Cryptococcosis Associated With Biologic Therapy: A Narrative Review

Abstract Cryptococcus is an opportunistic fungal pathogen that can cause disseminated infection with predominant central nervous system involvement in patients with compromised immunity. Biologics are increasingly used in the treatment of neoplasms and autoimmune/inflammatory conditions and the prevention of transplant rejection, which may affect human defense mechanisms against cryptococcosis. In this review, we comprehensively investigate the association between cryptococcosis and various biologics, highlighting their risks of infection, clinical manifestations, and clinical outcomes. Clinicians should remain vigilant for the risk of cryptococcosis in patients receiving biologics that affect the Th1/macrophage activation pathways, such as tumor necrosis factor α antagonists, Bruton tyrosine kinase inhibitors, fingolimod, JAK/STAT inhibitors (Janus kinase/signal transducer and activator of transcription), and monoclonal antibody against CD52. Other risk factors—such as age, underlying condition, and concurrent immunosuppressants, especially corticosteroids—should also be taken into account during risk stratification.

Members of the Cryptococcus neoformans/gattii species complex are basidiomycetous fungal pathogens that are environmental saprophytes and the etiologic agents of the potentially fatal human fungal infection cryptococcosis.Clinical manifestation ranges from asymptomatic pulmonary infection to disseminated central nervous system (CNS) infection [1].Cryptococcosis has become a major global health concern since the HIV pandemic in the 1980s, with most cases occurring in adults infected with HIV who live in sub-Saharan Africa.A recent modeling study estimated 152 000 cases of cryptococcal meningitis occurring among people with HIV per annum, resulting in 112 000 cryptococcosis-related deaths [2].Besides advanced HIV, other risk factors include hematopoietic stem cell or solid organ transplantation, hematologic malignancies, organ failure, sarcoidosis, primary immunodeficiencies affecting T-cell immunity, autoantibody against the granulocytemacrophage colony-stimulating factor (GM-CSF), and iatrogenic immunosuppression (eg, corticosteroids) [3].
With advances in the medical treatment of cancer and autoimmune and inflammatory diseases, including wider availability of solid organ and hematopoietic stem cell transplantation and an expanding variety of immunomodulatory agents, the number of patients who are immunocompromised and at risk of opportunistic infections is increasing.In addition, recent modeling studies have demonstrated global warming as a major driver of the expansion in the ecologic niches of pathogenic cryptococci [4].Coupled with the changing patterns of human behaviors and increasing numbers of susceptible hosts, the incidence of cryptococcosis is expected to rise in the next decades.In this review, we describe the pathobiology of cryptococcosis and review the risks of infection conferred by different biological agents used in clinical practice.are acquired in early childhood [8].Primary pulmonary cryptococcosis usually results in asymptomatic or subclinical infection in individuals who are immunocompetent [8,9] but can result in pneumonia in patients who are immunocompromised [10].In immunocompetent hosts, cryptococci are either cleared by the immune system after initial infection or establish a latent stage in immune cells, primarily macrophages, that can reactivate later in life due to immune dysregulation [11,12].
Besides pulmonary infection, disseminated infection involving the skin, soft tissue, bone, joint, liver, lymph nodes, peritoneum, urogenital tract, adrenal, eyes, and especially the CNS can occur, particularly in immunosuppressed hosts [3].Cryptococcal entry into the CNS compartment is postulated to occur through 1 or a combination of 3 mechanisms: paracytosis with the aid of fungal metalloproteases, transcytosis through binding between hyaluronic acid and CD44 on the endothelium, and a "Trojan horse" mechanism by hijacking host phagocytes to cross the blood-brain barrier [1].

Virulence Factors
The C neoformans/gattii species complex expresses several virulence factors to enable host invasion and survival.The yeast cells are surrounded by a fungal capsule of various thickness, which is predominantly composed of the polysaccharide glucuronoxylomannan.Glucuronoxylomannan plays a pivotal role in immune modulation through inhibition of phagocytosis, phagosomal acidification, antigen presentation, T-lymphocyte proliferation and humoral response, induction of macrophage apoptosis, and induction of an immunetolerant state [13][14][15][16][17].The capsule size determines early macrophage control of infection and subsequent intracellular proliferation [18].The production of melanin, regulated by Cryptococcosis pathogenesis and the impact of major categories of biologics in this review [1,6,7].Pathogenetic cryptococci elaborate various virulence factors to help establish infection and dissemination, especially to the central nervous system.For a detailed description of the impact of specific biologics on cryptococcosis, refer to the corresponding sections on TNF-α blockers, Bruton tyrosine kinase inhibitors, fingolimod, and others.CXCL1, chemokine (C-X-C motif) ligand 1; GM-CSF, granulocytemacrophage colony-stimulating factor; IFN-γ, interferon γ; IL, interleukin; JAK/STAT, Janus kinase/signal transducer and activator of transcription; TNF-α, tumour necrosis factor-α.Image created with BioRender.com.
During in vivo infection, dramatic changes in cryptococcal cellular morphology have been observed, resulting in the formation of "titan cells," which are 5-to 10-fold larger than typical cryptococcal yeast cells, are polypoid with a thickened cell wall and tightly compacted capsule, and form approximately 5% to 20% of the fungal cells in the infected lungs of mice [25][26][27][28].Titan cell formation impairs phagocytosis and skews the inflammatory response to a Th2-type response [29], promoting the establishment of the initial pulmonary infection, stress adaptation, brain dissemination, and mortality [27][28][29][30].
As the predominant resident phagocytic cells in the lung, alveolar macrophages play an essential role in the human immune response to cryptococcal invasion, including receptor-mediated phagocytosis, secretion of chemokines and cytokines, and antigen presentation, as well as serving as a reservoir for latency [37].The ability of macrophages to contain cryptococcal invasion depends on macrophage polarization and activation status, which are influenced by the cytokine microenvironment [38].Interferon γ (IFN-γ), tumor necrosis factor α (TNF-α), and GM-CSF signaling stimulates M1 polarization, which is essential for macrophage fungicidal activity [38,39].Yet, IL-4 stimulation differentially induces M2 polarization, which is associated with deficient anticryptococcal activity and disease progression [40].Conceptually, treatments that impair M1 polarization, such as antagonists to TNF-α or JAK/STAT inhibitors (Janus kinase/signal transducer and activator of transcription) that impair IFN-γ signaling, are associated with increased risks of cryptococcosis, among a population of patients who are often already predisposed to infection due to their underlying disease or concomitant immunosuppressants.
T-cell responses after cryptococcal infection are stimulated by activated dendritic cells, which respond to fungal pathogen-associated molecular patterns such as β-glucan, chitin, and glucuronoxylomannan.Activated CD4+ T cells secrete IL-12 and IL-23 to activate the T helper 1 (Th1) cells, which in turn produce IFN-γ to "superactivate" macrophages to enhance intraphagocytic killing.However, massive accruement of pathologic cryptococcal antigen-specific Th2 cells was demonstrated in the lungs following in vivo infection, which was coordinated by lung-resident CD11b+ conventional dendritic cells and induced by cleavage of chitin by the host chitotriosidase [41].

ASSOCIATION BETWEEN BIOLOGICS AND CRYPTOCOCCOSIS
We conducted a literature search on PubMed using combinations of an individual drug name and "cryptococcosis," "cryptococcal," or "cryptococcus" for publications related to cryptococcosis and biologics .Articles containing the relevant search terms that were published from 1990 to 20 January 2024 were included for title and abstract screening.Eligible articles that contained case-level data on at least 1 individual who was receiving biologics and was diagnosed with cryptococcosis were retrieved for full-text review.References of articles containing primary data were also reviewed for additional publications that might contain patient information.Non-English-language articles, cases whose demographic and clinical details were not available, as well as data reported only in abstracts of conference proceedings or scientific meetings were excluded (Supplementary Figure 1).The list of biologics according to therapeutic targets and disease groups is summarized in Supplementary Table 1.Only biologics approved by the US Food and Drug Administration (FDA) as of 20 January 2024 were included.The definitions of proven or probable cryptococcosis followed the 2020 EORTC/ MSGERC consensus definitions (European Organization for Research and Treatment of Cancer/Mycoses Study Group Education and Research Consortium) [155].Infection was deemed "disseminated" if there was fungemia or the infection involved at least 2 noncontiguous sites.

TNF-α Antagonists
TNF-α is a pleiotropic cytokine that is predominantly produced by cells of the monocytic lineage.It is synthesized as membrane-associated or soluble forms, and it signals through TNF receptors 1 and 2 to regulate a range of biologic activities, including inflammation, cell proliferation, host defense, and cell survival [156].Due to the prominent role of TNF-α in the proinflammatory cascade, therapeutic targeting of the TNF pathway has been harnessed to treat various inflammatory and autoimmune conditions.Despite the revolutionary success in tackling TNF-mediated pathogenesis, the use of TNF-α antagonists has been associated with an increased risk of opportunistic infections.Due to the inhibition of the formation and maintenance of granulomas [157], TNF-α inhibition increases the risk of infection by intracellular pathogens that are normally contained by granulomatous inflammation, most notably, tuberculosis, histoplasmosis, and coccidioidomycosis [158][159][160].
The risk of opportunistic infection is not equally elevated across all TNF-α antagonists.Infliximab binds to monomer and trimer forms of soluble TNF and assembles more stable complexes with soluble and transmembrane TNF, whereas etanercept binding is restricted to the trimer form, creates less stable complexes, and demonstrates lower avidity to transmembrane TNF than infliximab [161].These differences in pharmacodynamics underlie the lower risk of opportunistic infection conferred by etanercept as compared with antibodymediated TNF-α neutralizers such as infliximab and adalimumab, as demonstrated by data collected through the Adverse Event Reporting System of the FDA [162].In addition, patients who develop opportunistic infections while undergoing treatment with infliximab typically manifest earlier than those taking etanercept [163].The only study that yielded a cryptococcosis-specific risk calculation was a retrospective case-control study conducted among patients with rheumatoid arthritis who developed cryptococcosis from a single center in Taiwan over a 14-year period [164].Though the number of cryptococcosis cases with current use of TNF-α antagonists was small, exposure to adalimumab (n = 3) was significantly associated with increased risks of cryptococcosis (adjusted odds ratio, 4.50; 95% CI, 1.03-19.66;P = .046)while the crude odds ratio (1.61; 95% CI, .33-7.77;P = .55)for etanercept (n = 2) did not reach statistical significance.

Ibrutinib and Other Bruton Tyrosine Kinase Inhibitors
Ibrutinib is a small molecule inhibitor approved for the treatment of various lymphoid neoplasms, such as chronic lymphocytic leukemia (CLL) [165,166], Waldenstrom macroglobulinemia [167], mantle cell lymphoma [168], and follicular lymphoma [169].Early-onset opportunistic fungal infections have been associated with the use of ibrutinib [170], most notably cases of invasive aspergillosis with frequent involvement of the CNS [171].
Susceptibility to infection in patients treated with ibrutinib has been linked to altered B-cell receptor signaling and inhibition of IL-2-inducible kinases [172] as well as to impairments in neutrophil and monocyte functionality [173,174].Of note, a significant number of cases of invasive fungal infections, including cryptococcosis, in patients treated with ibrutinib occurred in heavily pretreated cases with relapsed or refractory disease [82,175].During experimental C neoformans infection with Bruton tyrosine kinase (BTK)-deficient mice, Szymczak et al found that X-linked immunodeficient mice carrying a Btk mutation were unable to contain C neoformans lung infection after intranasal inoculation and experienced disseminated disease [176].In contrast, Messina et al found no differences in disease severity among BTK knockout mice as compared with wild type ones [177].In addition, the administration of ibrutinib at doses replicating human exposure did not affect infection severity [177].Collectively, these animal models and clinical data suggest that increased susceptibility to cryptococcosis in patients with BTK inhibitors (BTKis) may reflect a high net state of immunosuppression rather than sole linkage to receipt of ibrutinib [178].Two more recent BTKis with greater specificity, acalabrutinib and zanubrutinib, are increasingly used in the treatment CLL due to better cardiovascular tolerability vs ibrutinib [179,180].Whether these newer BTKis are associated with the same off-target effects leading to increased susceptibility to fungal infections such as cryptococcosis is as yet unknown.Of note, however, 7 cases of cryptococcosis were reported in a pooled safety analysis of 6 studies totaling 779 patients receiving zanubrutinib [181].
We identified 28 cases of proven/probable cryptococcosis occurring in patients receiving BTKis, almost exclusively with ibrutinib (2 cases with acalabrutinib and 1 with zanubrutinib; Table 2).Only 2 cases were due to C gattii [88,91].The median age was 74 years, and 79% (22/28) were male.The main indication for receipt of BTKis was CLL (17/28, 61%), followed by mantle cell lymphoma (6/28, 21%).The median duration of treatment before onset of cryptococcosis was 4.5 months, and 18 of 28 (64.3%)cases occurred within the first 6 months of treatment.The BTKi was used as first-line therapy after diagnosis in only 29% of cases with available data (7/24) and was given with concurrent immunosuppressive treatment in 25% (6/24) of cases.The main presentations of infection were cryptococcal meningitis (10/28), pulmonary infections (9/28; including       single nodule, n = 2; multiple nodules, n = 2; consolidations, n = 3; pleural empyema, n = 1), and disseminated infections (7/28).In these reports, the BTKi was inconstantly discontinued after cryptococcosis in 65% (11/17) of patients with available data, indicating a need for clearer guidelines regarding the management of these biologics after the onset of opportunistic fungal infections.With the increasing treatment options available for these lymphoid neoplasms, discontinuation of BTKis may be a reasonable approach until more data emerge.

Fingolimod
Fingolimod (FTY720) is a first-in-class oral disease-modifying medication that was approved by the FDA in 2010 for the treatment of patients with relapsing forms of multiple sclerosis.It acts by interacting with sphingosine 1-phosphate receptors to prevent lymphocyte egress from lymphoid tissues, thereby reducing autoreactive lymphocyte infiltration into the CNS [182].Fingolimod induces a rapid and reversible reduction in lymphocyte counts, which remains stable during chronic treatment at 28% and 24% of baseline values at 24 months with 0.5 and 1.25 mg, respectively [183].Specifically, patients treated with fingolimod showed a significant reduction in circulating CD4+ T cells, and activation of T cells in the presence of fingolimod led to a subinflammatory phenotype with reduced production of IFN-γ, granzyme B, IL-17, GM-CSF, and TNF-α [184].These perturbations in lymphocyte number and function, which predominantly impair the activation of Th1 pathways, may underlie the increased risk of cryptococcosis in patients with multiple sclerosis treated by fingolimod.

Other Biologics Associated With Cryptococcosis
In addition to the aforementioned biologics that have been shown to be associated with major risks of cryptococcosis, we identified several other biologics with ≥3 cases of treatment-associated cryptococcosis reported (Table 4).These include inhibitors of the JAK/STAT pathway, anti-CD52 antagonists, anti-CD20 antagonists, and IL-6 inhibitors.The JAK/ STAT signaling pathway functions downstream of >50 cytokines and growth factors, including key players in anticryptococcal immunity, such as IFN-γ and GM-CSF [188].STAT1 deletion resulted in a shift from Th1 to Th2 cytokine response, pronounced lung inflammation, and defective classical macrophage activation in murine models of cryptococcosis [189].There have been 12 cases of ruxolitinib-associated cryptococcosis; most of them (8/12, 67%) did not receive other concomitant immunosuppressants, indicating that ruxolitinib per se leads to increased susceptibility to cryptococcosis.Consistent with this, in a retrospective cohort study, baricitinib (odds ratio, 12.4; 95% CI, 6.4-24.1;P < .0001),not dexamethasone, was associated with the development of cryptococcosis [190].
The anti-CD52 agent alemtuzumab is indicated in the treatment of CLL, T-cell lymphoma, and relapsing-remitting multiple sclerosis and has been used in solid organ and hematopoietic stem cell transplantation for induction therapy and acute organ rejection [191][192][193][194][195]. Alemtuzumab selectively targets CD52, which is expressed on the surface of B and T lymphocytes, leading to sustained lymphocyte depletion [196].Use of alemtuzumab has been associated with a range of opportunistic infections in patients with hematologic malignancies and solid organ transplantation [197][198][199][200].Among 547 patients with solid organ transplantation who received alemtuzumab for induction or rejection therapy, 62 (11%) experienced at least 1 opportunistic infection at a median 84 days after treatment initiation, including 2 cases of cryptococcosis [199].Among 357 patients with CLL or cutaneous T-cell lymphoma, 33 experienced opportunistic fungal infections, including 2 cases of cryptococcosis [200].In our review of 6 reported cases of cryptococcosis with individual case details, all occurred in heavily pretreated patients with hematologic malignancies (including 4/6 with CLL), and 5 of 6 presented with disseminated disease.Similarly, although we identified several cases of cryptococcosis in patients being treated with rituximab (anti-CD20) and tocilizumab (anti-IL-6), almost all of them received concomitant corticosteroids and/or chemotherapeutic agents, suggesting that susceptibility to cryptococcosis in these populations more likely reflected an overall degree of  Cryptococcosis Associated With Biologic Therapy • OFID • 9    [202][203][204][205].Among HIV-seronegative cohorts with cryptococcosis, prior corticosteroid use was reported in up to 28% to 48% of patients [206][207][208][209], although the dose and duration were often not specified.Prior high-dose corticosteroid use, defined as the equivalent of ≥20 mg/d of prednisone for ≥60 days prior to diagnosis of cryptococcosis, has been associated with a higher likelihood of dissemination (41% vs 18%, P = .002)among patients with pulmonary cryptococcosis [210], and corticosteroid usage was associated with a higher 30-day mortality in a recent observational study from Japan [208].
Since biologics are most likely to be initiated in patients with autoimmune conditions, neoplasms, and transplantation, other immunosuppressants and immunomodulatory agents, especially those affecting the T-cell activation and proliferation pathways, play a role in mediating the risk of cryptococcosis.For example, in our identified cases, purine analogues such as fludarabine and cytarabine were often given to patients with hematologic malignancies.In addition to corticosteroids, transplant recipients are likely receiving calcineurin inhibitors, mycophenolate mofetil, and/or mTOR inhibitors (mammalian target of rapamycin), all of which affect T-cell activation and differentiation [211][212][213].Therefore, the overall risk of infection is a product of the interaction between biologics and the host, as well as between biologics and Cryptococcus species.
There are limitations to this study.First, cases whose demographics and clinical details were not available were excluded from the analysis.Second, there is inherent difficulty in attributing causality to the biologics, as many patients in the literature review had underlying hematologic or rheumatologic conditions that impaired the immunity and they received concomitant or recent immunosuppressants, which all contributed to the increased risk of infection.The current study did not aim to address the causality of each biologic from a mechanistic point of view.Third, the manifestations of cryptococcosis may mimic other conditions.As noted in our series, different groups of biologics appeared to be associated with specific manifestations, such as the relatively high percentage of pulmonary cryptococcosis with TNF-α antagonists and skin and soft tissue infections with fingolimod.Disseminated disease most often occurred in patients receiving concomitant immunosuppressants and those with advanced age.However, there were significant differences in the exhaustivity in the diagnostic workup, which was based on the discretion of treating physicians and limited by the systematic availability of diagnostic tools.The apparent high percentage of some non-CNS forms of cryptococcosis associated with certain biologics may be partially attributed to the heterogeneity of the diagnostic workup.

CONCLUSION
In conclusion, biologics, especially those blocking the Th1-macrophage activation pathways, impart a substantially increased risk of cryptococcosis among patient populations who are already susceptible to opportunistic infections due to their underlying conditions or concomitant immunosuppressants.With the increasing number and variety of biologicsexpanding from the treatment of autoimmune diseases and neoplasms to novel therapeutics for atopy and metabolic diseases-clinicians must be vigilant of the risks, as lack of suspicion may lead to diagnostic delays and poorer outcomes.Knowledge of the association between biologic therapies and cryptococcosis, including the underlying mechanism of immune susceptibility and clinical manifestations, will help clinicians stratify the risks of cryptococcal infection and individualize the management plans for their patients.More data are needed to guide the management of cryptococcal infection in patients receiving biologic therapy, especially regarding the continuation or resumption of biologics during and after antifungal therapy.

Figure 1 .
Figure 1.Cryptococcosis pathogenesis and the impact of major categories of biologics in this review[1,6,7].Pathogenetic cryptococci elaborate various virulence factors to help establish infection and dissemination, especially to the central nervous system.For a detailed description of the impact of specific biologics on cryptococcosis, refer to the corresponding sections on TNF-α blockers, Bruton tyrosine kinase inhibitors, fingolimod, and others.CXCL1, chemokine (C-X-C motif) ligand 1; GM-CSF, granulocytemacrophage colony-stimulating factor; IFN-γ, interferon γ; IL, interleukin; JAK/STAT, Janus kinase/signal transducer and activator of transcription; TNF-α, tumour necrosis factor-α.Image created with BioRender.com.

Table 4 . Continued
instead of the independent effect of the biologics.Other biologics with rare cases of treatment-associated cryptococcosis are included in Supplementary Table2.
a Dual infection with disseminated histoplasmosis.b Dual infection with Mycobacterium haemophilum.c Dual infection with Mycobacterium tuberculosis.Cryptococcosis Associated With Biologic Therapy • OFID • 11 immunosuppression