Autoantibodies Neutralizing GM-CSF in HIV-Negative Colombian Patients Infected with Cryptococcus gattii and C. neoformans.

BACKGROUND: Cryptococcosis is a life-threatening disease caused by Cryptococcus neoformans or C. gattii. Neutralizing autoantibodies (auto-Abs) against granulocyte-macrophage colony-stimulating factor (GM-CSF) in otherwise healthy adults with cryptococcal meningitis have been described since 2013. We searched for neutralizing auto-Abs in sera collected from Colombian patients with non-HIV-associated cryptococcosis in a retrospective national cohort from 1997 to 2016. METHODS: We reviewed clinical and laboratory records and assessed the presence of neutralizing auto-Abs against GM-CSF in 30 HIV negative adults with cryptococcosis (13 caused by C. gattii and 17 caused by C. neoformans). RESULTS: We detected neutralizing auto-Abs against GM-CSF in the sera of 10 out of 13 (77%) patients infected with C. gattii and one out of 17 (6%) patients infected with C. neoformans. CONCLUSIONS: We report eleven Colombian patients diagnosed with cryptococcosis who had auto-Abs that neutralize GM-CSF. Among these patients, ten were infected with C. gattii and only one with C. neoformans.

Association between neuroimaging and clinical outcomes in individuals with central nervous system cryptococcosis.

BACKGROUND: The radiological manifestations of central nervous system (CNS) cryptococcosis are diverse and often subtle. There is heterogeneity on how different neuroimaging patterns impact prognosis. This study aims to assess the association between the neuroimaging and clinical outcomes of CNS cryptococcosis. METHODS: All patients with CNS cryptococcosis between July 2017 and April 2023 who underwent brain magnetic resonance imaging (MRI) were included. The primary outcome was mortality during hospitalisation. Secondary outcomes were readmission, ventricular shunting, duration of hospitalisation and time to the first negative cerebrospinal fluid culture. We compared the outcomes for each of the five main radiological findings on the brain MRI scan. RESULTS: We included 46 proven CNS cryptococcosis cases. The two main comorbidity groups were HIV infection (20, 43%) and solid organ transplantation (10, 22%), respectively. Thirty-nine patients exhibited at least one radiological abnormality (85%), with the most common being meningeal enhancement (34, 74%). The mortality rates occurred at 11% (5/46) during hospitalisation. We found no significant disparities in mortality related to distinct radiological patterns. The presence of pseudocysts was significantly associated with the need for readmission (p = .027). The ventricular shunting was significantly associated with the presence of pseudocysts (p = .005) and hydrocephalus (p = .044). CONCLUSION: In this study, there is no association between brain MRI findings and mortality. Larger studies are needed to evaluate this important issue.

Cryptococcosis Associated With Biologic Therapy: A Narrative Review.

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.

Antigen titers in cryptococcal meningitis: what determines how fast they fall?

Follow-up of previously healthy patients surviving cryptococcal meningitis found that cryptococcal antigen could be detected for more than one year in serum from 38 of 44 (86%) patients and in CSF from 20 of 31 patients (67%), far beyond the time of culture conversion. The speed of titer decline, measured as the number of days for a two fold drop in titer to occur, was slower in serum than in CSF. Speed of decline of antigen titers was much slower in serum and CSF for patients infected with C. gattii than C. neoformans. The speed of decline in CSF and serum titers was also much slower in patients who had received a ventriculoperitoneal shunt for increased intracranial pressure. The variable and extraordinarily slow rate of clearance in our patients did not appear to reflect differences in disease control but rather differences in species and shunting for increased intracranial pressure.

Cryptococcal Meningitis in an Immunocompetent Patient With Underlying Risk Factors.

Cryptococcal meningitis, a severe fungal infection of the central nervous system, is usually found in immunocompromised patients, especially those with human immunodeficiency virus/acquired immunodeficiency syndrome. Its occurrence in immunocompetent individuals is rare and the presentation can be nonspecific. We present a case of cryptococcal meningitis in a young, immunocompetent male with a known history of intravenous drug abuse who was also found to have hepatitis C during admission. Induction therapy with amphotericin B and flucytosine was completed for 14 days. This shorter duration was considered as he had a good clinical response with rapid improvement in mental status and intracranial pressure with an extraventricular drain and negative repeat cerebrospinal fluid (CSF) culture. However, during the consolidation phase with fluconazole, the patient developed new neurologic symptoms and the induction phase had to be re-initiated for a total of 28 days. The patient likewise required the re-placement of an extraventricular drain and the creation of a ventriculoperitoneal shunt due to persistent CSF accumulation and increased intracranial pressure. He was eventually discharged on fluconazole for a planned consolidation phase of eight weeks, followed by a prolonged maintenance phase, but the patient was lost to follow-up.

Cryptococcosis at the university hospital of Marseille: A case series.

Cryptococcosis is a fungal infection burdened by a high case-fatality rate in immunocompromised patients. Once limited to human immunodeficiency virus (HIV)-infected patients, the epidemiology of cryptococcosis has evolved in recent years and new risk factors have emerged. It is therefore essential to identify these risk factors in order to improve prevention and therapeutic efficacy. We conducted a retrospective observational study including all cases of cryptococcosis between January 2016 and December 2022, diagnosed at the University Hospital of Marseille. During the study period 15 cases of cryptococcosis were diagnosed. Six patients were HIV-infected. Nine patients had one or more comorbidities including liver cirrhosis, type 2 diabetes mellitus, primary immunodeficiency disorder, chronic lymphocytic leukemia and solid organ transplantation. Ten patients had central nervous system cryptococcosis, four had pulmonary cryptococcosis and one patient had extra-pulmonary disseminated cryptococcosis. Of the three patients with liver cirrhosis, two patients died with a post-mortem diagnosis. Our data suggest that emerging risk factors are probably underestimated by clinicians. It emphasizes the need for cryptococcal antigenemia as part of syndromic investigation of any unexplained fever or neurological symptoms in an at-risk patient. Early diagnosis and treatment are essential for patient's survival.

Diagnostic Study of Nodular Pulmonary Cryptococcosis Based on Radiomic Features Captured from CT Images.

BACKGROUND: Radiomics can quantify pulmonary nodule characteristics non-invasively by applying advanced imaging feature algorithms. Radiomic textural features derived from Computed Tomography (CT) imaging are broadly used to predict benign and malignant pulmonary nodules. However, few studies have reported on the radiomics-based identification of nodular Pulmonary Cryptococcosis (PC). OBJECTIVE: This study aimed to evaluate the diagnostic and differential diagnostic value of radiomic features extracted from CT images for nodular PC. METHODS: This retrospective analysis included 44 patients with PC (29 males, 15 females), 58 with Tuberculosis (TB) (39 males, 19 females), and 60 with Lung Cancer (LC) (20 males, 40 females) confirmed pathologically. Models 1 (PC vs. non-PC), 2 (PC vs. TB), and 3 (PC vs. LC) were established using radiomic features. Models 4 (PC vs. TB) and 5 (PC vs. LC) were established based on radiomic and CT features. RESULTS: Five radiomic features were predictive of PC vs. non-PC model, but accuracy and Area Under the Curve (AUC) were 0.49 and 0.472, respectively. In model 2 (PC vs. TB) involving six radiomic features, the accuracy and AUC were 0.80 and 0.815, respectively. Model 3 (PC vs. LC) with six radiomic features performed well, with AUC=0.806 and an accuracy of 0.76. Between the PC and TB groups, model 4 combining radiomics, distribution, and PI, showed AUC=0.870. In differentiating PC from LC, the combination of radiomics, distribution, PI, and RBNAV achieved AUC=0.926 and an accuracy of 0.90. CONCLUSION: The prediction models based on radiomic features from CT images performed well in discriminating PC from TB and LC. The individualized prediction models combining radiomic and CT features achieved the best diagnostic performance.

Cryptococcal Pleuritis in an Immunocompetent Patient: A Case Report and Literature Review.

Cryptococcosis, primarily an opportunistic infection, often occurs in immunocompromised patients but can also affect immunocompetent individuals. Cryptococcosis typically manifests in the lungs, but pleurisy is rare, particularly in immunocompetent patients. This report details a case of cryptococcal pleuritis in a 74-year-old immunocompetent male with a history of heart failure, presenting initially with pleural effusion. Diagnostic challenges arose due to the initial absence of intrapulmonary lesions. The diagnosis was eventually established through a surgical biopsy and tissue culture, revealing Cryptococcus neoformans. This case underscores the complexity of diagnosing cryptococcal infections, particularly in immunocompetent patients, and highlights the need for considering cryptococcosis in differential diagnoses of lymphocyte-predominant exudative pleural effusions.

Infectious Diseases Physician Management of Cryptococcal Meningitis in North America-Is Single High-Dose Liposomal Amphotericin B Being Used?

Background: Several recent randomized trials have been conducted in resource-limited settings for cryptococcal meningitis that have rapidly innovated international guidelines. The 2010 Infectious Diseases Society of America (IDSA) cryptococcal meningitis guideline has not been updated with recent trials. The 2022 AMBITION-cm trial found that a single 10-mg/kg dose of liposomal amphotericin B plus daily flucytosine and fluconazole for 2 weeks was noninferior to 1 week of amphotericin B deoxycholate with flucytosine. It is unknown whether physicians in high-resource settings are using this regimen or more traditional regimens. Methods: We developed an electronic survey in June 2023 to better understand whether physician members of the IDSA Emerging Infections Network (EIN) and Mycoses Study Group Education and Research Consortium (MSG-ERC) had used the AMBITION-cm induction regimen, would use the regimen in hypothetical clinical scenarios, and what perceived barriers to use existed. Results: A total of 242 of 561 (43%) physicians responded to the survey, of whom 205 provided care for persons with cryptococcal meningitis in the last year. Overall, 29 (14%) had used the AMBITION-cm regimen, and 176 (86%) had not. In various hypothetical clinical scenarios, only ∼10% of 209 respondents selected the AMBITION-cm regimen as preferred. Perceived barriers to uptake included the applicability of trials performed in low-resource settings to high-resource settings, that the regimen is not recommended in the 2010 IDSA guidelines, and the applicability to persons without HIV. Conclusions: Most respondents had not used the single-dose liposomal amphotericin B regimen, but the regimen is being used. Further study of this regimen in other patient populations and settings is necessary.

Cryptococcosis-a systematic review to inform the World Health Organization Fungal Priority Pathogens List.

Cryptococcosis causes a high burden of disease worldwide. This systematic review summarizes the literature on Cryptococcus neoformans and C. gattii infections to inform the World Health Organization's first Fungal Priority Pathogen List. PubMed and Web of Science were used to identify studies reporting on annual incidence, mortality, morbidity, antifungal resistance, preventability, and distribution/emergence in the past 10 years. Mortality rates due to C. neoformans were 41%-61%. Complications included acute renal impairment, raised intracranial pressure needing shunts, and blindness. There was moderate evidence of reduced susceptibility (MIC range 16-32 mg/l) of C. neoformans to fluconazole, itraconazole, ketoconazole, voriconazole, and amphotericin B. Cryptococcus gattii infections comprised 11%-33% of all cases of invasive cryptococcosis globally. The mortality rates were 10%-23% for central nervous system (CNS) and pulmonary infections, and ∼43% for bloodstream infections. Complications described included neurological sequelae (17%-27% in C. gattii infections) and immune reconstitution inflammatory syndrome. MICs were generally low for amphotericin B (MICs: 0.25-0.5 mg/l), 5-flucytosine (MIC range: 0.5-2 mg/l), itraconazole, posaconazole, and voriconazole (MIC range: 0.06-0.5 mg/l). There is a need for increased surveillance of disease phenotype and outcome, long-term disability, and drug susceptibility to inform robust estimates of disease burden.

A sandwich chemiluminescent magnetic microparticle immunoassay for cryptococcal antigen detection.

BACKGROUND: Cryptococcosis is a global invasive mycosis associated with significant morbidity and mortality. Cryptococcal antigen (CrAg) testing from serum and cerebrospinal fluid (CSF) has been regarded as a gold standard for early diagnosis. This study aimed to develop and validate a rapid and sensitive sandwich chemiluminescent magnetic microparticle immunoassay (CMIA) for quantitative detection of CrAg in sera. RESEARCH DESIGN AND METHODS: CMIA is based on magnetic beads modified with capture antibodies and biotinylated antibodies and Streptavidin-polyHRP, where biotinylated antibodies functioned as the recognition element and Streptavidin-polyHRP as the signal component. Assay parameters were first optimized, and then assay performances were evaluated. RESULTS: Under optimized conditions, the total runtime of the CMIA was 22 min. The assay had a wide linear range (2 -10,000 ng/mL) and high analytical sensitivity (0.24 ng/mL), together with acceptable reproducibility, accuracy, and stability. Besides, it exhibited no cross-reactivity with other pathogens. Importantly, the assay showed 92.91% (95% CI, 80.97-93.02%) overall qualitative agreement with a commercial ELISA kit in a retrospective cohort of 55 cases with confirmed cryptococcal infection, and 72 controls without evidence of invasive fungal disease (IFD). CONCLUSION: These results demonstrated that the present study paved a novel strategy for reliable quantitative detection of CrAg in sera.

Inhibition of Microbicidal Activity of Canine Macrophages DH82 Cell Line by Capsular Polysaccharides from Cryptococcus neoformans.

Cryptococcus neoformans is a lethal fungus that primarily affects the respiratory system and the central nervous system. One of the main virulence factors is the capsule, constituted by the polysaccharides glucuronoxylomannan (GXM) and glucuronoxylomanogalactan (GXMGal). Polysaccharides are immunomodulators. One of the target cell populations for modulation are macrophages, which are part of the first line of defense and important for innate and adaptive immunity. It has been reported that macrophages can be modulated to act as a "Trojan horse," taking phagocytosed yeasts to strategic sites or having their machinery activation compromised. The scarcity of information on canine cryptococcosis led us to assess whether the purified capsular polysaccharides from C. neoformans would be able to modulate the microbicidal action of macrophages. In the present study, we observed that the capsular polysaccharides, GXM, GXMGal, or capsule total did not induce apoptosis in the DH82 macrophage cell line. However, it was possible to demonstrate that the phagocytic activity was decreased after treatment with polysaccharides. In addition, recovered yeasts from macrophages treated with polysaccharides after phagocytosis could be cultured, showing that their viability was not altered. The polysaccharides led to a reduction in ROS production and the mRNA expression of IL-12 and IL-6. We observed that GXMGal inhibits MHC class II expression and GXM reduces ERK phosphorylation. In contrast, GXMGal and GXM were able to increase the PPAR-γ expression. Furthermore, our data suggest that capsular polysaccharides can reduce the microbicidal activity of canine macrophages DH82.

Global trends in Cryptococcus and its interactions with the host immune system: a bibliometric analysis.

Objectives: This manuscript undertakes a systematic examination of the research landscape concerning global Cryptococcus species and their dynamism with the host immune system spanning the past decade. It furnishes a detailed survey of leading knowledge institutions and critical focal points in this area, utilizing bibliometric analysis. Methods: VOSviewer and CiteSpace software platforms were employed to systematically analyze and graphically depict the relevant literature indexed in the WoSCC database over the preceding ten years. Results: In the interval between October 1, 2013, and October 1, 2023, a corpus of 795 publications was amassed. The primary research institutions involved in this study include Duke University, the University of Minnesota, and the University of Sydney. The leading trio of nations, in terms of publication volume, comprises the United States, China, and Brazil. Among the most prolific authors are Casadevall, Arturo; Wormley, Floyd L., Jr.; and Olszewski, Michal A., with the most highly cited author being Perfect, Jr. The most esteemed journal is Mbio, while Infection and Immunity commands the highest citation frequency, and the Journal of Clinical Microbiology boasts the most significant impact factor. Present research foci encompass the intricate interactions between Cryptococcus pathogenesis and host immunity, alongside immune mechanisms, complications, and immunotherapies. Conclusion: This represents the first exhaustive scholarly review and bibliometric scrutiny of the evolving landscapes in Cryptococcus research and its interactions with the host immune system. The analyses delineated herein provide insights into prevailing research foci and trajectories, thus furnishing critical directions for subsequent inquiries in this domain.

Semisynthetic Glycoconjugate Vaccine Candidates against Cryptococcus neoformans.

Cryptococcus neoformans is a fungus classified by the World Health Organization as a critically important pathogen, which poses a significant threat to immunocompromised individuals. In this study, we present the chemical synthesis and evaluation of two semisynthetic vaccine candidates targeting the capsular polysaccharide glucuronoxylomannan (GXM) of C. neoformans. These semisynthetic glycoconjugate vaccines contain an identical synthetic decasaccharide (M2 motif) antigen. This antigen is present in serotype A strains, which constitute 95% of the clinical cryptococcosis cases. This synthetic oligosaccharide was conjugated to two proteins (CRM197 and Anthrax 63 kDa PA) and tested for immunogenicity in mice. The conjugates elicited a specific antibody response that bound to the M2 motif but also exhibited additional cross-reactivity toward M1 and M4 GXM motifs. Both glycoconjugates produced antibodies that bound to GXM in ELISA assays and to live fungal cells. Mice immunized with the CRM197 glycoconjugate produced weakly opsonic antibodies and displayed trends toward increased median survival relative to mice given a mock PBS injection (18 vs 15 days, p = 0.06). These findings indicate promise, achieving a successful vaccine demands further optimization of the glycoconjugate. This antigen could serve as a component in a multivalent GXM motif vaccine.

In Vivo Modeling of Cryptococcus neoformans Infection and Collection of Murine Samples.

In vivo models provide advantages to study the progression of disease and to identify potential biomarkers to detect and monitor infections. For the human fungal pathogen Cryptococcus neoformans, murine intranasal models aim to recapitulate natural infection from inhalation of desiccated fungal cells from the environment and permit monitoring of disease over time. In this chapter, we describe the establishment of a murine model for cryptococcosis and the subsequent collection of organs, tissues, and fluids for sampling. These samples may support novel diagnostic strategies and opportunities to monitor dissemination of the fungal cells throughout the host and propose new treatment options to combat disease.

Proteomic Profiling of Samples Derived from a Murine Model Following Cryptococcus neoformans Infection.

Proteomic profiling provides in-depth information about the regulation of diverse biological processes, activation of and communication across signaling networks, and alterations to protein production, modifications, and interactions. For infectious disease research, mass spectrometry-based proteomics enables detection of host defenses against infection and mechanisms used by the pathogen to evade such responses. In this chapter, we outline protein extraction from organs, tissues, and fluids collected following intranasal inoculation of a murine model with the human fungal pathogen Cryptococcus neoformans. We describe sample preparation, followed by purification, processing on the mass spectrometer, and a robust bioinformatics analysis. The information gleaned from proteomic profiling of fungal infections supports the detection of novel biomarkers for diagnostic and prognostic purposes.

Models for Inducing Experimental Cryptococcosis in Mice.

Cryptococcus neoformans and Cryptococcus gattii are the predominant etiological agents of cryptococcosis, a particularly problematic disease in immunocompromised individuals. The increased clinical use of immunosuppressive drugs, the inherent ability of Cryptococcus species to suppress and evade host immune responses, and the emergence of drug-resistant yeast support the need for model systems that facilitate the design of novel immunotherapies and antifungals to combat disease progression. The mouse model of cryptococcosis is a widely used system to study Cryptococcus pathogenesis and the efficacy of antifungal drugs in vivo. In this chapter, we describe three commonly used strategies to establish cryptococcosis in mice: intranasal, intratracheal, and intravenous inoculations. Also, we discuss the methodology for delivering drugs to mice via intraperitoneal injection.

Interaction Between Macrophages and Cryptococcus neoformans: Distinguishing Phagocytosed Versus External Fungi.

The interaction between macrophages and Cryptococcus neoformans is crucial in the pathogenesis of cryptococcosis. These phagocytes are important immune effectors, but also a niche in which facultative intracellular parasites, such as C. neoformans, thrive. Consequently, phagocytosis of cryptococcal cells and its outcomes are very frequently studied. One major issue with several of the tests used for this, however, is that macrophage-C. neoformans interaction does not always result in phagocytosis, as fungi may be attached to the external surface of the phagocyte. The most used methodologies to study phagocytosis of cryptococcal cells have varying degrees of precision in separating fungi that are truly internalized from those that are outside macrophages. Here we describe two assays to measure phagocytosis that can differentiate internal from external C. neoformans cells.

Measuring Laccase Activity and Melanin Production in Cryptococcus neoformans.

Melanin is a complex dark pigment synthetized by the phenoloxidase enzyme laccase in Cryptococcus neoformans. In vitro, this enzyme oxidizes exogenous catecholamines to produce melanin that may be secreted or incorporated into the fungal cell wall. This pigment has multiple roles in C. neoformans virulence during its interaction with different hosts and probably also in protecting fungal cells in the environment against predation and oxidative and radiation stresses, among others. However, it is important to note that laccase also has melanin-independent roles in C. neoformans interactions with host cells. In this chapter, we describe a quantitative laccase assay and a method for evaluating the kinetics of melanin production in C. neoformans colonies.