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The role of glucocorticoids in the treatment of bacterial or fungal meningitis is controversial. In this trial, adjunctive dexamethasone therapy in patients with HIV-associated cryptococcal meningitis did not confer a benefit and was associated with increased adverse events. Cryptococcal meningitis associated with human immunodeficiency virus (HIV) infection is estimated to cause more than 600,000 deaths each year, the vast majority in sub-Saharan Africa and in South and Southeast Asia. 1 Among patients receiving combination antifungal therapy with amphotericin B and either flucytosine or fluconazole, mortality remains more than 30% at 10 weeks, and survivors often have substantial disability. 2 , 3 There is a pressing need to improve outcomes. However, no new anticryptococcal agents are currently close to approval for clinical use, so innovative strategies are needed. Adjunctive treatments, such as glucocorticoids, have shown some benefit in central nervous system (CNS) . . .

Abstract Background We performed a phase 2 noninferiority trial examining the early fungicidal activity (EFA) of 3 short-course, high-dose liposomal amphotericin B (L-AmB) regimens for cryptococcal meningitis (CM) in Tanzania and Botswana. Methods Human immunodeficiency virus (HIV)-infected adults with CM were randomized to (i) L-AmB 10 mg/kg on day 1 (single dose); (ii) L-AmB 10 mg/kg on day 1 and 5 mg/kg on day 3 (2 doses); (iii) L-AmB 10 mg/kg on day 1 and 5 mg/kg on days 3 and 7 (3 doses); or (iv) L-AmB 3 mg/kg/day for 14 days (control). All patients also received oral fluconazole 1200 mg/day for 14 days. Primary endpoint was mean rate of clearance of cerebrospinal fluid cryptococcal infection (EFA). Noninferiority was defined as an upper limit of the 2-sided 95% confidence interval (CI) of difference in EFA between intervention and control <0.2 log10 colony-forming units (CFU)/mL/day. Results Eighty participants were enrolled. EFA for daily L-AmB was -0.41 log10 CFU/mL/day (standard deviation, 0.11; n = 17). Difference in mean EFA from control was -0.11 (95% CI, -.29 to .07) log10 CFU/mL/day faster with single dose (n = 16); -0.05 (95% CI, -.20 to .10) log10 CFU/mL/day faster with 2 doses (n = 18); and -0.13 (95% CI, -.35 to .09) log10 CFU/mL/day faster with 3 doses (n = 18). EFA in all short-course arms was noninferior to control. Ten-week mortality was 29% (n = 23) with no statistical difference between arms. All arms were well tolerated. Conclusions Single-dose 10 mg/kg L-AmB was well tolerated and led to noninferior EFA compared to 14 days of 3 mg/kg/day L-AmB in HIV-associated CM. Induction based on a single 10 mg/kg L-AmB dose is being taken forward to a phase 3 clinical endpoint trial. Clinical Trials Registration ISRCTN 10248064. This phase 2 randomized trial showed that single high-dose (10 mg/kg) liposomal amphotericin B (L-AmB) treatment was well tolerated and noninferior in terms of fungal clearance to standard 14-day L-AmB (3 mg/kg) treatment in patients with HIV-associated cryptococcal meningitis.

We evaluated the prevalence of serum and meningeal cryptococcosis in asymptomatic outpatients with advanced HIV disease (CD4 < 200 cells/mm3) in a cross-sectional screening context in Kinshasa clinics (DRC). Lumbar puncture (LP) was performed in patients with positive serum cryptococcal antigen (CrAg) test, and Cryptococcus spp. isolated from cerebrospinal fluid (CSF) were identified by MALDI-TOF-MS, and characterized using serotyping-PCR, ITS-sequencing and multilocus sequence typing (MLST). The genetic profiles obtained were then compared with those of isolates previously described in symptomatic patients in the same clinics. Forty-seven patients with advanced HIV disease out of 262 included were positive for serum CrAg (18%, 95% CI: 14.2–24.3). The prevalence of asymptomatic cryptococcal meningitis (CM) was then measured at 50% among patients with positive serum CrAg test who consented to LP (19/38). Only four CSF samples were culture positive and all were characterized as Cryptococcus neoformans , molecular type VNI and belonging to two different sequence types (ST): ST93 (3/4) and ST63 (1/4). While ST93 is also the main genomic profile described in advanced HIV disease patients with symptomatic CM in Kinshasa clinics, ST63 has not yet been identified in DRC before. It is likely that future studies involving a large number of strains will be necessary before any definitive conclusions can be drawn on the involved strains in asymptomatic patients.

Background. The standard therapy for human immunodeficiency virus (HIV)–associated cryptococcal meningitis of amphotericin B (AmB; 0.7 mg/kg per day) plus flucytosine frequently takes >2 weeks to sterilize the cerebral spinal fluid, and acute mortality remains high. A dosage range for AmB of 0.7–1 mg/kg per day is noted in current guidelines, but there are no data comparing 0.7 mg/kg per day with 1 mg/kg per day. Methods. Sixty-four HIV-seropositive, antiretroviral therapy–naive patients in Cape Town, South Africa, who experienced their first episode of cryptococcal meningitis during the period May 2005–June 2006 were randomized to receive either (1) AmB, 0.7 mg/kg per day, plus flucytosine, 25 mg/kg 4 times per day (group 1; 30 patients); or (2) AmB, 1 mg/kg per day, plus flucytosine, 25 mg/kg 4 times per day (group 2; 34 patients). Regimens were given for 2 weeks, followed by treatment with oral fluconazole. The primary outcome measure was early fungicidal activity, as determined by results of serial, quantitative cerebral spinal fluid cryptococcal cultures. Secondary outcome measures were safety and mortality. The median duration of follow-up was 1 year. Results. Early fungicidal activity was significantly greater for group 2 than for group 1 (mean±SD, −0.56±0.24 vs. −0.45±0.16 log cfu/mL of cerebral spinal fluid per day; P=.02). The incidence of renal impairment did not significantly differ between the 2 groups. Anemia was associated with female sex and, less strongly, with membership in group 2. Renal impairment and anemia reversed after the regimen was switched to fluconazole. Two- and 10-week mortality rates were 6% and 24%, respectively, with no difference between groups. Conclusions. AmB, 1 mg/kg per day, plus flucytosine is more rapidly fungicidal than is standard-dose AmB plus flucytosine. Because of its size, this study provides limited data on any difference in toxicity between the regimens, but toxicities were manageable and reversible. Clinical trials registration number. ISRCTN68133435 (http://www.controlled-trials.com).

It frequently takes more than 2 weeks for drug treatments for cryptococcal meningitis to sterilise cerebrospinal fluid (CSF). In-vitro and animal studies lend support to the use of combinations of amphotericin B, flucytosine, and fluconazole for treatment of cryptococcosis. We compared the fungicidal activity of combinations of these drugs for initial treatment of patients with cryptococcal meningitis. 64 patients with a first episode of HIV-associated cryptococcal meningitis were randomised to initial treatment with: amphotericin B (0·7 mg/kg daily); amphotericin B plus flucytosine (100 mg/kg daily); amphotericin B plus fluconazole (400 mg daily); or triple therapy with amphotericin B, flucytosine, and fluconazole. Our primary endpoint was fungicidal activity, measured by the rate of reduction in CSF cryptococcal colony-forming units (CFU) from serial quantitative CSF cultures on days 3, 7, and 14 of treatment. Baseline CSF CFU counts were an important prognostic factor. Clearance of cryptococci from the CSF was exponential and was significantly faster with amphotericin B plus flucytosine than with amphotericin B alone (p=0·0006), amphotericin B plus fluconazole (pp=0·02), or triple therapy (p=0·02). At these doses, amphotericin B plus flucytosine is the most rapidly fungicidal regimen. Quantification of CSF cultures provides a powerful new means to accurately assess the fungicidal activity of new treatment regimens for cryptococcal meningitis.

Cryptococcal meningitis (CM) causes an estimated 180,000 deaths annually, predominantly in sub-Saharan Africa, where most patients receive fluconazole (FLC) monotherapy. While relapse after FLC monotherapy with resistant strains is frequently observed, the mechanisms and impact of emergence of FLC resistance in human CM are poorly understood. Heteroresistance (HetR) - a resistant subpopulation within a susceptible strain - is a recently described phenomenon in Cryptococcus neoformans (Cn) and Cryptococcus gattii (Cg), the significance of which has not previously been studied in humans. A cohort of 20 patients with HIV-associated CM in Tanzania was prospectively observed during therapy with either FLC monotherapy or in combination with flucytosine (5FC). Total and resistant subpopulations of Cryptococcus spp. were quantified directly from patient cerebrospinal fluid (CSF). Stored isolates underwent whole genome sequencing and phenotypic characterization. Heteroresistance was detectable in Cryptococcus spp. in the CSF of all patients at baseline (i.e., prior to initiation of therapy). During FLC monotherapy, the proportion of resistant colonies in the CSF increased during the first 2 weeks of treatment. In contrast, no resistant subpopulation was detectable in CSF by day 14 in those receiving a combination of FLC and 5FC. Genomic analysis revealed high rates of aneuploidy in heteroresistant colonies as well as in relapse isolates, with chromosome 1 (Chr1) disomy predominating. This is apparently due to the presence on Chr1 of ERG11, which is the FLC drug target, and AFR1, which encodes a drug efflux pump. In vitro efflux levels positively correlated with the level of heteroresistance. Our findings demonstrate for what we believe is the first time the presence and emergence of aneuploidy-driven FLC heteroresistance in human CM, association of efflux levels with heteroresistance, and the successful suppression of heteroresistance with 5FC/FLC combination therapy. This work was supported by the Wellcome Trust Strategic Award for Medical Mycology and Fungal Immunology 097377/Z/11/Z and the Daniel Turnberg Travel Fellowship.

Human opportunistic fungi C. neoformans are a global health concern as they cause severe pneumonia and cryptococcal meningitis in tropical and subtropical regions. Though the occurrence of infection has reduced from 2014 to 2022, the mortality rate remains high, and it is considered a neglected tropical disease. More studies are needed to elucidate the genomic architecture and functional traits required to underpin the disease management of Cryptococcus neoformans . This study showcases the first complete genome sequence of an Indian isolate of Cryptococcus, identified as subsp. grubii containing 4.8% of virulence genes in a total genome. A comparative virulome analysis revealed that study genomes Cn, H99 and JEC21 harbor crucial virulence genes associated with infection severity. While all study strains possess genes promoting antifungal resistance (AFR), most lack specific SNPs defining AFR. The whole genome Single Nucleotide Polymorphism (SNP) analysis categorized the strains as hyper- and non-hypermutators. The phylogenomic and pan-genome analysis revealed that the study strains share a core genome, highlighting evolutionary relatedness. The study shows gene expansion and contraction between the study strains. This pioneering study presents Cryptococcus ’s first comprehensive genomic analysis from India, integrating genetic variations, virulome, resistome, and pan-genome dynamics. The study highlights the necessity of bioinformatic tools for efficient genome analysis and large-scale comparative genome research.

Cryptococcus is the most common fungus causing high-morbidity and -mortality human meningitis. This encapsulated yeast has a unique propensity to travel to the central nervous system to produce disease. Cryptococcus neoformans is a major human central nervous system (CNS) fungal pathogen causing considerable morbidity and mortality. In this study, we provide the widest view to date of the yeast transcriptome directly from the human subarachnoid space and within cerebrospinal fluid (CSF). We captured yeast transcriptomes from C. neoformans of various genotypes in 31 patients with cryptococcal meningoencephalitis as well as several Cryptococcus gattii infections. Using transcriptome sequencing (RNA-seq) analyses, we compared the in vivo yeast transcriptomes to those from other environmental conditions, including in vitro growth on nutritious media or artificial CSF as well as samples collected from rabbit CSF at two time points. We ranked gene expressions and identified genetic patterns and networks across these diverse isolates that reveal an emphasis on carbon metabolism, fatty acid synthesis, transport, cell wall structure, and stress-related gene functions during growth in CSF. The most highly expressed yeast genes in human CSF included those known to be associated with survival or virulence and highlighted several genes encoding hypothetical proteins. From that group, a gene encoding the CMP1 putative glycoprotein (CNAG_06000) was selected for functional studies. This gene was found to impact the virulence of Cryptococcus in both mice and the CNS rabbit model, in agreement with a recent study also showing a role in virulence. This transcriptional analysis strategy provides a view of regulated yeast genes across genetic backgrounds important for human CNS infection and a relevant resource for the study of cryptococcal genes, pathways, and networks linked to human disease. IMPORTANCE Cryptococcus is the most common fungus causing high-morbidity and -mortality human meningitis. This encapsulated yeast has a unique propensity to travel to the central nervous system to produce disease. In this study, we captured transcriptomes of yeasts directly out of the human cerebrospinal fluid, the most concerning site of infection. By comparing the RNA transcript levels with other conditions, we gained insights into how the basic machinery involved in metabolism and environmental responses enable this fungus to cause disease at this body site. This approach was applied to clinical isolates with diverse genotypes to begin to establish a genotype-agnostic understanding of how the yeast responds to stress. Based on these results, future studies can focus on how these genes and their pathways and networks can be targeted with new therapeutics and possibly classify yeasts with bad infection outcomes.

Fungal dissemination into the bloodstream is a critical step leading to invasive fungal infections. Here, using intravital imaging, we show that Kupffer cells (KCs) in the liver have a prominent function in the capture of circulating Cryptococcus neoformans and Candida albicans , thereby reducing fungal dissemination to target organs. Complement C3 but not C5, and complement receptor CRIg but not CR3, are involved in capture of C. neoformans . Internalization of C. neoformans by KCs is subsequently mediated by multiple receptors, including CR3, CRIg, and scavenger receptors, which work synergistically along with C5aR signaling. Following phagocytosis, the growth of C. neoformans is inhibited by KCs in an IFN-γ independent manner. Thus, the liver filters disseminating fungi from circulation via KCs, providing a mechanistic explanation for the enhanced risk of cryptococcosis among individuals with liver diseases, and suggesting a therapeutic strategy to prevent fungal dissemination through enhancing KC functions. Patients with liver diseases are at increased risk of fungal infections. Here the authors show that Kupffer cells are critical for the filtration of fungi out of the blood and thereby for liver-mediated protection against disseminating fungal infection.

Cryptococcus neoformans var. grubii is the causative agent of cryptococcal meningitis, a significant source of mortality in immunocompromised individuals, typically human immunodeficiency virus/AIDS patients from developing countries. Despite the worldwide emergence of this ubiquitous infection, little is known about the global molecular epidemiology of this fungal pathogen. Here we sequence the genomes of 188 diverse isolates and characterize the major subdivisions, their relative diversity, and the level of genetic exchange between them. While most isolates of C. neoformans var. grubii belong to one of three major lineages (VNI, VNII, and VNB), some haploid isolates show hybrid ancestry including some that appear to have recently interbred, based on the detection of large blocks of each ancestry across each chromosome. Many isolates display evidence of aneuploidy, which was detected for all chromosomes. In diploid isolates of C. neoformans var. grubii (serotype AA) and of hybrids with C. neoformans var. neoformans (serotype AD) such aneuploidies have resulted in loss of heterozygosity, where a chromosomal region is represented by the genotype of only one parental isolate. Phylogenetic and population genomic analyses of isolates from Brazil reveal that the previously “African” VNB lineage occurs naturally in the South American environment. This suggests migration of the VNB lineage between Africa and South America prior to its diversification, supported by finding ancestral recombination events between isolates from different lineages and regions. The results provide evidence of substantial population structure, with all lineages showing multi-continental distributions; demonstrating the highly dispersive nature of this pathogen.