a bit like TB, like it'd be indistinguishable from TB on the chest x ray. And, and I think if you have crypto in the chest, you need to think about whether there's crypto in the brain and you should do a lot of puncturing your patients.

Alyssa: 26:22

And generally with pulmonary diseases, so my understanding is that Crypto Caucus is in acquired through inhalation, and then you might either get like a primary pulmonary infection or. Your immune system clears it or it, establishes latent disease. or latent infection that can then reactivate when patients present with pulmonary cryptococcus, is that more a kind of primary pulmonary disease as opposed to a quiescence?

Jeremy: 26:52

Yeah, it's a really good question. I'm not sure we, I'm not sure we know the answer. I expect it's a mix, a mix of those. If it was Gatti and you asked me to put money on it, I would suggest it's more likely a recent.

Alyssa: 27:03

Okay.

Jeremy: 27:05

Certainly with Nia Formans, We think it's, we think it's generally, certainly in HIV patients, it's recrudescence of old infection because patients take their strains around the world with them. So, this work really was, came a long time ago from looking at immigrants in France who, if they were from sub Saharan Africa, they were more likely to have serotype A disease. And if they were French HIV patients, they were more likely to have serotype D disease.

Alyssa: 27:32

And it's thought that that acquisition often occurs quite early on, isn't it? Like in childhood and then. So,

Jeremy: 27:41

it's all around us. Yeah. and then, otherwise, the skin disease can occur and this looks like little, ulcerated disease or can start as a pet pill and you sometimes see that in as part of disease in meningitis as well. So if you're HIV patient has a pet pill that then ulcerates, Certainly in Southeast Asia that we would be thinking about crypto disease or T mycosis, formerly known as Posis. In France, there's quite a lot of, skin disease reported, which is due to serotype D and which, may in part represent inoculation as much as essence. but yeah, I think the other thing just to remember is that, these ancient infections, um, TB, syphilis, crypto, they break mimics. They can cause disease anywhere you want. And as we know, with the first identified case of cryptococcal disease, it wasn't meningitis, so it can cause disease anywhere, really.

Alyssa: 28:37

Mainly, it has this predilection for the CNS, the main disease that we're going to be seeing is this cryptococcal meningoencephalitis, which has varying presentations depending on level of immunosuppression and whether it's cryptococcus neoformans or gatii, and then pulmonary disease. and cutaneous disease, which can either be more representative of localized like inoculation or associated with more disseminated disease in association with meningitis. but it can affect any body site, but I guess those are the most common. Okay.

Jeremy: 29:19

our HIV patients, essentially all of them are blood culture positive at presentation. And when you start antifungal treatment, those blood cultures rapidly become negative, but they're nearly all blood culture positive.

Alyssa: 29:34

That's really interesting because my understanding is that. blood culture positivity in disseminated disease was quite rare and was actually, is quite a poor prognostic marker. So that's interesting to know that the majority of yours were Yeah.

Jeremy: 29:50

I don't know if that's a reflection of later presentation to hospital in low income settings where you're paying. For everything, but certainly in Vietnam and my, colleagues who work in Africa and are African patients, we had very high rates of blood culture positivity

Alyssa: 30:05

Okay.

Jeremy: 30:06

patients. Yeah.

Alyssa: 30:08

Super. So I guess that brings us on nicely to diagnostics and we've already talked about the importance cSF sampling what's your, favorite, Cryptococcal diagnostic

Jeremy: 30:20

Well, despite its similarities with TB in terms of presentation, that's pulmonary disease or meningitis, the great difference is that it's much easier. to identify the organism. So TB is such a struggle, to show organisms in sputum or on CSF smear. You need highly trained and highly experienced, lab scientists with the time to spend a long time looking at a smear. Crypto is easy to spot. we don't even need sophisticated stuff to do that. A microscope slide, a cover slip, a bit of ink doesn't really matter. what color it is, and it will show up in, in fact, it will show up with any stained crypto really. and we counter stain, look at it down the microscope and you can see these beautiful cells with their capsules, their budding, their oval, their spherical. And you can see the vacuoles inside the cells, this very characteristic appearance. So, in terms of what's to, if I could only have one test, that would actually be one of these fantastic antigen tests that have been developed over the last, 15 years or so. I think Emmy was the company, didn't invent antigen testing, but their test is super sensitive and it's a lateral flow test, a bit like a pregnancy test. And, just a brilliant test, super sensitive, super specific. And probably, more sensitive than culture and smear in terms of diagnosing works CSF and, credit to them. They sell it for 2 a test in low income, a very low amount in low income countries. So, a brilliant test, that has made the diagnosis easy. You can do this test at the bedside with a patient. and show that your patient has a cryptococcal disease. Otherwise of course, blood culture, as we mentioned earlier, is useful if you, and if you feel for some reason you can't do a lumbar puncture, maybe your patient's got a sixth node lesion or something and you're anxious about doing a lumbar puncture. But the gold standard, of course, is to obtain CSF we would do, our usual microscopy with our stains, including, an India ink stain, and our antigen testing and put it up for culture. And when you do your lumbar puncture, patients have, a normal or elevated protein. a normal or low glucose, and some white cells and often not many white cells whatsoever. And those are probably lymphocytes. So the differential diagnosis would given the history, which is usually that of a sort of insidious onset. And I think the median duration of symptoms in our patients enrolled in our trials was about two weeks, is very similar to how TB meningitis might present. And that's a great challenge in low resource settings is trying to distinguish, cryptococcal meningitis from TB meningitis and also co infection. And we would see co infection, In about 1 in 200 of our patients, we would prove TB infection and Crypto infection through culture., Of course, the great advance has been improvement in a diagnostic test, both in TB and in Crypto. And, And of course, now with these modern PCR based, uh, multiplex diagnostics, I think some of them have crypto, crypto diagnostics on there as well,

Callum: 33:38

I mean, when you're culturing this, so you said earlier on that it grows better at 30 degrees, because I think generally when we're culturing fungi, we would set up plates and culture them at two different temperatures, so at 1337

Jeremy: 33:50

Yeah.

Callum: 33:50

on a Saburo dextrose agar.

Jeremy: 33:52

Yeah.

Callum: 33:53

Um, so. Is there anything else that, that we should, any other special plates that are used? You said it's quite easy

Jeremy: 33:59

Uh, it'll grow. It'll grow on anything. Unless you want an antifungal. Unless there's an antifungal in the agar, of course. But yeah, it'll grow.

Callum: 34:10

It's a blood egg or anything like that. Wow. Okay. Well, that's a good change from TB, And then I guess when they grow, they look, they like cream colored,

Jeremy: 34:18

Yeah. Creamy brown, smooth, uh, sometimes quite mucoid looking colonies, varying sizes. it can take, you may not see any growth after 24 hours. Just hang on, keep it in there. We usually boil our colonies up for a week. Because some of them are, but some of them are quite slow growing

Callum: 34:36

okay, that's an important point. So keep them for a bit longer, because I think a lot of labs, when you're looking for yeasts, will go for about five days or 48

Jeremy: 34:45

for, I don't think 48 hours is long enough.

Callum: 34:47

So I guess the key is telling the lab the travel history and, and that you're suspecting it so that we can do these, do things slightly differently. And then you mentioned the bird seed agar, so I've popped a link to the Adelaide Mycology website in the show notes, and there's a really beautiful image. They look very similar to the candida on the plate, apart from this differentiation. I guess that's useful. But then microscopy differentiates, so there's that classic appearance, of the large capsule. So I guess if you see it in a blood culture, it would be pretty obvious as well that it's

Jeremy: 35:17

if you stain it outta CSF for blood, it will have a big fat capsule on it. If you've grown it on blood a or sub, it may not express its capsule at all. So don't cut off by a lack of a capsule, when it's been grown on another plate. So, I think there's a picture in the, in your notes of, which I presume will be available to everybody. Is that right

Alyssa: 35:36

Yes. Yeah. For

Jeremy: 35:41

of CSF or grow on a plate that induces capsule production.

Callum: 35:48

And you said any ink will work, so India ink is like this sort of purplish ink, but I think you just take it out of a Parker pen? Is that, am I getting mixed up

Jeremy: 35:57

it doesn't, I, it's just a counterstain. It just has to be dark.

Callum: 36:01

Right. Because it's this mysterious thing, India ink, when I first learned about it, it must be some special stain, so that's interesting to know, just to get dark.

Jeremy: 36:09

call it India, an ink in India, I don't think they call it something.

Callum: 36:13

Do they call it the UK

Jeremy: 36:15

they're somewhat calling it China ink or something, I think, but I might be wrong.

Callum: 36:20

We'll do a on the origin of the naming of the ink,

Jeremy: 36:23

principle is just counter saving. So yeah, you could use your fountain pen. I never travel without a fountain pen. In case you come across some strange piece of India ink. That's what I

Callum: 36:35

I feel like that's the sort of story you'd read in a paper, my mycology specialist doctor diagnoses cryptococcus on plane using fountain pen. real wilderness medicine,

Alyssa: 36:45

And then once you've got your cultured isolate, we talked that, I guess looking at it on the plate, it tends to be more kind of muco than Canada. If we use a different, either like bird seed data though, you can see the mein, which Canada doesn't have. But I think that the most reliable way of, speci it now is probably. Moldy tuff.

Jeremy: 37:08

Yeah.

Callum: 37:08

When you have access to it,

Alyssa: 37:09

pudding when you have access to it.

Callum: 37:11

When you, I guess, yeah, yeah. This is a luxury tool, isn't

Jeremy: 37:14

I mean, there are classical ways of distinguishing between Gatti and Neoformant, which is to go on CGB, Agar, and I think the Gatti turns it. blue and the neoformans doesn't

Alyssa: 37:26

So is that like a cremogenic?

Jeremy: 37:28

yeah, it's a chromogenic that is that it's converted by an enzyme that Gatti produces that the neoformans doesn't, canine glycine bro. Blue Canna Glo.

Callum: 37:39

Cool. Never heard of that before.. So we were just talking about identification. So, in histopathology, I guess sometimes if it's not suspected we might find this in histopathology,

Jeremy: 37:51

so just adding in silver stains for fungi would make it clearer and, yeah, and you would expect to see, the with capsules with budding yeast oval to spherical cells, very rarely disease has been described due to capsule deficient cells or at least cells that can't be bothered to express their capsule in immunodeficient people. Absolutely tiny handful of case reports of disease due to apparently a capsular strains,

Callum: 38:21

then we've put in antimicrobial susceptibility testing here,

Jeremy: 38:25

the two main methods that are used are the CLSI protocol from the US or the ucast, methodology. There's also some commercial tests available, like the yeast one, which are a lot less of a fiddle. These are broth, micro dilute,, tests. And the used one is, has a color indicator, which is nice. So you can Kate your strains and you can measure their. Susceptibility there really is no role for it in at the point of diagnosis because we measured susceptibility at the point of diagnosis has no impact on predicting outcome to treatment. So susceptibility testing is sometimes recommended in patients who've had a relapsed disease and then it might alter how you treat but in the first instance there's no there's really no benefit.

Alyssa: 39:08

Okay, that's interesting. Okay.

Callum: 39:15

Start treatment empirically, I think when we have had cases we've asked for susceptibility testing, but that's really interesting to hear that

Jeremy: 39:22

We've published on it Lucy O'Connor was the first author. It's in CID and we've shown it has no impact on outcome and it has no, this is, and these are data from randomized controlled trials, so the high quality data with patients sampled, at the same times, not only does it not have any impact on clinical outcome, which of course is the most important thing, but it has no impact on the rate of clearance of use from CSF either. So I think that's reassuring that you are probably. don't need to do it. It's just not going to help you make a good decision about treating that patient. What's really important is that you choose the best combination of drugs that you can based on data from RCTs, which I'm sure we're going to come on to and that you get them into your patient. Yeah.

Alyssa: 40:09

Yeah.

Callum: 40:10

And why do you think that is? Why do you think it has no relation between the AST data and the, is it because of the capsule interfering with how that works in vitro or,

Jeremy: 40:20

Really good question. I suppose the most obvious answer is that our model of testing is no good. It's not a good model of what's happening in humans. And I think, I kind of believe that because if you look at, amphotericin in vitro, it kills crypto rapidly., But if you look at what happens to the patients, well, we clear their blood, the blood counts has become negative rapidly in the order of three to four days, but the CSF takes care of it. age is to become negative. half the patients are still CSF culture positive two weeks into treatment. And I think it's telling us something about how, where our brains are going in the body and how the yeast is evading drugs and immune system and so on. So I think, the model that we have for susceptibility testing isn't very relevant to what's going on in the brain. Caveat about that testing is that in relapse patients, then it is felt that there may be a role for susceptibility testing in patients with relapse. I don't know if that's going to make a difference, but that's probably where we would reach for

Alyssa: 41:29

Yeah. Okay.

Callum: 41:30

that's that's huge. This is not something that I really have experience dealing with, so that's a really important point, So, I think now we've learned so much there. I'll maybe just summarize what we've talked about so far. And we're going to have a slightly, new format. Or you might say a new format, this episode. Where we'll split off the treatment. and have a bit of a gate, between the two parts. So hopefully that discussion has made this caucus a little bit cryptic. We've talked about the, We've talked a little bit about the, what the Crypto Caucus is, its taxonomy. Why, it's pathogenic mechanisms and how it evades immune response, where about we find it, um, the differences, between the two, the sort of different, serotypes. we've talked about the risk factors, particularly HIV but many others. And then we've gone through some of the clinical syndromes as well. we talked about the pathogenic mechanisms, which were the capsular polysaccharides, growth at 37 and melanin production. And then we talked about the clinical syndromes which were the Cryptococcal Meningeal Encephalitis, Pneumonia, Cutaneous and then other sites. With our main differential being TB Meningitis or TB in other cases. And then we talked about the lab diagnostics in particular the crags of the Cryptococcal Antigen and how useful that is. CSF sampling, and if you can only get one test, it would be the paired CSF serum glucose. But other things are important, like opening pressure and protein. And then we talked about the appearance in direct microscopies, or a halo sign. Culture, looking like an agar, best at 30 degrees. Molditoff, and a little bit about the role of antifungal susceptibility testing. any closing thoughts on that section, you'll get a second chance at closing thoughts, so there could be closing thought one. so Jeremy, any closing thoughts on that section before we move on

Jeremy: 43:20

I think you've enabled us to segue very easily into treatment.

Callum: 43:25

Oh, okay, well we'll join you next week for our discussion of management of cryptococcal disease. Thank you both Jeremy and

Jeremy: 43:37

Thank you.

Alyssa: 43:38

Thank you.

43:40

This Mycology series of episodes is sponsored by the British Society for Medical Mycology. The 59th BSMM annual conference is due to take place in Norwich from the seventh to the 9th of September, and aims to bring together clinicians and researchers in the field of medical mycology. Registration is only two 40 for trainees and includes accommodation and meals. Abstract submission is now open and we welcome abstracts on research, clinical cases and auditing QI projects in medical. Visit www.bsm.org to register and submit your abstract. Now that the episode's done, we hope you learn, had lots of fun. So go forth and treat people with some of what you now know.