The Evolution of Sexual Reproduction and the Mating-Type Locus: Links to Pathogenesis of Cryptococcus Human Pathogenic Fungi.

Cryptococcus species utilize a variety of sexual reproduction mechanisms, which generate genetic diversity, purge deleterious mutations, and contribute to their ability to occupy myriad environmental niches and exhibit a range of pathogenic potential. The bisexual and unisexual cycles of pathogenic Cryptococcus species are stimulated by properties associated with their environmental niches and proceed through well-characterized signaling pathways and corresponding morphological changes. Genes governing mating are encoded by the mating-type (MAT) loci and influence pathogenesis, population dynamics, and lineage divergence in Cryptococcus. MAT has undergone significant evolutionary changes within the Cryptococcus genus, including transition from the ancestral tetrapolar state in nonpathogenic species to a bipolar mating system in pathogenic species, as well as several internal reconfigurations. Owing to the variety of established sexual reproduction mechanisms and the robust characterization of the evolution of mating and MAT in this genus, Cryptococcus species provide key insights into the evolution of sexual reproduction.

Comparative genomics of the closely related fungal genera Cryptococcus and Kwoniella reveals karyotype dynamics and suggests evolutionary mechanisms of pathogenesis.

In exploring the evolutionary trajectories of both pathogenesis and karyotype dynamics in fungi, we conducted a large-scale comparative genomic analysis spanning the Cryptococcus genus, encompassing both global human fungal pathogens and nonpathogenic species, and related species from the sister genus Kwoniella. Chromosome-level genome assemblies were generated for multiple species, covering virtually all known diversity within these genera. Although Cryptococcus and Kwoniella have comparable genome sizes (about 19.2 and 22.9 Mb) and similar gene content, hinting at preadaptive pathogenic potential, our analysis found evidence of gene gain (via horizontal gene transfer) and gene loss in pathogenic Cryptococcus species, which might represent evolutionary signatures of pathogenic development. Genome analysis also revealed a significant variation in chromosome number and structure between the 2 genera. By combining synteny analysis and experimental centromere validation, we found that most Cryptococcus species have 14 chromosomes, whereas most Kwoniella species have fewer (11, 8, 5, or even as few as 3). Reduced chromosome number in Kwoniella is associated with formation of giant chromosomes (up to 18 Mb) through repeated chromosome fusion events, each marked by a pericentric inversion and centromere loss. While similar chromosome inversion-fusion patterns were observed in all Kwoniella species with fewer than 14 chromosomes, no such pattern was detected in Cryptococcus. Instead, Cryptococcus species with less than 14 chromosomes showed reductions primarily through rearrangements associated with the loss of repeat-rich centromeres. Additionally, Cryptococcus genomes exhibited frequent interchromosomal translocations, including intercentromeric recombination facilitated by transposons shared between centromeres. Overall, our findings advance our understanding of genetic changes possibly associated with pathogenicity in Cryptococcus and provide a foundation to elucidate mechanisms of centromere loss and chromosome fusion driving distinct karyotypes in closely related fungal species, including prominent global human pathogens.

Alternative TSS use is widespread in Cryptococcus fungi in response to environmental cues and regulated genome-wide by the transcription factor Tur1.

Alternative transcription start site (TSS) usage regulation has been identified as a major means of gene expression regulation in metazoans. However, in fungi, its impact remains elusive as its study has thus far been restricted to model yeasts. Here, we first re-analyzed TSS-seq data to define genuine TSS clusters in 2 species of pathogenic Cryptococcus. We identified 2 types of TSS clusters associated with specific DNA sequence motifs. Our analysis also revealed that alternative TSS usage regulation in response to environmental cues is widespread in Cryptococcus, altering gene expression and protein targeting. Importantly, we performed a forward genetic screen to identify a unique transcription factor (TF) named Tur1, which regulates alternative TSS (altTSS) usage genome-wide when cells switch from exponential phase to stationary phase. ChiP-Seq and DamID-Seq analyses suggest that at some loci, the role of Tur1 might be direct. Tur1 has been previously shown to be essential for virulence in C. neoformans. We demonstrated here that a tur1Δ mutant strain is more sensitive to superoxide stress and phagocytosed more efficiently by macrophages than the wild-type (WT) strain.

Epistatic genetic interactions govern morphogenesis during sexual reproduction and infection in a global human fungal pathogen.

Cellular development is orchestrated by evolutionarily conserved signaling pathways, which are often pleiotropic and involve intra- and interpathway epistatic interactions that form intricate, complex regulatory networks. Cryptococcus species are a group of closely related human fungal pathogens that grow as yeasts yet transition to hyphae during sexual reproduction. Additionally, during infection they can form large, polyploid titan cells that evade immunity and develop drug resistance. Multiple known signaling pathways regulate cellular development, yet how these are coordinated and interact with genetic variation is less well understood. Here, we conducted quantitative trait locus (QTL) analyses of a mapping population generated by sexual reproduction of two parents, only one of which is unisexually fertile. We observed transgressive segregation of the unisexual phenotype among progeny, as well as a large-cell phenotype under mating-inducing conditions. These large-cell progeny were found to produce titan cells both in vitro and in infected animals. Two major QTLs and corresponding quantitative trait genes (QTGs) were identified: RIC8 (encoding a guanine-exchange factor) and CNC06490 (encoding a putative Rho-GTPase activator), both involved in G protein signaling. The two QTGs interact epistatically with each other and with the mating-type locus in phenotypic determination. These findings provide insights into the complex genetics of morphogenesis during unisexual reproduction and pathogenic titan cell formation and illustrate how QTL analysis can be applied to identify epistasis between genes. This study shows that phenotypic outcomes are influenced by the genetic background upon which mutations arise, implicating dynamic, complex genotype-to-phenotype landscapes in fungal pathogens and beyond.

Innate phase production of IFN-γ by memory and effector T cells expressing early activation marker CD69 during infection with Cryptococcus deneoformans in the lungs.

Cryptococcus deneoformans is a yeast-type fungus that causes fatal meningoencephalitis in immunocompromised patients and evades phagocytic cell elimination through an escape mechanism. Memory T (Tm) cells play a central role in preventing the reactivation of this fungal pathogen. Among these cells, tissue-resident memory T (TRM) cells quickly respond to locally invaded pathogens. This study analyzes the kinetics of effector T (Teff) cells and Tm cells in the lungs after cryptococcal infection. Emphasis is placed on the kinetics and cytokine expression of TRM cells in the early phase of infection. CD4+ Tm cells exhibited a rapid increase by day 3, peaked at day 7, and then either maintained their levels or exhibited a slight decrease until day 56. In contrast, CD8+ Tm cells reached their peak on day 3 and thereafter decreased up to day 56 post-infection. These Tm cells were predominantly composed of CD69+ TRM cells and CD69+ CD103+ TRM cells. Disruption of the CARD9 gene resulted in reduced accumulation of these TRM cells and diminished interferon (IFN) -γ expression in TRM cells. TRM cells were derived from T cells with T cell receptors non-specific to ovalbumin in OT-II mice during cryptococcal infection. In addition, TRM cells exhibited varied behavior in different tissues. These results underscore the importance of T cells, which produce IFN-γ in the lungs during the early stage of infection, in providing early protection against cryptococcal infection through CARD9 signaling.

Description of Cryptococcosis Following SARS-CoV-2 Infection: A Disease Survey Through the Mycosis Study Group Education and Research Consortium (MSG-19).

BACKGROUND: Invasive fungal infections have been described throughout the COVID-19 pandemic. Cryptococcal disease after infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been reported in several isolated case reports and 1 larger case series. We sought to describe cryptococcal infections following SARS-CoV-2 through establishing a database to investigate underlying risk factors, disease manifestations, and outcomes. METHODS: We created a crowdsourced call for cases solicited through the Mycoses Study Group Education and Research Consortium, the Centers for Disease Control and Prevention Emerging Infectious Diseases Network, and infectious diseases Twitter groups. Data were collected in a web-based and secure REDCap survey without personal identifiers. RESULTS: Sixty-nine cases were identified and submitted by 29 separate institutional sites. Cryptococcosis was diagnosed a median of 22 days (interquartile range, 9-42 days) after SARS-CoV-2 infection. Mortality among those with available follow-up was 72% (26/36) for the immunocompetent group and 48% (15/31) for the immunocompromised group (likelihood ratio, 4.01; P = .045). We observed a correlation between disease manifestation (central nervous system infection, proven/probable disseminated disease, and respiratory) and mortality (P = .002). CONCLUSIONS: The mortality rate of 59% for patients with cryptococcosis following SARS-CoV-2 is higher than that of modern Cryptococcus cohorts. There was an association between immunocompromised status and cryptococcal disease manifestations as well as mortality. Moreover, our series emphasizes the need for clinical and laboratory assessment of opportunistic infections beyond 30 days when concerning symptoms develop.

Macrophages-derived exo-miR-4449 induced by Cryptococcus affects HUVEC permeability and promotes pyroptosis in BEAS-2B via the HIC1 pathway.

Macrophages have recently been discovered to assume a significant role in the progression of cryptococcosis. However, the potential involvement of macrophage-derived exosomes in the pathogenesis of cryptococcosis remains uncertain. In this study, we investigated the changes of microRNAs in macrophage exosomes (exo-miRNAs) in cryptococcal infections and the role of markedly altered exo-miRNAs in the modulation of Human Umbilical Vein Endothelial Cells (HUVEC) permeability and ROS accumulation and pyroptosis in Human Bronchial Epithelioid Cells (BEAS-2B). Techniques such as microarray analysis and real-time quantitative PCR were used to detect different exo-miRNAs and to screen for the most highly expressed exo-miRNAs. Then its mimics were transfected into HUVEC to study its effect on the monolayer permeability of HUVEC. Finally, the relationship between this exo-miRNAs and the ROS accumulation and pyroptosis was verified by bioinformatics analysis. The results showed that five exo-miRNAs were overexpressed and two exo-miRNAs were reduced, among which, exo-miR-4449 was expressed at the highest level. Exo-miR-4449 could be internalized by HUVEC and enhanced its monolayer permeability. Moreover, exo-miR-4449 was found to promote ROS accumulation and pyroptosis in BEAS-2B through HIC1 pathway. Thus, exo-miR-4449 plays an important role in the pathogenesis of cryptococcosis and holds promise as a significant biomarker for treatment.

The significance of low titer serum cryptococcal antigen testing from 2017 to 2023 performed in a tertiary care center.

Several false positive low serum cryptococcal antigen (SCrAg) reports by lateral flow assay (LFA) were identified in late 2016 at our tertiary care hospital. After the recall and correction of the problem in the reagent, we studied the significance of SCrAg LFA ≤ 1:10 from January 2017 to October 2023. Of 20 patients with 31 samples of SCrAg LFA ≤ 1:10, 14 patients (70%) were classified as true positives, four (20%) were indeterminate, and only two (10%) patients were false positives. If a new SCrAg LFA ≤ 1:10 is detected, it should be repeated, and additional workup should be pursued.

We studied the significance of low serum cryptococcal antigen (SCrAg) titer lateral flow assay (LFA) ≤ 1:10 from January 2017 to October 2023. Of 20 patients with SCrAg LFA ≤ 1:10, only two patients (10%) were false positives. If a new SCrAg ≤ 1:10 is detected, it should be repeated, and additional workup should be done.

Molecular epidemiological investigation of Cryptococcus spp. isolated from cats in Japan using multi-locus sequence typing.

Cryptococcosis is an important fungal infection for both humans and cats, but molecular epidemiological studies on strains isolated from cats are limited. We conducted multi-locus sequence typing analysis and antifungal susceptibility testing of 14 Cryptococcus spp. strains from domestic cats in Japan and one strain isolated from a cat in Singapore. All 14 strains from domestic cats in Japan were identified as Cryptococcus neoformans molecular type VNI. The sequence types (STs) included eight cases of ST5, five cases of ST31, and one novel ST. VNI ST5 is the most frequently isolated strain in Japanese patients as well, while there are no records of VNI ST31 being isolated from Japanese patients. The Singaporean cat strain was identified as C. gattii VGIIb (C. deuterogattii), ST7. We compared these results with strains previously reported to have been isolated from cats. This comparison suggested that molecular types of Cryptococcus spp. isolated from cats may differ depending on the country. In the antifungal susceptibility testing of C. neoformans, one strain each exceeded the epidemiological cutoff value (ECV) for amphotericin B and 5-fluorocytosine, while two strains exceeded the ECV for fluconazole. This study reveals the molecular epidemiology of Cryptococcus spp. isolated from cats with cryptococcosis in Japan. It suggests that investigating Cryptococcus spp. carried by cats, which share close living environments with humans, may contribute to the health of both cats and human populations.

Cryptococcosis is an important fungal disease in both humans and cats. We genotyped strains isolated from cats with cryptococcosis in Japan. Our findings revealed that the most common genotype infecting both cats and humans in Japan is identical.

Glucocorticoids as a risk factor for infection and adverse outcomes in non-HIV and non-transplant patients with cryptococcal meningitis.

BACKGROUND: Cryptococcal meningitis (CM), an opportunistic fungal infection affecting immunocompromised hosts, leads to high mortality. The role of previous exposure to glucocorticoids as a risk factor and as an outcome modulator has been observed, but systematic studies are lacking. OBJECTIVE: The primary aim of this study is to evaluate the impact of glucocorticoid use on the clinical outcomes, specifically mortality, of non-HIV and non-transplant (NHNT) patients diagnosed with CM. METHODS: We queried a global research network to identify adult NHNT patients with CM based on ICD codes or recorded specific Cryptococcus CSF lab results with or without glucocorticoid exposure the year before diagnosis. We performed a propensity score-matched analysis to reduce the risk of confounding and analysed outcomes by glucocorticoid exposure. We used a Cox proportional hazards model for survival analysis. RESULTS: We identified 764 patients with a history of glucocorticoid exposure and 1267 patients without who developed CM within 1 year. After propensity score matching of covariates, we obtained 627 patients in each cohort. The mortality risk in 1 year was greater in patients exposed to prior glucocorticoids (OR: 1.3, CI: 1.2-2.0, p = 0.002). We found an excess of 45 deaths among CM patients with previous glucocorticoid use (7.4% increased absolute risk of dying within 1 year of diagnosis) compared to CM controls without glucocorticoid exposure. Hospitalisation, intensive care unit admission, emergency department visits, stroke and cognitive dysfunction also showed significant, unfavourable outcomes in patients with glucocorticoid-exposed CM compared to glucocorticoid-unexposed CM patients. CONCLUSIONS: Previous glucocorticoid administration in NHNT patients seems to associate with 1-year mortality after CM adjusted for possible confounders related to demographics, comorbidities and additional immunosuppressive medications. Serial CrAg screening might be appropriate for higher-risk patients on glucocorticoids after further cost-benefit analyses.

Epigenetic dynamics of centromeres and neocentromeres in Cryptococcus deuterogattii.

Deletion of native centromeres in the human fungal pathogen Cryptococcus deuterogattii leads to neocentromere formation. Native centromeres span truncated transposable elements, while neocentromeres do not and instead span actively expressed genes. To explore the epigenetic organization of neocentromeres, we analyzed the distribution of the heterochromatic histone modification H3K9me2, 5mC DNA methylation and the euchromatin mark H3K4me2. Native centromeres are enriched for both H3K9me2 and 5mC DNA methylation marks and are devoid of H3K4me2, while neocentromeres do not exhibit any of these features. Neocentromeres in cen10Δ mutants are unstable and chromosome-chromosome fusions occur. After chromosome fusion, the neocentromere is inactivated and the native centromere of the chromosome fusion partner remains as the sole, active centromere. In the present study, the active centromere of a fused chromosome was deleted to investigate if epigenetic memory promoted the re-activation of the inactive neocentromere. Our results show that the inactive neocentromere is not re-activated and instead a novel neocentromere forms directly adjacent to the deleted centromere of the fused chromosome. To study the impact of transcription on centromere stability, the actively expressed URA5 gene was introduced into the CENP-A bound regions of a native centromere. The introduction of the URA5 gene led to a loss of CENP-A from the native centromere, and a neocentromere formed adjacent to the native centromere location. Remarkably, the inactive, native centromere remained enriched for heterochromatin, yet the integrated gene was expressed and devoid of H3K9me2. A cumulative analysis of multiple CENP-A distribution profiles revealed centromere drift in C. deuterogattii, a previously unreported phenomenon in fungi. The CENP-A-binding shifted within the ORF-free regions and showed a possible association with a truncated transposable element. Taken together, our findings reveal that neocentromeres in C. deuterogattii are highly unstable and are not marked with an epigenetic memory, distinguishing them from native centromeres.

Antifungal efficacy of photodynamic therapy on Cryptococcus and Candida species is enhanced by Streptomyces spp. extracts in vitro.

The research on actinobacteria isolated from traditional medicinal plants is limited. Here, four new Streptomyces isolates (Ha1, Pp1, UzK and UzM) were obtained from the rhizospheres of Helianthus annuus, Pongamia pinnata and Ziziphus mauritiana, frequently utilized in Indian traditional medicine. The Streptomyces isolates aqueous extracts were studied alone against the growth of the Cryptococcus neoformans H99 reference strain, the fluconazole-tolerant T1-5796 and 89-610 strains, three histone deacetylase (HDAC) genes mutant strains, C. gattii NIH198, Candida albicans, C. glabrata, C. parapsilosis and C. tropicalis to determine minimum inhibitory concentration (MIC). Next, the extracts were employed in combination with aluminium-phthalocyanine chloride nanoemulsion-mediated photodynamic therapy to evaluate a possible interaction. We demonstrated that the C. neoformans T1-5796 fluconazole-tolerant strain was more severely inhibited by the Pp1 isolate extract (MIC: 6 mg mL-1) than H99, which was not inhibited. Growth inhibition of the HDAC null mutants was more prominent for the extract of the UzM isolate, showing inhibition at 2 mg mL-1. The UzM extract was also the most effective in hindering the Candida species proliferation, with MIC values ranging from 10 to 40 mg mL-1. The four Streptomyces extracts, especially UzK and UzM, significantly enhanced the antifungal effect of the photodynamic therapy. Our results indicate these Streptomyces isolates as sources of novel metabolites which could potentiate the effect of photodynamic therapy in controlling yeasts superficial infections.

Trends in the Incidence of Disseminated Cryptococcosis in Japan: A Nationwide Observational Study, 2015-2021.

BACKGROUND: Cryptococcus species can cause severe disseminated infections in immunocompromised hosts. This study investigated the epidemiological features and trends in disseminated cryptococcosis in Japan. METHODS: We used publicly available Infectious Diseases Weekly Reports to obtain data on the incidence of disseminated cryptococcosis in Japan from 2015 to 2021. Patient information, including age, sex, and regional and seasonal data, were extracted. The Joinpoint regression program was used to determine the age-adjusted incidence rate (AAR) per 100,000 population, annual percentage change (APC), and average APC (AAPC). RESULTS: A total of 1047 cases of disseminated cryptococcosis were reported, of which those aged ≥ 70 years accounted for 68.8%. The AAR in men was significantly higher than that in women (median: 0.13 vs. 0.09: p = 0.0024). APC for the overall cases increased by 9.9% (95% confidence interval [95% CI] - 5.4-27.7) from 2015 to 2018 and then decreased by 3.3% (95% CI - 15.5-10.7) from 2018 to 2021. AAPC for the entire study period was 3.1% (95% CI - 1.5-8.0), indicating a possible increase in its number, although not statistically significant. In terms of regional distribution, the average AAR was highest in Shikoku District (0.17) and lowest in Hokkaido District (0.04). Northern Japan exhibited a significantly lower median AAR (median [interquartile range]: 0.06 [0.05, 0.08]) than the Eastern (0.12 [0.12, 0.13]), Western (0.11 [0.10, 0.13]), and Southern (0.14 [0.12, 0.15]) regions. No seasonal variation in incidence was observed. CONCLUSION: The prevalence of disseminated cryptococcosis has not increased in Japan. Geographically, the incidence is lower in Northern Japan. Further investigations that incorporate detailed clinical data are required.

Association of Antibody Immunity With Cryptococcal Antigenemia and Mortality in a South African Cohort With Advanced Human Immunodeficiency Virus Disease.

BACKGROUND: Asymptomatic cryptococcal antigenemia (positive blood cryptococcal antigen [CrAg]) is associated with increased mortality in individuals with human immunodeficiency virus (HIV) even after adjusting for CD4 count and despite receiving antifungal treatment. The association of antibody immunity with mortality in adults with HIV with cryptococcal antigenemia is unknown. METHODS: Cryptococcal capsular glucuronoxylomannan (GXM)- and naturally occurring ß-glucans (laminarin, curdlan)-binding antibodies were measured in blood samples of 197 South Africans with HIV who underwent CrAg screening and were followed up to 6 months. Associations between antibody titers, CrAg status, and all-cause mortality were sought using logistic and Cox regression, respectively. RESULTS: Compared with CrAg-negative individuals (n = 130), CrAg-positive individuals (n = 67) had significantly higher IgG1 (median, 6672; interquartile range [IQR], 4696-10 414 vs 5343, 3808-7722 µg/mL; P = .007), IgG2 (1467, 813-2607 vs 1036, 519-2012 µg/mL; P = .01), and GXM-IgG (1:170, 61-412 vs 1:117, 47-176; P = .0009) and lower curdlan-IgG (1:47, 11-133 vs 1:93, 40-206; P = .01) titers. GXM-IgG was associated directly with cryptococcal antigenemia adjusted for CD4 count and antiretroviral therapy use (odds ratio, 1.64; 95% confidence interval [CI], 1.21 to 2.22). Among CrAg-positive individuals, GXM-IgG was inversely associated with mortality at 6 months adjusted for CD4 count and tuberculosis (hazard ratio, 0.50; 95% CI, .33 to .77). CONCLUSIONS: The inverse association of GXM-IgG with mortality in CrAg-positive individuals suggests that GXM-IgG titer may have prognostic value in those individuals. Prospective longitudinal studies to investigate this hypothesis and identify mechanisms by which antibody may protect against mortality are warranted.

Outpatient Cryptococcal Antigen Screening Is Associated With Favorable Baseline Characteristics and Improved Survival in Persons With Cryptococcal Meningitis in Uganda.

BACKGROUND: It is unknown whether persons with symptomatic cryptococcal meningitis detected during routine blood cryptococcal antigen (CrAg) screening have better survival than persons presenting with overt meningitis. METHODS: We prospectively enrolled Ugandans with HIV and cryptocococcal meningitis from December 2018 to December 2021. Participants were treated with amphotericin-based combination therapy. We compared outcomes between persons who were CrAg screened then referred to hospital with those presenting directly to the hospital with symptomatic meningitis. RESULTS: Among 489 participants with cryptococcal meningitis, 40% (194/489) received blood CrAg screening and were referred to hospital (median time to referral 2 days; interquartile range [IQR], 1-6). CrAg-screened persons referred to hospital had lower 14-day mortality than non-CrAg-screened persons who presented directly to hospital with symptomatic meningitis (12% vs 21%; hazard ratio, .51; 95% confidence interval, .32-.83; P = .006). Fewer CrAg-screened participants had altered mental status versus non-CrAg-screened participants (29% vs 41%; P = .03). CrAg-screened persons had lower quantitative cerebrospinal fluid (CSF) culture burden (median [IQR], 4570 [11-100 000] vs 26 900 [182-324 000] CFU/mL; P = .01) and lower CSF opening pressures (median [IQR], 190 [120-270] vs 225 [140-340] mmH2O; P = .004) compared with non-CrAg-screened persons. CONCLUSIONS: Survival from cryptococcal meningitis was higher in persons with prior CrAg screening than those without CrAg screening. Altered mental status was the most potent predictor for mortality in a multivariate model. We suggest that CrAg screening detects cryptococcal meningitis at an earlier stage, as evidenced by a favorable baseline risk profile and notably fewer persons with altered mental status.

Cryptococcal Antigenemia in Advanced Human Immunodeficiency Virus Disease: Pathophysiology, Epidemiology, and Clinical Implications.

Cryptococcal antigen (CrAg) is detectable in blood prior to the onset of symptomatic cryptococcal meningitis (CM), a leading cause of death among people with advanced human immunodeficiency virus (HIV) disease globally. Highly sensitive assays can detect CrAg in blood, and screening people with HIV with low CD4 counts, followed by preemptive antifungal treatment, is recommended and widely implemented as part of a global strategy to prevent CM and end cryptococcal-related deaths. Cryptococcal antigenemia encompasses a spectrum of conditions from preclinical asymptomatic infection (cerebrospinal fluid [CSF] CrAg-negative) through subclinical (CSF CrAg-positive without overt meningism) to clinical symptomatic cryptococcal disease, usually manifesting as CM. In this review, we summarize current understanding of the pathophysiology, risk factors for, and clinical implications of cryptococcal antigenemia within this spectrum. We also provide an update on global prevalence, recommended screening and treatment strategies, and future considerations for improving outcomes among patients with cryptococcal antigenemia.

Cryptococcus tetragattii Meningitis Associated with Travel, Taiwan.

Meningitis caused by Cryptococcus tetragattii fungus is rare and has been found in specific geographic regions. We report a case of meningitis caused by C. tetragattii (molecular type VGIV) in an immunocompetent patient in Taiwan. The patient had traveled to Egypt and was positive for granulocyte-macrophage colony-stimulating factor autoantibody.

Tocilizumab as a Potential Adjunctive Therapy to Corticosteroids in Cryptococcal Post-infectious Inflammatory Response Syndrome (PIIRS): a Report of Two Cases.

PURPOSE: Non-HIV cryptococcal meningoencephalitis (CM) in previously healthy individuals is often complicated by a post-infectious inflammatory response syndrome (c-PIIRS) characterized by neurologic deterioration after appropriate antifungal therapy with sterilization of CSF fungal cultures. c-PIIRS results from an excessive inflammatory response to fungal antigens released during fungal lysis, mediated by IFN-γ, IL-6, and activated T-helper cells, leading to immune-mediated host damage that responds to pulse-corticosteroid taper therapy (PCT). Typically, oral steroids may take up to a year to taper, and occasionally, patients will be refractory to steroid therapy or may demonstrate high-risk lesions such as those involving intracranial arteries. Also, patients can have problematic side effects from prolonged corticosteroids. Hence, appropriate adjunctive agents are needed to reduce corticosteroid doses in the treatment of c-PIIRS. Due to a possible role of IL-6 in pathogenesis, IL-6 receptor blockade by tocilizumab may be useful in the treatment of c-PIIRS. METHODS: Two previously healthy patients with non-HIV cPIIRS were seen at the NIH. Due to concerns for intracranial vascular rupture in an area of inflammation (Patient 1) and intractable symptoms on high-dose oral corticosteroids (Patient 2) with evidence of persistent CSF inflammation, patients were treated with 4-8 mg/kg tocilizumab every 2 weeks while maintained on a constant dose of prednisone. RESULTS: Two patients exhibited rapid immunological improvement following treatment with tocilizumab. Patient 1 remained vascularly stable, and Patient 2 had near resolution of headaches with improvement in mental status as evidenced by improved MOCA score. The two had improved CSF inflammatory parameters and no significant side effects. Both CSF cultures remained negative throughout treatment. CONCLUSIONS: Tocilizumab may be a safe adjunctive treatment for CM-related PIIRS suggesting further study.

Development and validation of a machine learning model to predict prognosis in HIV-negative cryptococcal meningitis patients: a multicenter study.

PURPOSE: To predict prognosis in HIV-negative cryptococcal meningitis (CM) patients by developing and validating a machine learning (ML) model. METHODS: This study involved 523 HIV-negative CM patients diagnosed between January 1, 1998, and August 31, 2022, by neurologists from 3 tertiary Chinese centers. Prognosis was evaluated at 10 weeks after the initiation of antifungal therapy. RESULTS: The final prediction model for HIV-negative CM patients comprised 8 variables: Cerebrospinal fluid (CSF) cryptococcal count, CSF white blood cell (WBC), altered mental status, hearing impairment, CSF chloride levels, CSF opening pressure (OP), aspartate aminotransferase levels at admission, and decreased rate of CSF cryptococcal count within 2 weeks after admission. The areas under the curve (AUCs) in the internal, temporal, and external validation sets were 0.87 (95% CI 0.794-0.944), 0.92 (95% CI 0.795-1.000), and 0.86 (95% CI 0.744-0.975), respectively. An artificial intelligence (AI) model was trained to detect and count cryptococci, and the mean average precision (mAP) was 0.993. CONCLUSION: A ML model for predicting prognosis in HIV-negative CM patients was built and validated, and the model might provide a reference for personalized treatment of HIV-negative CM patients. The change in the CSF cryptococcal count in the early phase of HIV-negative CM treatment can reflect the prognosis of the disease. In addition, utilizing AI to detect and count CSF cryptococci in HIV-negative CM patients can eliminate the interference of human factors in detecting cryptococci in CSF samples and reduce the workload of the examiner.

Genetic diversity and microevolution in clinical Cryptococcus isolates from Cameroon.

Cryptococcal meningitis is the second most common cause of death in people living with HIV/AIDS, yet we have a limited understanding of how cryptococcal isolates change over the course of infection. Cryptococcal infections are environmentally acquired, and the genetic diversity of these infecting isolates can also be geographically linked. Here, we employ whole genome sequences for 372 clinical Cryptococcus isolates from 341 patients with HIV-associated cryptococcal meningitis obtained via a large clinical trial, across both Malawi and Cameroon, to enable population genetic comparisons of isolates between countries. We see that isolates from Cameroon are highly clonal, when compared to those from Malawi, with differential rates of disruptive variants in genes with roles in DNA binding and energy use. For a subset of patients (22) from Cameroon, we leverage longitudinal sampling, with samples taken at days 7 and 14 post-enrollment, to interrogate the genetic changes that arise over the course of infection, and the genetic diversity of isolates within patients. We see disruptive variants arising over the course of infection in several genes, including the phagocytosis-regulating transcription factor GAT204. In addition, in 13% of patients sampled longitudinally, we see evidence for mixed infections. This approach identifies geographically linked genetic variation, signatures of microevolution, and evidence for mixed infections across a clinical cohort of patients affected by cryptococcal meningitis in Central Africa.

Cryptococcal meningitis, caused by Cryptococcus, results in approximately half a million deaths per year globally. We compare clinical Cryptococcus samples from Cameroon and Malawi to explore the genetic diversity of these isolates. We find instances of mixed-strain infections and identify genetic variants arising in Cryptococcus over disease.