Ibrutinib-associated cutaneous mucormycosis due to an Apophysomyces species: report of a case and review of the literature

The use of ibrutinib, a Bruton tyrosine kinase inhibitor, has been associated with invasive fungal infections (IFIs). We describe a case of Apophysomyces infection associated with long-term use of ibrutinib for the treatment of chronic lymphocytic leukemia as well as perform a literature review of Mucormycosis infections in patients on ibrutinib. Our review found that the onset of IFI can occur within months to years of starting tyrosine kinase inhibitors. These reports provide a more complete picture of the risk of IFI while patients are on ibrutinib. Our case also demonstrates the utility of molecular techniques in the diagnosis of IFI, as the diagnosis was made using 28S rDNA/internal transcribed spacer PCR.


Introduction
Ibrutinib, a Bruton tyrosine kinase (BTK) inhibitor, is an effective therapy in chronic lymphocytic leukemia (CLL) and mantel cell lymphoma and has increasingly replaced chemotherapy for the treatment of CLL over the last 10 years. 1,2This the time of this presentation, he was receiving IVIG monthly.He lives in Houston, Texas, has no recent travel history, and enjoys doing yard work.On physical examination, there was erythema and warmth on the right forearm without involvement of his elbow or wrist.The patient was diagnosed with cellulitis and discharged on a 10-day course of trimethoprim/sulfamethoxazole.Four days later, the patient presented again with persistent erythema and a new ulcerated lesion with eschar in the center (Figure 1).He reported subjective fevers the day prior to presentation but had no chest pain, cough, abdominal pain, shortness of breath, nausea, vomiting, diarrhea, or night sweats.He had tachycardia to 100/min with a neutrophilpredominant leukocytosis to 11,600/mm 3 (normal range: 3500-10,000/mm 3 ).Based on his presentation with mild tachycardia, worsening right forearm lesion, presumed insufficient treatment of cellulitis, and elevated white blood cell count, the patient was admitted to the hospital.He was empirically treated with cefepime 2 g Q8H and vancomycin 750 mg Q12H, while ibrutinib was held.
Throughout his hospital course, the erythema resolved but the eschar persisted.Blood culture and histological examination of biopsy specimens were negative, as were cultures for acid-fast bacteria, anaerobic bacteria, Nocardia, and fungi.Tests for Cryptococcus, Histoplasma, Aspergillus, HIV, methicillin-resistant Staphylococcus aureus, and serum 1→3,β-d-glucan were also negative.A computed tomographic scan with contrast and magnetic resonance imaging of the arm showed only cellulitis/myositis with no drainable abscess.Tissue from the lesion was sent to the University of Washington Department of Laboratory Medicine and Pathology for PCR targeting 28S ribosomal DNA (rDNA) and internal transcribed spacer (ITS) regions along with sequencing.Meanwhile, the patient was discharged with cefpodoxime 300 mg twice a day (BID) to complete a 2-week course of antibiotics for suspected concurrent cellulitis and posaconazole (300 mg BID for 1 day, followed by 300 mg daily) and infectious disease clinic follow-up in 1 week.Ibrutinib continued to be held.
At his outpatient infectious disease follow-up 1 week after discharge, the eschar had continued to grow but was non-tender.The patient and wife had been dressing the wound with meta-honey and gauze with no improvement.The patient had not yet begun to take the posaconazole since the medicine had not been obtained.He had no pain, swelling, or drainage and no fevers or chills.The patient and his wife were advised to continue local wound care with meta-honey and start the posaconazole while awaiting the molecular results.
One day after his outpatient infectious disease follow-up, 28S rDNA and ITS PCR sequencing results returned and revealed an Apophysomyces species DNA.The patient was admitted and completed a 7-day course of liposomal amphotericin B (3 mg/kg daily).He was then transitioned to posaconazole 300 mg BID for 1 day, followed by 300 mg daily.The patient also underwent debridement and full-thickness skin graft.Tissue specimens from the debridement showed non-septate fungal hyphal elements consistent with a Mucorales species (Figure 2).However, specific fungal cultures had no growth.Hyphae were present in the deep advancing portions of the ulcer.Posaconazole was stopped 3 months later following the resolution of the lesion.At the time of subsequent outpatient follow-up 9 months later, his arm lesion had completely healed (Figure 3).

Discussion
Apophysomyces mode of transmission Fungi in the order Mucorales usually reside in decaying vegetation and soil.These organisms can spread during natural disaster events when people sustain lacerations or traumatic inoculation of uprooted vegetation and other debris.Several cases of Apophysomyces infections, specifically, have been reported following injuries suffered in tsunamis and tornadoes.During the 2004 Southeast Asian tsunami in Thailand and Sri Lanka, there were two case reports of survivors sustaining multiple soft-tissue injuries and laceration wounds who presented with large areas of full-thickness skin and fat necrosis in the buttock and thigh, respectively.Although these two cases occurred in different geographical locations (one patient was in Sri Lanka and the other was in Thailand), the patients had strikingly similar hospital courses.Despite treatment with empiric antibiotics and surgical debridement, both remained clinically infected.For these cases, histopathology and cultures eventually revealed

TherapeuTic advances in infectious disease
Apophysomyces elegans, and the patients were treated with a prolonged course of liposomal amphotericin B resulting in gradual resolution of infection.Their wounds were also treated with further surgical debridement and skin grafting. 5,6other case series reported 13 patients who developed necrotizing cutaneous mucormycosis following the 2011 EF5 tornado in Joplin, Missouri.These patients sustained a variety of wounds including fractures, blunt trauma, and penetrating wounds.On multivariate analysis, cutaneous mucormycosis was associated with penetrating trauma and an increased number of wounds. 7Interestingly, Apophysomyces trapeziformis was found in all 13 patients using 28S rDNA sequencing. 7Our case, however, is an unusual occurrence of Apophysomyces infection in the absence of known direct penetrating trauma to facilitate its seeding.Instead, our patient's gardening hobby might have predisposed him to Apophysomyces infection in the context of incidental penetrating wounds or laceration.

Mechanism of immune system impairment in BTK dysregulation
The main and most well-known mechanism of immunodeficiency in CLL patients is hypogammaglobinemia which is present in 85% of CLL patients, although defects in cell-mediated processes also occur. 8As a result, the predominant pathogens to which CLL patients are at risk are typically encapsulated bacteria which are primarily targeted and cleared by antibody-mediated processes. 9Patients with CLL may be at risk for other opportunistic pathogens, but these infections are not usually present unless the patients have received other immunosuppressive therapies such as corticosteroids, rituximab, or fludarabine.Fungal infections are rare and usually associated with recurrent neutropenia following monoclonal antibody therapies, or receipt of the agents mentioned previously. 8,10Our patient did not have recurrent neutropenia nor prior monoclonal antibody therapy.In the absence of other risk factors, this patient's ibrutinib therapy likely predisposed him to an IFI.
BTKs are known to play a role in sensing infection via their interaction with Toll-like receptors on human dendritic cells and macrophages, promoting transcription of inflammatory cytokines and interferons. 11Microbes reported to be recognized by BTK include Listeria monocytogenes, Staphylococcus aureus, dengue virus, and Aspergillus. 10A substantial portion of current literature on BTK function has focused on cases of X-linked agammaglobulinemia (XLA), as various genes coding for the BTK protein are mutated in XLA, although IFI rarely occurs in patients with XLA. 11,12It is hypothesized that the predisposition to IFI in patients receiving ibrutinib results from a combination of defects in infection recognition, innate immune response dysregulation including off-target effects on BTK-related immune kinases, increased susceptibility to monocyte apoptosis, and impaired development and effector functions of myeloid phagocytes. 4,12terature review on the association between BTK inhibitors and mucormycosis An increasing number of cases of BTK inhibitor (BTKi)-associated mucormycosis have been reported.Five BTKi, namely ibrutinib, acalabrutinib, zanubrutinib, tirabrutinib, and orelabrutinib, are approved for the treatment of multiple B-cell malignancies in humans. 13In January 2023, pirtobrutinib was also approved in the United States for the treatment of mantel cell lymphoma. 14By using the names of these six BTKi and by typing the keywords 'name of BTKi' and 'mucormycosis' at the same time into the search engine of the National Center for Biotechnology Information or NCBI databases, 14 case reports of BTKi-associated mucormycosis were found.In addition, another case by Castro et al., although not found in the NCBI databases, was also included since it was mentioned in the case by Sittig et al.Most noticeably, all 15 cases involved patients treated with ibrutinib as the BTKi of choice (Table 1).No cases of mucormycosis were reported in patients treated with acalabrutinib, zanubrutinib, tirabrutinib, orelabrutinib, or pirtobrutinib.In 10 cases, speciation was performed to guide therapy.These included one case of Mucor irregularis, four cases of Rhizomucor species, two cases of Rhizopus species, two cases of Lichtheimia species, and one case of Cunninghamella bertholletiae (Table 1).Our case is the first to report the involvement of Apophysomyces in the development of cutaneous mucormycosis after ibrutinib treatment.
These patients presented with a wide range of organ involvement including CNS, lungs, sinuses, liver, kidney, spleen, pancreas, vertebrae, thyroid gland, and skin (Table 1).The time between initiation of BTKi therapy to the onset of mucormycosis in these cases ranges from the shortest interval of 25 days to the longest interval of 3 years (Table 1).Rogers et al. reported that the time from ibrutinib initiation to OI development ranges from 0.36 to 52.0 months with a median time of 4.68 months. 3Our patient's cutaneous mucormycosis developed 3 years after beginning ibrutinib therapy.
In addition, although our patient's ibrutinib dose was decreased due to cardiac adverse effects, it has been shown that ibrutinib dosage is not associated with infection risk.In a retrospective study in 2018 on 378 patients who received ibrutinib for the treatment of lymphoid cancer, 11.4% of these patients developed serious infections with 37.2% of these infections involving IFI. 30 The infection risk analysis between patients with serious infections and those without any infection showed that both the daily and cumulative ibrutinib dose was not associated with infection risk. 30rthermore, it was found that the majority of patients on ibrutinib with IFI did not have the classic risk factors predisposing to fungal infection including neutropenia, lymphopenia, and corticosteroid therapy. 30 addition to our patient, six of the reported cases were treatment naïve prior to ibrutinib (Table 1), strengthening the association between ibrutinib initiation and mucormycosis development.Most prominently, of these six cases that had no prior therapies, four received no concurrent therapy besides ibrutinib (Table 1).Similarly, our patient received ibrutinib monotherapy for his CLL treatment, strengthening the association between the BTKi and his cutaneous mucormycosis caused by Apophysomyces.

The importance of speciation and the role of 28S rDNA/ITS PCR and sequencing to characterize 'difficult-to-identify' fungal species
Cultures for the Mucorales are frequently negative even when there is microscopic evidence of fungal hyphae in tissue specimens.A retrospective review of 929 cases of mucormycosis from 1940 to 2003 reported a culture positivity rate of only 50%. 31Although histological examination can suggest the presence of these organisms, microscopic appearance is not always sufficiently accurate to differentiate among various filamentous fungi and attempts at improved culture methods for the Mucorales have not been implemented successfully in clinical microbiology laboratories. 32The initial histological evaluation of our patient's biopsy was negative.The limitations posed by fungal cultures and the potential errors in identification using histologic morphologies highlight the need for alternative diagnostic methods to differentiate invasive fungal pathogens.Such differentiation has therapeutic relevance as only posaconazole and isavuconazole, among the azoles, have reliable activity against Mucorales species and are the drugs of choice for oral treatment.
16S rRNA sequencing is well established as a genetic method to identify poorly described, rarely isolated, novel, or culture-negative bacteria.This mode of sequencing is particularly valuable in bacterial phylogeny and taxonomy studies due to the presence of 16S rRNA in almost all bacteria, its unchanged function over evolutionary journals.sagepub.com/home/taiBID, twice a day; BTKi, Bruton tyrosine kinase inhibitor; CLL, chronic lymphocytic leukemia; CNS, central nervous system; IV, intravenous; R-ICE, rituximab, ifosfamide, carboplatin, etoposide; TID, three times a day.

Table 1.
(Continued) time, and its sequence size being large enough for analytic tests. 33Similar techniques incorporating 28S rRNA and ITS sequencing have been used for fungi considered to be 'difficult to identify'. 34[37]

Conclusion
This case shows that mucormycosis due to Apophysomyces can occur in the context of immune system impairment caused by prolonged ibrutinib treatment.Despite initial negative cultures and biopsy, this pathogen was correctly identified using 28S rDNA and ITS sequencing.

Figure 1 .
Figure 1.Progression of the patient's lesion in the right forearm at the second presentation.There was a new 3.5 cm × 3.5 cm eschar with surrounding erythema that was tender to palpation.

Figure 2 .
Figure 2. Histological sections of tissues are removed during debridement.(a) It demonstrates non-septate fungal hyphae in the central necrosis (arrow right) and within a giant cell (arrow top left) (400×).(b) It shows a higher magnification of the giant cells with the hyphal fragments (400×).(c) It shows staining of fungal hyphae with the Gomori methenamine silver stain (400×).

Figure 3 .
Figure 3. Appearance of right forearm lesion 9 months after debridement with full-thickness skin graft and 3 months of posaconazole therapy.