Hi everyone. Welcome to Febrile, a cultured podcast about all things infectious disease. We use consult questions to dive into ID clinical reasoning, diagnostics and antimicrobial management. I'm Sara Dong, your host and a Med Peds ID doc. Today's episode of Below the Belt is led by a team from Australia. First up, we have Dr. Morgan Hui. Morgan is a basic physician's trainee with an interest in infectious diseases.
He is currently working as an ID registrar at Peninsula Health in Melbourne, Australia. Hi, it's Morgan. Very keen to be here today. Dr. Jonathan Darby is an infectious diseases and general medicine physician. He is the head of General Medicine at St. Vincent's Hospital, Melbourne, and an Associate Professor at the University of Melbourne. Hi. It's great to be here with you today.
Dr. Max Olenski is an infectious diseases and general medicine physician with an interest in tropical medicine and infections in immunocompromised hosts. He is the current Perioperative Medicine Unit Clinical Lead at Peninsula Health and a Sessional Academic with Monash University in Melbourne. Hi, it's Max Olenski here. Thanks so much for having us today. Really excited for this podcast.
Dr. Catriona Halliday is the Principal Hospital Scientist in charge of the Clinical Mycology Reference Lab at the University for Clinical Pathology and Medical Research and New South Wales Health Pathology based at Westmead Hospital in Sydney. She is actively involved in teaching both scientific and medical staff in medical mycology, and has a strong interest in culture independent tests to aid in the rapid diagnosis of invasive fungal infections and antifungal drug susceptibility surveillance.
Hi, it's Catriona Halliday, and nice to be here too. Wonderful. Thank you guys so much for coming. We are just gonna quickly start by asking you to share a little piece of culture, because we call Febrile everyone's favorite cultured podcast. So really just sharing a little something non-medical that you like, whether that's pop culture or hobbies, um, or other interests that you have. So what have you guys been enjoying recently?
I had the pleasure of going to see recently for International Women's Day, I went to see the RBG: Of Many, One, um, production about Ruth Bader Ginsburg's life, which was fantastic. It's one woman show, I think it's tour of the states, uh, and it's come back time and time again in, in, uh, Australia. That's cool. I haven't seen that. I was gonna say that in recent times, my daily routine has, uh, incorporated the New York Times puzzles.
I, I sort of churn through them every morning while I'm having my morning coffee. And also I've been doing the cryptid crossword of late to try and get my creative juices flowing. Excellent. Love a good puzzle. Uh, Morgan. Uh, since Covid, I think I've been picking up golf after lockdown. Um, not that I play well, but it's enjoyable to get outdoors. And then in the same vein, uh, I've been watching a lot of golf, YouTube, especially being between studying. It's very easy watching.
Um, and it's just something to turn my brain off at night, which is quite good. Yeah, the turning your brain off is usually a, a common theme on people's suggestions. Um, and rounding us out, Jonathan. Well, I saw you like travel and food. So yesterday at weekend Australian time, I said to my kids, I'm gonna cook dinner, and they requested bao buns, so I made bao buns yesterday, which was good fun. Labor of love takes most of the day, a lot of work. was very nice. Yeah, very nice. Amazing.
I love it. Well, thank you guys for sharing. Um, all right, well, Morgan is in charge today and is gonna take us through the case. So I'll hand it over. Thank you. So today, our case involves a 49-year-old male who presents to the renal outpatient department with a four month history of a slow growing, painless right elbow lesion. He's adherent with his immunosuppression and a systems review is unrevealing.
His past medical history includes a renal transplant in 2017 for primary focal segmental glomerular sclerosis. His pre-transplant assessment did not reveal any infections for which prophylaxis was afforded. His early post-transplant course was complicated by pulmonary aspergillosis and he was treated with a six month course of voriconazole with clinical, biochemical, and radiological resolution.
At the time of seeing him, he has a stable allograft function, currently on low dose prednisolone, everolimus, and tacrolimus, and his comorbidities include well controlled hypertension, dyslipidemia, steroid induced osteoporosis, and GORD (gastro-oesophageal reflux disease). On examination his vitals were normal alongside a normal cardio respiratory exam. His abdominal examination revealed a non-tender right lower quadrant mass underlying a hockey stick scar, consistent with a renal allograft.
He has a 1.5 centimeter non-tender mobile lump on the lateral aspect of his right elbow, not tethered to the underlying skin without notable discoloration, overlying skin changes, induration, nor sinus. There were no other masses on examination of his lymph node stations. At this point, is there anything else that you'd want to ask our patients and what differentials do you think that you'd entertain? Thanks Morgan.
So just to summarize, this is a middle aged gentleman who's a renal allograft recipient with stable function on immunosuppression. His post-transplant course was complicated in the early stages, uh, with pulmonary aspergillosis, but he seems to have recovered from that with therapy, uh, and presents now with an incidental painless lump on his elbow. Some important questions that come to mind really are clarification surrounding exposures.
Um, for instance, what he does for work, what sort of hobbies he has, um, has he had any animal contact, or, a little bit more about his sexual history. Uh, what else is important would be, um, whether there are other locoregional infections or constitutional symptoms of note. The differential diagnosis for a painless lump in a transplant recipient runs the gamut from a foreign body reaction to things like a lipoma or other soft tissue neoplasms, and this includes lymphoma.
But considering this is through the lens of an infectious disease approach, I think it's important to entertain various sorts of infections. And likewise, we'd entertain things like run of the mill bacterial skin, soft tissue infections, like a furuncle or carbuncle. But syphilis and cat scratch disease also appear amongst the differentials.
Considering a significant history, fungal etiologies need to be entertained, and this includes disseminated aspergillosis alongside sporotrichosis, and, um, more rare things like mucormycosis or rhizopus infection.
Um, and then the fine print would be things like mycobacterial infection, viral or parasitic infections, things like TB or non tuberculous mycobacteria (NTM), um, or cysticercosis or trichinellosis but those sort of things would really come to, uh, the fore if his, um, exposure history was compatible.
So taking our case a bit further, his social history revealed that the patient immigrated from the Philippines to Australia 18 years previously, where he returns to visit family and friends every six to 12 months, but not in recent times. He's in a long-term heterosexual, monogamous relationship and works in maintenance at an aged care facility.
He's a sporadic gardener with no additional epidemiological exposures, and does not report any recent animal nor insect contact, nor recent locoregional infections. He consumes no alcohol nor smokes tobacco. On further history when pressed, he thinks that he might have had the onset following a seamlessly innocuous traumatic injury against a doorknob without skin breach, and no set associated systemic upset.
His routine lab results were unremarkable with white cell count, LFTs, and inflammatory markers within normal limits, a stable renal function compared to previous, a non-reactive RPR and a negative QuantiFERON gold with a good mitogen response. Ultrasound sonography of the lesion revealed two small circumscribed ovoid masses suggestive of reactive lymphadenopathy.
This was clinically corroborated upon surgical review and was resected en bloc with tissue set up for histopathological assessment alongside routine mycobacterial and fungal cultures. No organisms were seen from the Gram and Zeihl-Neelson stains, nor subsequently cultured . However, filamentous fungi were noted on fluorescent staining. Histopathological examination revealed multinucleated giant cells, and possible copper penny, aka Medlar bodies.
In addition, there was a suggestion of a brown micro colony of hyphal elements with suspicion for an underlying deep mycosis, and the specimen was sent for further culture and identification. I guess, now, what do you think is meant by deep mycoses and how are they differentiated? Yeah. Thanks for that update, Morgan, on the case. Um, to, to just take a step back. This was a subcutaneous lesion and it, it didn't have any changes on the skin, which would make us think of an inoculation injury.
But we were certainly taken by the history of his previous Aspergillus infection in the lung and wondered if this was a site of disseminated infection. And there is, as you've pointed out, Max, a broad differential in a immunosuppressed transplant patient with any lesion. But it was an unusual site. It was an unusual appearance with relatively few other additional symptoms, just that he brought this mass like lesion to our attention.
And so to be honest, I hadn't really used the term deep mycosis in clinical practice before. Um, I know it's, uh, been raised here and has been talked about in, in some literature, but really when we think about these lesions, we have a broad differential from, from fungal to atypical mycobacteria to other organisms which may be indolent and slow growing in a transplant patient.
But this suggestion here with the, the pigmented nature of the fungal elements certainly makes us concerned about a group of fungi, which we classify in the dematiaceous mould group. Um, this is an unusual finding, so this doesn't happen every day where you find this on a biopsy and sometimes has been found in further other organ sites such as brain or lung or subcutaneous skin and soft tissue.
So the, the overarching term we would sort of use for this would be a phaeohyphomycosis, if it is related to a pigmented mold. Actually had to look up the word "phaeo", 'cause I've always wondered where that came from. And, and that's, uh, Greek word for dusky. So gray or, or black forming. And this is thought to be the melanin in this group of fungal infections' structure that causes that, that appearance. So there's a few terms we use here.
And then the other term that we would consider is this term of chromoblastomycosis, where a chronic infection, usually due to inoculation but it could be dissemination in an appropriate host such as this with dissemination. Um, there is a well-known entity in the tropics where these patients have a crusted verrucous like lesion on the skin with chromoblastomycosis. And there's a range of specific species which are fairly geographically distinct depending on where a patient may have spent time.
And so with this individual, whilst living in residential suburban Australia, we did note that his history is from the Philippines and traveling back and forth there fairly frequently with some sparse gardening. So we were concerned about this family of organisms from that basic microbiological description before we had a formal culture and identification from our mycology reference laboratory.
The next step in his workup was the sterile tissue was sent to a diagnostic mycology laboratory for specialized fungal cultures. Uh, from here, Catriona will take us through exactly what happens in the lab to get our diagnosis. Thanks very much Morgan, and thank you Jon for that nice history and my, I've now learned what phaeo means in the phaeohyphomyosis. I've got a case at the moment, so that's good. Um, okay.
So when the sample was sent to us for culture, we already knew that there were fungal elements seen histologically . When we receive samples in the fungal lab, it's really important that any tissue samples aren't actually ground up.
We, we just make them into small pieces and put them onto the various agar and, um, we don't grind them up because if this fungus was likely to be, or we sometimes we don't even know if it will be, a mucormycete, this particular group of fungi are really, really fragile. Um, and grinding would destroy the hyphae and, and so the organism would be non-viable. So as a rule, no tissue samples are ground up in a mycology laboratory.
So these, sterile pieces of tissue were put onto a variety of different media. We usually use a couple of different media, a very general purpose media with no antibiotics such as Sabouroud dextrose agar, and then we use more nutritious media such as a Sabouraud dextrose agar base, which might have something like brain, heart infusion and some antibiotics like chloramphenicol and gentamicin in them to suppress any bacterial growth.
So following inoculation, we then use plates, but some labs do use slopes. The plates, we seal them with parafilm, and then we put them in an incubator at 30 degrees, rather than 35 degrees, which is what would be used for most of the bacterial growth. And 30 degrees, I think most fungi are environmental organisms and they just do better for growing at a slightly lower temperature. We keep these plates for four weeks and look at them every couple of days.
And so the reason they're para filmed is, is both to, to keep the lids closed, but also prevents dehydration when they're put in the incubators for, for quite a long time. So that's the reason we use that lower temperature. And, in this particular case, after about 10 days, we noticed a small amount of growth of a, of a dark gray fungus growing directly out of that tissue. So as soon as we get positive culture, we would then always work in a biological safety cabinet.
We would then put that organism and subculture it onto a general purpose Saboroud agar with no antibiotics in it. And we'll also prepare a slide culture by inoculating a potato dextrose agar plate. In order to identify fungi in the conventional way, you, you really need to see how those fungi are growing in their natural state.
And so for that, you, you can use macroscopic appearance, but the macroscopic appearance can change depending on the media that you might be using, how much light there might be exposed, you know, to, and things like that. The macroscopic appearance can be a bit of a guide, but that microscopic appearance is what we need to identify something to the genus and ideally the species level. So you need to use a media such as potato dextrose agar to encourage that sporulation.
Other media that could be used for microscopy to prepare slide cultures is cornmeal agar, which is probably a little bit less nutritious and more encouraging of sporulation. So in this particular case, again, we grew a very gray, velvety fungus with radial grooves. It changed and became a bit darker with age on the Sabourouds plate. And there was a bit of a dark, exudate or, droplets forming directly outta that colony.
The reverse of the colony was also dark, so the fact that it was dark on the reverse as well as on the surface suggests that the organism does contain melanin, which fits with what we've seen histologically. Unfortunately the microscopic appearance of this fungus from the slide culture was not very helpful. We just got hyphae. We didn't see any conidia forming, and so if you don't see conidia forming out of that hyphae, we can't identify it using conventional methods.
Fortunately, we have the ability to overcome this problem and perform DNA sequencing, which is, is actually now considered the gold standard for identification of fungi. There's a couple of different barcoding genes for fungal identification that we rely upon, the internal transcribed space region, as well as some organisms might identify better with something like the elongation factor gene.
But for this particular case, we used DNA sequencing of the internal transcribed spacer region as well as the D1/D2 region of the 28S ribosomal DNA. We did two different PCRs. We sent that pCR product off the sequencing and ran the sequence results against gen bank data databases using a, a BLASTn algorithm. And I'd never had encountered the answer that we got. But the answer we did get from both the ITS sequencing was 99.4% identity to an organism called Falciformispora lignatalis.
And I might have butchered that pronunciation, but I'm happy for someone else to correct it. Um, and then the next closest match was another species within that Falciformispora, uh, species, but it was further away at only 96% identity. So we were pretty confident that the organism we'd come across was this F.lignatalis.
We did try and do antifungal susceptibility testing for this patient, but again, with antifungal susceptibility testing, you must have sporulation and, uh, we didn't manage to induce sporulation and therefore we weren't able to perform antifungal susceptibility testing in this particular case. Was it far enough away to call it catrionelis or not quite?
It makes me feel better that you also doubt your pronunciation like, never come across this organism before and I'm yet, but I, and I haven't even come across that genus before. So, Thank you. Thank you so much. Uh, I guess what do we make of this and the significance of this organism? Would we call it a deep mycosis? Yeah. And is it how this syndrome typically manifests itself?
Um, well, as we've said already, uh, this is an unusual organism and I think it's been implied as well that the world of mycology is forever changing and it is difficult to keep up with this kaleidoscopic landscape of fungal nomenclature. But this syndrome is indeed, um, consistent with what we call mycetoma. And whilst I am no budding mycologist, uh, I have done my research and we'll talk a little bit about mycetoma now.
It is a chronic granulomatous subcutaneous infection caused by several species of fungi as well as soil inhabiting bacteria. Which is endemic within this so-called mycetoma belt, um, which spans from 15 degrees south to 30 degrees north of the equator and really within the tropics of the world. Sporadic cases have been reported worldwide, including in temperate regions, and it's really quite rare in Australia.
And when it does occur, it occurs in the northern states and territories, which approach the tropics. We make a distinction between actinomycetoma and eumycetoma, the former being caused by soil inhabiting bacteria, and the latter from fungis, such as in this instance. And the typical presentation is usually with the triad of tumour, uh, sinus tracts, uh, and macroscopic grains, which are essentially colonies or aggregates of the infectious organism.
Uh, it can extend to adjacent structures including bone, muscle, lymphatics. And the classic description from a long, long time ago was that of Madura foot, named after a region in India called Madura, where people would've stepped on this, these fungal organism, and it was quite disfiguring.
Risk factors as you might gather are typically environmental or related to occupation, and thus it had previously been known or sometimes is known as an implantation mycosis, uh, and risk factors also included, such as in this instance, states of immunocompromised, whether they be inherited or acquired. The main age for this sort of syndrome is in the thirties and typically occurs in males with changing epidemiology based on an itinerant population in other regions around the world.
It usually involves the, the feet, as an implantation mycosis where workers might be not wearing shoes and exposed to thorns and other things within the soil, and less likely to occur in parts of the torso, the arms and other parts of the body. Common organisms for mycetoma itself, or eumycetoma I should say. Madurella mycetomatis, which is the, the classic one, but it's worthwhile noting that eumycetoma itself is the minority of cases of mycetoma.
It's 35% of cases of mycetoma across the board and within eumycetoma, Madurella mycetomatis, uh, accounts for 75% of eumycetoma. Followed by Falciforma species such as the other ones that Katrina had identified as being similar to the Falciforma lignatalis which was identified in this case. And they tend to differ by local epidemiology including climate, vegetation, rainfall, and, and different soil types.
Little is known about the incubation period between inoculation and clinical manifestations, given that many patients do don't recall a specific predisposing injury, such as in this instance. It's worth noting that there are atypical presentations that typically affect immunocompromised hosts, whereby the classic triad might not be present. There are numerous case reports, uh, out there that highlight a link with the tropics in transplant recipients or those who are immunocompromised.
For instance, patients who have migrated from their country of origin to a place that is not within the Mycetoma belt, and some 40 years later receiving a transplant and then having this fungus identified from various parts of the body. But there's yeah, um, really quite interesting cases out there. So beyond clinical suspicion, if someone came in with a similar presentation, how would you go about diagnosing mycetoma?
Uh, it's hard not to have a talk like this without saying that clinical impression and an index of suspicion are paramount. Uh, and they are. Um, so the presence of that triad are certainly things that would give you an indication, but beyond that, in terms of clinching the diagnosis, it's a composite of growing the organism, and pursuing culture. Whether that be with a fine needle aspirate of the growth, wherever it may be on the body, uh, with subsequent inoculation under plates.
I might ask you at this instance, Catriona, I know you mentioned that you would tend to culture these only at 30 degrees, but from my reading, sometimes these get inoculated at both 30 degrees and uh, 37 degrees for a number of weeks because different organisms within this eumycetoma syndrome tend to grow predominantly different, uh, temperatures. Is that fair to say? Or something you don't always do? Yeah, it is a fair enough question.
I guess we're guided by standards for recommending what conditions we do grow fungi at. And certainly the guideline we used is the CLSI guideline, which the people in the states would be very well aware of and, and they make the suggestion that whilst you can put things at two different temperatures and some, some labs do put things at two different temperatures. Every time you put more and more plates out, you dilute how much sample you actually have to be able to try and, and grow things.
So for fungal work, the general recommendation is you can just use 30 degrees. Uh, but, you've gotta remember, there's also plates that get set up for bacterial cultures, and there's no reason why many of these fungi wouldn't actually grow on some of the bacterial plates as well, and that is incubated at the higher temperature. So we have in my own lab times when we, we get something growing, but it's also growing on the bacterial plates as well. Great, thanks for that.
So, beyond culture, the other things that are in establishing a diagnosis include histopathology with evidence of chronic granulomatous reaction, and possibly the presence of a grain, which given how rare it is, ought to be discussed directly with histopathology at your own lab.
Um, given that this has not seen a great deal, and indeed when we returned to the histopathologist and asked them was that bunch of fungi you saw in a conglomerate consistent with a grain, and when they look through textbook, they confirm that indeed was the case. So, uh, good have good relationships with the various disciplines around the hospital.
And imaging is used sometimes to determine extent of disease, whether it's invading in surrounding tissues, uh, but also to suggest whether or not, there may be improvements to help guide duration of therapy. I might ask Catriona, just lastly, is there anything in particular, you would say about sensitivities?
Um, I know we weren't unable to achieve sporulation in this instance, but given this is , this was a rare fungus and something you hadn't quite encountered before, clinically, um, or at a lab level, if you were even able to achieve sporulation, would there be much to be gleaned from undertaking sensitivity testing? Yeah, so definitely. We would, uh, try and do susceptibility testing on any fungus that grew from a, um, and was deemed to be significant.
In this particular case, we, because we couldn't get s sporulation, we couldn't do susceptibility testing. If we had been able to do susceptibility testing, all we would be able to give would be a value of, of what the, in minimal inhibitory concentration was for that particular isolate. Um, but that can often give clinicians some confidence that whatever drug they've decided to treat with, you know, is likely to be effective or not.
There's certainly no break points available for any of these unusual fungi. We, we really only have break points available for one organism and one drug for Aspergillus fumigatus with voriconazole, but, and we don't really have breakpoint for anything else, so we are really only gonna be able to give you a number, which may give you confidence as to which drug can be used.
Uh. From my experience with testing other dematiaceous fungi which grow in, you know, from cases like this, quite often itraconazole is actually really a, um, it suggests that it is quite an effective drug against these dark, slow growing molds. Uh, so. I'm not a clinician, but that is kind of my experience in the lab side of things.
As you say, I think as clinicians we quite enjoy, uh, MICs that are low numbers, but appreciate that these are just environmental cutoffs and uh, it really comes down to trying to pick what you think is gonna be the most, um, effective agent. I think it'd be worth emphasizing that the biopsy is ideally an excisional biopsy because it's unusual to get either histological diagnosis or an effective culture on either an FNA.
Um, sometimes a core biopsy, but in this instance, we were fortunate enough to have the surgical team perform a full excision, and I think that's really led to a, an expedited diagnosis in this case. I completely agree with that, Jon. And it's the same as if we'd have to go down the path of using, if we hadn't managed to grow it, if we'd gone down the path of having to use PCR directly from the specimen.
Your, um, ability to get a, a positive meaningful result from a fine needle aspirate is, is quite, quite a lot lower than it would be if you get a nice, proper tissue specimen. Jon, how did you go on to choose your antifungal therapy in this instance? Yeah, so that was obviously a little bit complicated in the sense that we didn't have the full susceptibility data to guide us.
And in these type of infections, there's no prospective randomized clinical trials to determine the most effective drug therapy. But we're used to dealing in these, let's call it gray zones, for the theme today. Um, in ID where we have to treat based on what's being reported to work and what we have familiarity with. I think the surgical adjunctive therapy is really important in these cases. If we can get local control with the surgical excision, then that should be emphasized.
However, we still wanted to pursue some antimicrobial therapy in this case. Just recalling the case that it is a transplant patient on tacrolimus and everolimus. There are a lot of drug, drug drug interactions to consider and reviewing, you know, all of the literature in the past, a lot of the cases have been treated perhaps in more resource limited settings with ketoconazole, which we would rarely use in our setting currently.
Um, and then itraconazole has certainly been used as Catriona alluded to. Many of the newer studies where there have been MICs reported, have reported low values to the newer triazole, such as voriconazole or posaconazole. We did have some experience with voriconazole in the past with this patient, but perhaps given that that was not that long ago, in a time interval, we considered a, a new agent perhaps being a better option to switch to. So we decided to use posaconazole.
This certainly had some significant drug interactions and really reviewing this carefully with our nephrology colleagues and looking at the drug interactions. There's a significant interaction with the cytochrome P450 enzyme subset, 3A4. And so we had to look at that and the posaconazole was expected to significantly interact with tacrolimus so that the initial dose of the tacrolimus had to be about a third of the baseline dosing, and the everolimus had to be about 50% of the baseline dosing.
So the treatment was certainly initiated in close consultation with the nephrology team. We did that at a time when they were ready and able to do drug levels quite frequently, and the tacrolimus actually had to come down further. So this particular patient was on 0.5 milligrams bd, which eventually went down to 0.05 milligrams BD in a step by sequential fashion, as the levels stayed very high.
So whilst we look at reports of what is expected, it's a lesson that you always have to individualize these drug drug interactions. There was no ongoing imaging really for this particular site 'cause we could clinically feel it and palpate it very nicely. I. Uh, we did review a cerebral image just for the completeness, uh, because some of these organisms have been known to disseminate to the central nervous system and that was normal.
Uh, and we did repeat his pulmonary scan, which had the stable pulmonary nodules from his previous pulmonary aspergillosis with no change. So we elected to treat for six months. Uh, this was in the time where we had another worldwide epidemic going on with, with Covid, and so it was quite challenging in terms of monitoring and clinic visits, but we were able to get the patient in and ensure that he remained symptom free after successfully completing the treatment.
Thanks to Morgan, John Max and Catriona for joining Febrile today. Don't forget to check out the website febrilepodcast.com, where you can find the Consult Notes, which are written supplements to the episodes with links to references, our library of ID infographics, and a link to our merch store. Febrile is produced with support from the Infectious Diseases Society of America. Please reach out if you have any suggestions for future shows or wanna be more involved with Febrile.
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