Attributable mortality of candidemia - Results from the ECMM Candida III multinational European Observational Cohort Study.

INTRODUCTION: Despite antifungal advancements, candidaemia still has a high mortality rate of up to 40%. The ECMM Candida III study in Europe investigated the changing epidemiology and outcomes of candidaemia for better understanding and management of these infections. METHODS: In this observational cohort study, participating hospitals enrolled the first ten consecutive adults with blood culture-proven candidemia. Collected data included patient demographics, risk factors, hospital stay duration (follow-up of 90 days), diagnostic procedures, causative Candida spp., management details, and outcome. Controls were included in a 1:1 fashion from the same hospitals. The matching process ensured similarity in age (10-year range), primary underlying disease, hospitalization in intensive care versus non-ICU ward, and major surgery within 2 weeks before candidemia between cases and controls. Overall and attributable mortality were described, and a survival probability for cases and controls was performed. RESULTS: One hundred seventy-one pairs consisting of patients with candidemia and matched controls from 28 institutions were included. In those with candidemia, overall mortality was 40.4%. Attributable mortality was 18.1% overall but differed between causative Candida species (7.7% for Candida albicans, 23.7% for Candida glabrata/Nakaseomyces glabratus, 7.7% for Candida parapsilosis and 63.6% for Candida tropicalis). Regarding risk factors, the presence of a central venous catheter, total parenteral nutrition and acute or chronic renal disease were significantly more common in cases versus controls. Duration of hospitalization, and especially that of ICU stay, was significantly longer in candidemia cases (20 (IQR 10-33) vs 15 days (IQR 7-28); p = 0.004). CONCLUSIONS: Although overall and attributable mortality in this subgroup analysis of matched case/control pairs remains high, the attributable mortality appears to have decreased in comparison to historical cohorts. This decrease may be driven by improved prognosis of Candida albicans and Candida parapsilosis candidemia; whereas candidemia due to other Candida spp. exhibits a much higher attributable mortality.

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.

Global guideline for the diagnosis and management of cryptococcosis: an initiative of the ECMM and ISHAM in cooperation with the ASM.

Cryptococcosis is a major worldwide disseminated invasive fungal infection. Cryptococcosis, particularly in its most lethal manifestation of cryptococcal meningitis, accounts for substantial mortality and morbidity. The breadth of the clinical cryptococcosis syndromes, the different patient types at-risk and affected, and the vastly disparate resource settings where clinicians practice pose a complex array of challenges. Expert contributors from diverse regions of the world have collated data, reviewed the evidence, and provided insightful guideline recommendations for health practitioners across the globe. This guideline offers updated practical guidance and implementable recommendations on the clinical approaches, screening, diagnosis, management, and follow-up care of a patient with cryptococcosis and serves as a comprehensive synthesis of current evidence on cryptococcosis. This Review seeks to facilitate optimal clinical decision making on cryptococcosis and addresses the myriad of clinical complications by incorporating data from historical and contemporary clinical trials. This guideline is grounded on a set of core management principles, while acknowledging the practical challenges of antifungal access and resource limitations faced by many clinicians and patients. More than 70 societies internationally have endorsed the content, structure, evidence, recommendation, and pragmatic wisdom of this global cryptococcosis guideline to inform clinicians about the past, present, and future of care for a patient with cryptococcosis.

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.

The importance of antimicrobial resistance in medical mycology.

Prior to the SARS-CoV-2 pandemic, antibiotic resistance was listed as the major global health care priority. Some analyses, including the O'Neill report, have predicted that deaths due to drug-resistant bacterial infections may eclipse the total number of cancer deaths by 2050. Although fungal infections remain in the shadow of public awareness, total attributable annual deaths are similar to, or exceeds, global mortalities due to malaria, tuberculosis or HIV. The impact of fungal infections has been exacerbated by the steady rise of antifungal drug resistant strains and species which reflects the widespread use of antifungals for prophylaxis and therapy, and in the case of azole resistance in Aspergillus, has been linked to the widespread agricultural use of antifungals. This review, based on a workshop hosted by the Medical Research Council and the University of Exeter, illuminates the problem of antifungal resistance and suggests how this growing threat might be mitigated.

Evaluation of perturbed iron-homeostasis in a prospective cohort of patients with COVID-19.

Background: Marked reductions in serum iron concentrations are commonly induced during the acute phase of infection. This phenomenon, termed hypoferremia of inflammation, leads to inflammatory anemia, but could also have broader pathophysiological implications. In patients with coronavirus disease 2019 (COVID-19), hypoferremia is associated with disease severity and poorer outcomes, although there are few reported cohorts. Methods: In this study, we leverage a well characterised prospective cohort of hospitalised COVID-19 patients and perform a set of analyses focussing on iron and related biomarkers and both acute severity of COVID-19 and longer-term symptomatology. Results: We observed no associations between acute serum iron and long-term outcomes (including fatigue, breathlessness or quality of life); however, lower haemoglobin was associated with poorer quality of life. We also quantified iron homeostasis associated parameters, demonstrating that among 50 circulating mediators of inflammation IL-6 concentrations were strongly associated with serum iron, consistent with its central role in inflammatory control of iron homeostasis. Surprisingly, we observed no association between serum hepcidin and serum iron concentrations. We also observed elevated erythroferrone concentrations in COVID-19 patients with anaemia of inflammation. Conclusions: These results enhance our understanding of the regulation and pathophysiological consequences of disturbed iron homeostasis during SARS-CoV-2 infection.

Safety and Efficacy of Intermittent High-Dose Liposomal Amphotericin B Antifungal Prophylaxis in Haemato-Oncology: An Eight-Year Single-Centre Experience and Review of the Literature.

Triazoles remain first-line agents for antifungal prophylaxis in high-risk haemato-oncology patients, but their use is increasingly contraindicated due to drug-drug interactions and additive toxicities with novel treatments. In this retrospective, single-centre, observational study, we present our eight-year experience of antifungal prophylaxis using intermittent high-dose liposomal Amphotericin B (L-AmB). All adults identified through our Antifungal Stewardship Programme as receiving L-AmB prophylaxis at 7.5 mg/kg once-weekly between February 2012 and January 2020 were included. Adverse reactions, including infusion reactions, electrolyte loss, and nephrotoxicity, were recorded. 'Breakthrough' invasive fungal infection (IFI) occurring within four weeks of L-AmB was classified using European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group and the National Institute of Allergy and Infectious Diseases Mycoses Study Group (EORTC/MSG) criteria. Moreover, 114 courses of intermittent high-dose L-AmB prophylaxis administered to 92 unique patients were analysed. Hypokalaemia was the most common grade 3-4 adverse event, with 26 (23%) courses. Grade 3 nephrotoxicity occurred in 8 (7%) and reversed in all six patients surviving to 90 days. There were two (1.8%) episodes of breakthrough IFI, one 'probable' and one 'possible'. In this study, the largest evaluation of intermittent high-dose L-AmB prophylaxis conducted to date, toxicity was manageable and reversible and breakthrough IFI was rare. L-AmB prophylaxis represents a viable alternative for patients with a contraindication to triazoles.

Fluconazole Monotherapy Is a Suboptimal Option for Initial Treatment of Cryptococcal Meningitis Because of Emergence of Resistance.

Cryptococcal meningitis is a lethal disease with few therapeutic options. Induction therapy with fluconazole has been consistently demonstrated to be associated with suboptimal microbiological and clinical outcomes. Exposure to fluconazole causes dynamic changes in antifungal susceptibility, which are associated with the development of aneuploidy. The implications of this phenomenon for pharmacodynamics of fluconazole for cryptococcal meningitis are poorly understood. The pharmacodynamics of fluconazole were studied using a hollow-fiber infection model (HFIM) and a well-characterized murine model of cryptococcal meningoencephalitis. The relationship between drug exposure and both antifungal killing and the emergence of resistance was quantified. The same relationships were further evaluated in a recently described group of patients with cryptococcal meningitis undergoing induction therapy with fluconazole at 800 to 1,200 mg/day. The pattern of emergence of fluconazole resistance followed an "inverted U." Resistance amplification was maximal and suppressed at ratios of the area under the concentration-time curve for the free, unbound fraction of the drug to the MIC (fAUC:MIC) of 34.5 to 138 and 305.6, respectively. Emergence of resistance was observed in vivo with an fAUC:MIC of 231.4. Aneuploidy with duplication of chromosome 1 was demonstrated to be the underlying mechanism in both experimental models. The pharmacokinetic (PK)-pharmacodynamic model accurately described the PK, antifungal killing, and emergence of resistance. Monte Carlo simulations from the clinical pharmacokinetic-pharmacodynamic model showed that only 12.8% of simulated patients receiving fluconazole at 1,200 mg/day achieved sterilization of the cerebrospinal fluid (CSF) after 2 weeks and that 83.4% had a persistent subpopulation that was resistant to fluconazole. Fluconazole is primarily ineffective due to the emergence of resistance. Treatment with 1,200 mg/day leads to the killing of a susceptible subpopulation but is compromised by the emergence of resistance.IMPORTANCE Cryptococcal meningitis is a lethal disease with few treatment options. The incidence remains high and intricately linked with the HIV/AIDS epidemic. In many parts of the world, fluconazole is the only agent that is available for the initial treatment of cryptococcal meningitis despite considerable evidence that it is associated with suboptimal microbiological and clinical outcomes. Fluconazole has a fungistatic mode of action: it predominantly inhibits growth rather than causing fungal killing. Our work shows that the pattern of fluconazole activity is caused by the emergence of resistance in Cryptococcus not detected by standard susceptibility tests, with chromosomal duplication/aneuploidy as the main mechanism. Resistance emergence is related to drug exposure and occurs with the use of clinically relevant regimens. Hence, fluconazole (and potentially other agents that target 14-alpha-demethylase) is compromised by an intrinsic property that limits its effectiveness. However, this resistance may be potentially overcome by dosage escalation or the use of combination therapy.

Dynamic ploidy changes drive fluconazole resistance in human cryptococcal meningitis.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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. FUNDING: 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.

Effectiveness of an antifungal stewardship programme at a London teaching hospital 2010-16.

Background: The need for antifungal stewardship is gaining recognition with increasing incidence of invasive fungal infection (IFI) and antifungal resistance alongside the high cost of antifungal drugs. Following an audit showing suboptimal practice we initiated an antifungal stewardship programme and prospectively evaluated its impact on clinical and financial outcomes. Patients and methods: From October 2010 to September 2016, adult inpatients receiving amphotericin B, echinocandins, intravenous fluconazole, flucytosine or voriconazole were reviewed weekly by an infectious diseases consultant and antimicrobial pharmacist. Demographics, diagnosis by European Organization for Research and Treatment of Cancer (EORTC) criteria, drug, indication, advice, acceptance and in-hospital mortality were recorded. Antifungal consumption and expenditure, and candidaemia species and susceptibility data were extracted from pharmacy and microbiology databases. Results: A total of 432 patients were reviewed, most commonly receiving AmBisome® (35%) or intravenous fluconazole (29%). Empirical treatment was often unnecessary, with 82% having no evidence of IFI. Advice was given in 64% of reviews (most commonly de-escalating or stopping treatment) and was followed in 84%. Annual antifungal expenditure initially reduced by 30% (£0.98 million to £0.73 million), then increased to 20% above baseline over a 5 year period; this was a significantly lower rise compared with national figures, which showed a doubling of expenditure over the same period. Inpatient mortality, Candida species distribution and rates of resistance were not adversely affected by the intervention. Conclusions: Provision of specialist input to optimize antifungal prescribing resulted in significant cost savings without compromising on microbiological or clinical outcomes. Our model is readily implementable by hospitals with high numbers of at-risk patients and antifungal expenditure.

Leave no one behind: response to new evidence and guidelines for the management of cryptococcal meningitis in low-income and middle-income countries.

In 2018, WHO issued guidelines for the diagnosis, prevention, and management of HIV-related cryptococcal disease. Two strategies are recommended to reduce the high mortality associated with HIV-related cryptococcal meningitis in low-income and middle-income countries (LMICs): optimised combination therapies for confirmed meningitis cases and cryptococcal antigen screening programmes for ambulatory people living with HIV who access care. WHO's preferred therapy for the treatment of HIV-related cryptococcal meningitis in LMICs is 1 week of amphotericin B plus flucytosine, and the alternative therapy is 2 weeks of fluconazole plus flucytosine. In the ACTA trial, 1-week (short course) amphotericin B plus flucytosine resulted in a 10-week mortality of 24% (95% CI -16 to 32) and 2 weeks of fluconazole and flucytosine resulted in a 10-week mortality of 35% (95% CI -29 to 41). However, with widely used fluconazole monotherapy, mortality because of HIV-related cryptococcal meningitis is approximately 70% in many African LMIC settings. Therefore, the potential to transform the management of HIV-related cryptococcal meningitis in resource-limited settings is substantial. Sustainable access to essential medicines, including flucytosine and amphotericin B, in LMICs is paramount and the focus of this Personal View.

Transcriptional Profiling of Patient Isolates Identifies a Novel TOR/Starvation Regulatory Pathway in Cryptococcal Virulence.

Human infection with Cryptococcus causes up to a quarter of a million AIDS-related deaths annually and is the most common cause of nonviral meningitis in the United States. As an opportunistic fungal pathogen, Cryptococcus neoformans is distinguished by its ability to adapt to diverse host environments, including plants, amoebae, and mammals. In the present study, comparative transcriptomics of the fungus within human cerebrospinal fluid identified expression profiles representative of low-nutrient adaptive responses. Transcriptomics of fungal isolates from a cohort of HIV/AIDS patients identified high expression levels of an alternative carbon nutrient transporter gene, STL1, to be associated with poor early fungicidal activity, an important clinical prognostic marker. Mouse modeling and pathway analysis demonstrated a role for STL1 in mammalian pathogenesis and revealed that STL1 expression is regulated by a novel multigene regulatory mechanism involving the CAC2 subunit of the chromatin assembly complex 1, CAF-1. In this pathway, the global regulator of virulence gene VAD1 was found to transcriptionally regulate a cryptococcal homolog of a cytosolic protein, Ecm15, in turn required for nuclear transport of the Cac2 protein. Derepression of STL1 by the CAC2-containing CAF-1 complex was mediated by Cac2 and modulated binding and suppression of the STL1 enhancer element. Derepression of STL1 resulted in enhanced survival and growth of the fungus in the presence of low-nutrient, alternative carbon sources, facilitating virulence in mice. This study underscores the utility of ex vivo expression profiling of fungal clinical isolates and provides fundamental genetic understanding of saprophyte adaption to the human host.IMPORTANCECryptococcus is a fungal pathogen that kills an estimated quarter of a million individuals yearly and is the most common cause of nonviral meningitis in the United States. The fungus is carried in about 10% of the adult population and, after reactivation, causes disease in a wide variety of immunosuppressed individuals, including the HIV infected and patients receiving transplant conditioning, cancer therapy, or corticosteroid therapy for autoimmune diseases. The fungus is widely carried in the soil but can also cause infections in plants and mammals. However, the mechanisms for this widespread ability to infect a variety of hosts are poorly understood. The present study identified adaptation to low nutrients as a key property that allows the fungus to inhabit these diverse environments. Further studies identified a nutrient transporter gene, STL1, to be upregulated under low nutrients and to be associated with early fungicidal activity, a marker of poor clinical outcome in a cohort of HIV/AIDS patients. Understanding molecular mechanisms involved in adaptation to the human host may help to design better methods of control and treatment of widely dispersed fungal pathogens such as Cryptococcus.

Early versus delayed antiretroviral treatment in HIV-positive people with cryptococcal meningitis.

BACKGROUND: There remains uncertainty about the optimum timing of antiretroviral therapy (ART) initiation in HIV-positive people with cryptococcal meningitis. This uncertainty is the result of conflicting data on the mortality risk and occurrence of immune reconstitution inflammatory syndrome (IRIS) when ART is initiated less than four weeks after cryptococcal meningitis treatment is commenced. OBJECTIVES: To compare the outcomes of early initiation of ART (less than four weeks after starting antifungal treatment) versus delayed initiation of ART (four weeks or more after starting antifungal treatment) in HIV-positive people with concurrent cryptococcal meningitis. SEARCH METHODS: We searched the Cochrane Central Register of Controlled Trials (CENTRAL), MEDLINE, and Embase for trials published between 1 January 1980 and 7 August 2017. We additionally searched international trial registries, including ClinicalTrials.gov and WHO International Clinical Trials Registry Platform (ICTRP), and conference abstracts from the International AIDS Society (IAS) and the Conference on Retroviruses and Opportunistic Infections (CROI) for ongoing or unpublished studies between 2015 and 2017. We reviewed reference lists of included studies to identify additional studies. SELECTION CRITERIA: We included randomized controlled trials (RCTs) that compared early versus delayed ART initiation in HIV-positive people with cryptococcal meningitis. Children, adults, and adolescents from any setting were eligible for inclusion. DATA COLLECTION AND ANALYSIS: Two review authors independently applied the inclusion criteria and extracted data. We presented dichotomous outcomes as risk ratios (RR) with 95% confidence intervals (CIs). We presented time-to-death data as hazard ratios with 95% CIs. We assessed the certainty of the evidence using the GRADE approach. MAIN RESULTS: Four trials including 294 adult participants met the inclusion criteria of this review. Participants were predominantly from low- and middle-income countries. Two trials treated cryptococcal meningitis with amphotericin B and fluconazole; a third trial used fluconazole monotherapy; and the fourth trial did not specify the antifungal used.Early ART initiation may increase all-cause mortality compared to delayed ART initiation (RR 1.42, 95% CI 1.02 to 1.97; 294 participants, 4 trials; low-certainty evidence). Early ART initiation may reduce relapse of cryptococcal meningitis compared to delayed ART initiation (RR 0.27, 95% CI 0.07 to 1.04; 205 participants, 2 trials, low-certainty evidence). We are uncertain whether early ART initiation increases or reduces cryptococcal IRIS events compared to delayed ART initiation (RR 3.56, 95% CI 0.51 to 25.02; 205 participants, 2 trials; I2 = 54%; very low-certainty evidence). We are uncertain if early ART initiation increases or reduces virological suppression at six months compared to delayed ART initiation (RR 0.93, 95% CI 0.72 to 1.22; 205 participants, 2 trials; I2 statistic = 0%; very low-certainty evidence).We were unable to pool results related to rate of fungal clearance for the two trials that reported this outcome; individual trial results indicated that there was no difference in cerebrospinal fluid fungal clearance between trial arms. Similarly, we were unable to pool results on adverse events for the trials reporting on this outcome; individual trial results indicated no difference in the occurrence of grade 3 to 5 adverse events between trial arms.Three of the four included trials had an overall low or unclear risk of bias related to the primary outcome of all-cause mortality. However, we assessed one trial as at high risk of bias due to selective outcome reporting and other bias. This, in addition to the few clinical events and imprecision of effect estimates, led to downgrading of the evidence to low or very low certainty. AUTHORS' CONCLUSIONS: The results of this review are relevant to HIV-positive adults with cryptococcal meningitis in low- and middle-income countries. These data suggest a higher risk of mortality among people who initiate ART within four weeks of cryptococcal meningitis diagnosis. However, it is unclear if this higher mortality risk is related to cryptococcal meningitis-IRIS.

The Cryptococcus neoformans Titan cell is an inducible and regulated morphotype underlying pathogenesis.

Fungal cells change shape in response to environmental stimuli, and these morphogenic transitions drive pathogenesis and niche adaptation. For example, dimorphic fungi switch between yeast and hyphae in response to changing temperature. The basidiomycete Cryptococcus neoformans undergoes an unusual morphogenetic transition in the host lung from haploid yeast to large, highly polyploid cells termed Titan cells. Titan cells influence fungal interaction with host cells, including through increased drug resistance, altered cell size, and altered Pathogen Associated Molecular Pattern exposure. Despite the important role these cells play in pathogenesis, understanding the environmental stimuli that drive the morphological transition, and the molecular mechanisms underlying their unique biology, has been hampered by the lack of a reproducible in vitro induction system. Here we demonstrate reproducible in vitro Titan cell induction in response to environmental stimuli consistent with the host lung. In vitro Titan cells exhibit all the properties of in vivo generated Titan cells, the current gold standard, including altered capsule, cell wall, size, high mother cell ploidy, and aneuploid progeny. We identify the bacterial peptidoglycan subunit Muramyl Dipeptide as a serum compound associated with shift in cell size and ploidy, and demonstrate the capacity of bronchial lavage fluid and bacterial co-culture to induce Titanisation. Additionally, we demonstrate the capacity of our assay to identify established (cAMP/PKA) and previously undescribed (USV101) regulators of Titanisation in vitro. Finally, we investigate the Titanisation capacity of clinical isolates and their impact on disease outcome. Together, these findings provide new insight into the environmental stimuli and molecular mechanisms underlying the yeast-to-Titan transition and establish an essential in vitro model for the future characterization of this important morphotype.

Antifungal Combinations for Treatment of Cryptococcal Meningitis in Africa.

BACKGROUND: Cryptococcal meningitis accounts for more than 100,000 human immunodeficiency virus (HIV)-related deaths per year. We tested two treatment strategies that could be more sustainable in Africa than the standard of 2 weeks of amphotericin B plus flucytosine and more effective than the widely used fluconazole monotherapy. METHODS: We randomly assigned HIV-infected adults with cryptococcal meningitis to receive an oral regimen (fluconazole [1200 mg per day] plus flucytosine [100 mg per kilogram of body weight per day] for 2 weeks), 1 week of amphotericin B (1 mg per kilogram per day), or 2 weeks of amphotericin B (1 mg per kilogram per day). Each patient assigned to receive amphotericin B was also randomly assigned to receive fluconazole or flucytosine as a partner drug. After induction treatment, all the patients received fluconazole consolidation therapy and were followed to 10 weeks. RESULTS: A total of 721 patients underwent randomization. Mortality in the oral-regimen, 1-week amphotericin B, and 2-week amphotericin B groups was 18.2% (41 of 225), 21.9% (49 of 224), and 21.4% (49 of 229), respectively, at 2 weeks and was 35.1% (79 of 225), 36.2% (81 of 224), and 39.7% (91 of 229), respectively, at 10 weeks. The upper limit of the one-sided 97.5% confidence interval for the difference in 2-week mortality was 4.2 percentage points for the oral-regimen group versus the 2-week amphotericin B groups and 8.1 percentage points for the 1-week amphotericin B groups versus the 2-week amphotericin B groups, both of which were below the predefined 10-percentage-point noninferiority margin. As a partner drug with amphotericin B, flucytosine was superior to fluconazole (71 deaths [31.1%] vs. 101 deaths [45.0%]; hazard ratio for death at 10 weeks, 0.62; 95% confidence interval [CI], 0.45 to 0.84; P=0.002). One week of amphotericin B plus flucytosine was associated with the lowest 10-week mortality (24.2%; 95% CI, 16.2 to 32.1). Side effects, such as severe anemia, were more frequent with 2 weeks than with 1 week of amphotericin B or with the oral regimen. CONCLUSIONS: One week of amphotericin B plus flucytosine and 2 weeks of fluconazole plus flucytosine were effective as induction therapy for cryptococcal meningitis in resource-limited settings. (ACTA Current Controlled Trials number, ISRCTN45035509 .).

A Population Genomics Approach to Assessing the Genetic Basis of Within-Host Microevolution Underlying Recurrent Cryptococcal Meningitis Infection.

Recurrence of meningitis due to Cryptococcus neoformans after treatment causes substantial mortality in HIV/AIDS patients across sub-Saharan Africa. In order to determine whether recurrence occurred due to relapse of the original infecting isolate or reinfection with a different isolate weeks or months after initial treatment, we used whole-genome sequencing (WGS) to assess the genetic basis of infection in 17 HIV-infected individuals with recurrent cryptococcal meningitis (CM). Comparisons revealed a clonal relationship for 15 pairs of isolates recovered before and after recurrence showing relapse of the original infection. The two remaining pairs showed high levels of genetic heterogeneity; in one pair we found this to be a result of infection by mixed genotypes, while the second was a result of nonsense mutations in the gene encoding the DNA mismatch repair proteins MSH2, MSH5, and RAD5 These nonsense mutations led to a hypermutator state, leading to dramatically elevated rates of synonymous and nonsynonymous substitutions. Hypermutator phenotypes owing to nonsense mutations in these genes have not previously been reported in C. neoformans, and represent a novel pathway for rapid within-host adaptation and evolution of resistance to first-line antifungal drugs.

Genomic epidemiology of Cryptococcus yeasts identifies adaptation to environmental niches underpinning infection across an African HIV/AIDS cohort.

Emerging infections caused by fungi have become a widely recognized global phenomenon and are causing an increasing burden of disease. Genomic techniques are providing new insights into the structure of fungal populations, revealing hitherto undescribed fine-scale adaptations to environments and hosts that govern their emergence as infections. Cryptococcal meningitis is a neglected tropical disease that is responsible for a large proportion of AIDS-related deaths across Africa; however, the ecological determinants that underlie a patient's risk of infection remain largely unexplored. Here, we use genome sequencing and ecological genomics to decipher the evolutionary ecology of the aetiological agents of cryptococcal meningitis, Cryptococcus neoformans and Cryptococcus gattii, across the central African country of Zambia. We show that the occurrence of these two pathogens is differentially associated with biotic (macroecological) and abiotic (physical) factors across two key African ecoregions, Central Miombo woodlands and Zambezi Mopane woodlands. We show that speciation of Cryptococcus has resulted in adaptation to occupy different ecological niches, with C. neoformans found to occupy Zambezi Mopane woodlands and C. gattii primarily recovered from Central Miombo woodlands. Genome sequencing shows that C. neoformans causes 95% of human infections in this region, of which over three-quarters belonged to the globalized lineage VNI. We show that VNI infections are largely associated with urbanized populations in Zambia. Conversely, the majority of C. neoformans isolates recovered in the environment belong to the genetically diverse African-endemic lineage VNB, and we show hitherto unmapped levels of genomic diversity within this lineage. Our results reveal the complex evolutionary ecology that underpins the reservoirs of infection for this, and likely other, deadly pathogenic fungi.