C5a-licensed phagocytes drive sterilizing immunity during systemic fungal infection.

Systemic candidiasis is a common, high-mortality, nosocomial fungal infection. Unexpectedly, it has emerged as a complication of anti-complement C5-targeted monoclonal antibody treatment, indicating a critical niche for C5 in antifungal immunity. We identified transcription of complement system genes as the top biological pathway induced in candidemic patients and as predictive of candidemia. Mechanistically, C5a-C5aR1 promoted fungal clearance and host survival in a mouse model of systemic candidiasis by stimulating phagocyte effector function and ERK- and AKT-dependent survival in infected tissues. C5ar1 ablation rewired macrophage metabolism downstream of mTOR, promoting their apoptosis and enhancing mortality through kidney injury. Besides hepatocyte-derived C5, local C5 produced intrinsically by phagocytes provided a key substrate for antifungal protection. Lower serum C5a concentrations or a C5 polymorphism that decreases leukocyte C5 expression correlated independently with poor patient outcomes. Thus, local, phagocyte-derived C5 production licenses phagocyte antimicrobial function and confers innate protection during systemic fungal infection.

Fab-dimerized glycan-reactive antibodies are a structural category of natural antibodies.

Natural antibodies (Abs) can target host glycans on the surface of pathogens. We studied the evolution of glycan-reactive B cells of rhesus macaques and humans using glycosylated HIV-1 envelope (Env) as a model antigen. 2G12 is a broadly neutralizing Ab (bnAb) that targets a conserved glycan patch on Env of geographically diverse HIV-1 strains using a unique heavy-chain (VH) domain-swapped architecture that results in fragment antigen-binding (Fab) dimerization. Here, we describe HIV-1 Env Fab-dimerized glycan (FDG)-reactive bnAbs without VH-swapped domains from simian-human immunodeficiency virus (SHIV)-infected macaques. FDG Abs also recognized cell-surface glycans on diverse pathogens, including yeast and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike. FDG precursors were expanded by glycan-bearing immunogens in macaques and were abundant in HIV-1-naive humans. Moreover, FDG precursors were predominately mutated IgM+IgD+CD27+, thus suggesting that they originated from a pool of antigen-experienced IgM+ or marginal zone B cells.

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.

Donor-Derived Mycoplasma and Ureaplasma Infections in Lung Transplant Recipients: A Prospective Study of Donor and Recipient Respiratory Tract Screening and Recipient Outcomes.

Mycoplasma hominis and Ureaplasma species are urogenital mollicutes that can cause serious donor-derived infections in lung transplant recipients. Best practices for mollicute screening remain unknown. We conducted a single center prospective study analyzing lung transplants performed from 10/5/20 - 9/5/21 whereby donor and recipient bronchoalveolar lavage (BAL) samples obtained at time of transplant underwent mollicute screening via culture and polymerase chain reaction (PCR). Of 115 total lung transplants performed, 99 (86%) donors underwent combined mollicute BAL culture and PCR testing. The study cohort included these 99 donors and their matched recipients. In total, 18 (18%) of 99 donors screened positive via culture or PCR. Among recipients, 92 (93%) of 99 had perioperative BAL screening performed, and only 3 (3%) had positive results. After transplant, 9 (9%) recipients developed mollicute infection. Sensitivity of donor screening in predicting recipient mollicute infection was 67% (6/9) via culture and 56% (5/9) via PCR. Positive predictive value (PPV) for donor culture was 75% (6/8), compared to 33% (5/15) for PCR. Donor screening via culture predicted all serious recipient mollicute infections and had better PPV than PCR; however, neither screening test predicted all mollicute infections. Independent of screening results, clinicians should remain suspicious for post-transplant mollicute infection.

Risk factors, management, and clinical outcomes of invasive Mycoplasma and Ureaplasma infections after lung transplantation.

Mollicute infections, caused by Mycoplasma and Ureaplasma species, are serious complications after lung transplantation; however, understanding of the epidemiology and outcomes of these infections remains limited. We conducted a single-center retrospective study of 1156 consecutive lung transplants performed from 2010-2019. We used log-binomial regression to identify risk factors for infection and analyzed clinical management and outcomes. In total, 27 (2.3%) recipients developed mollicute infection. Donor characteristics independently associated with recipient infection were age ≤40 years (prevalence rate ratio [PRR] 2.6, 95% CI 1.0-6.9), White race (PRR 3.1, 95% CI 1.1-8.8), and purulent secretions on donor bronchoscopy (PRR 2.3, 95% CI 1.1-5.0). Median time to diagnosis was 16 days posttransplant (IQR: 11-26 days). Mollicute-infected recipients were significantly more likely to require prolonged ventilatory support (66.7% vs 21.4%), undergo dialysis (44.4% vs 6.3%), and remain hospitalized ≥30 days (70.4% vs 27.4%) after transplant. One-year posttransplant mortality in mollicute-infected recipients was 12/27 (44%), compared to 148/1129 (13%) in those without infection (P <.0001). Hyperammonemia syndrome occurred in 5/27 (19%) mollicute-infected recipients, of whom 3 (60%) died within 10 weeks posttransplant. This study highlights the morbidity and mortality associated with mollicute infection after lung transplantation and the need for better screening and management protocols.

Mucorales: A systematic review to inform the World Health Organization priority list of fungal pathogens.

The World Health Organization, in response to the growing burden of fungal disease, established a process to develop a fungal priority pathogens list (FPPL). This systematic review aimed to evaluate the epidemiology and impact of invasive fungal disease due to Mucorales. PubMed and Web of Science were searched to identify studies published between January 1, 2011 and February 23, 2021. Studies reporting on mortality, inpatient care, complications and sequelae, antifungal susceptibility, risk factors, preventability, annual incidence, global distribution, and emergence during the study time frames were selected. Overall, 24 studies were included. Mortality rates of up to 80% were reported. Antifungal susceptibility varied across agents and species, with the minimum inhibitory concentrations lowest for amphotericin B and posaconazole. Diabetes mellitus was a common risk factor, detected in 65%-85% of patients with mucormycosis, particularly in those with rhino-orbital disease (86.9%). Break-through infection was detected in 13.6%-100% on azole or echinocandin antifungal prophylaxis. The reported prevalence rates were variable, with some studies reporting stable rates in the USA of 0.094-0.117/10 000 discharges between 2011 and 2014, whereas others reported an increase in Iran from 16.8% to 24% between 2011 and 2015. Carefully designed global surveillance studies, linking laboratory and clinical data, are required to develop clinical breakpoints to guide antifungal therapy and determine accurate estimates of complications and sequelae, annual incidence, trends, and global distribution. These data will provide robust estimates of disease burden to refine interventions and better inform future FPPL.

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.

Aspergillus fumigatus-a systematic review to inform the World Health Organization priority list of fungal pathogens.

Recognizing the growing global burden of fungal infections, the World Health Organization established a process to develop a priority list of fungal pathogens (FPPL). In this systematic review, we aimed to evaluate the epidemiology and impact of invasive infections caused by Aspergillus fumigatus to inform the first FPPL. The pre-specified criteria of mortality, inpatient care, complications and sequelae, antifungal susceptibility, risk factors, preventability, annual incidence, global distribution, and emergence were used to search for relevant articles between 1 January 2016 and 10 June 2021. Overall, 49 studies were eligible for inclusion. Azole antifungal susceptibility varied according to geographical regions. Voriconazole susceptibility rates of 22.2% were reported from the Netherlands, whereas in Brazil, Korea, India, China, and the UK, voriconazole susceptibility rates were 76%, 94.7%, 96.9%, 98.6%, and 99.7%, respectively. Cross-resistance was common with 85%, 92.8%, and 100% of voriconazole-resistant A. fumigatus isolates also resistant to itraconazole, posaconazole, and isavuconazole, respectively. The incidence of invasive aspergillosis (IA) in patients with acute leukemia was estimated at 5.84/100 patients. Six-week mortality rates in IA cases ranged from 31% to 36%. Azole resistance and hematological malignancy were poor prognostic factors. Twelve-week mortality rates were significantly higher in voriconazole-resistant than in voriconazole-susceptible IA cases (12/22 [54.5%] vs. 27/88 [30.7%]; P = .035), and hematology patients with IA had significantly higher mortality rates compared with solid-malignancy cases who had IA (65/217 [30%] vs. 14/78 [18%]; P = .04). Carefully designed surveillance studies linking laboratory and clinical data are required to better inform future FPPL.

Pichia kudriavzevii (Candida krusei): A systematic review to inform the World Health Organisation priority list of fungal pathogens.

In response to the growing global threat of fungal infections, in 2020 the World Health Organisation (WHO) established an Expert Group to identify priority fungi and develop the first WHO fungal priority pathogen list (FPPL). The aim of this systematic review was to evaluate the features and global impact of invasive infections caused by Pichia kudriavzevii (formerly known as Candida krusei). PubMed and Web of Science were used to identify studies published between 1 January 2011 and 18 February 2021 reporting on the criteria of mortality, morbidity (defined as hospitalisation and length of stay), drug resistance, preventability, yearly incidence, and distribution/emergence. Overall, 33 studies were evaluated. Mortality rates of up to 67% in adults were reported. Despite the intrinsic resistance of P. kudriavzevii to fluconazole with decreased susceptibility to amphotericin B, resistance (or non-wild-type rate) to other azoles and echinocandins was low, ranging between 0 and 5%. Risk factors for developing P. kudriavzevii infections included low birth weight, prior use of antibiotics/antifungals, and an underlying diagnosis of gastrointestinal disease or cancer. The incidence of infections caused by P. kudriavzevii is generally low (∼5% of all Candida-like blood isolates) and stable over the 10-year timeframe, although additional surveillance data are needed. Strategies targeting the identified risk factors for developing P. kudriavzevii infections should be developed and tested for effectiveness and feasibility of implementation. Studies presenting data on epidemiology and susceptibility of P. kudriavzevii were scarce, especially in low- and middle-income countries (LMICs). Thus, global surveillance systems are required to monitor the incidence, susceptibility, and morbidity of P. kudriavzevii invasive infections to inform diagnosis and treatment. Timely species-level identification and susceptibility testing should be conducted to reduce the high mortality and limit the spread of P. kudriavzevii in healthcare facilities.

Fusarium species,Scedosporium species, and Lomentospora prolificans: A systematic review to inform the World Health Organization priority list of fungal pathogens.

Recognizing the growing global burden of fungal infections, the World Health Organization established a process to develop a priority list of fungal pathogens (FPPL). In this systematic review, we aimed to evaluate the epidemiology and impact of infections caused by Fusarium spp., Scedosporium spp., and Lomentospora prolificans to inform the first FPPL. PubMed and Web of Sciences databases were searched to identify studies published between January 1, 2011 and February 23, 2021, reporting on mortality, complications and sequelae, antifungal susceptibility, preventability, annual incidence, and trends. Overall, 20, 11, and 9 articles were included for Fusarium spp., Scedosporium spp., and L. prolificans, respectively. Mortality rates were high in those with invasive fusariosis, scedosporiosis, and lomentosporiosis (42.9%-66.7%, 42.4%-46.9%, and 50.0%-71.4%, respectively). Antifungal susceptibility data, based on small isolate numbers, showed high minimum inhibitory concentrations (MIC)/minimum effective concentrations for most currently available antifungal agents. The median/mode MIC for itraconazole and isavuconazole were ≥16 mg/l for all three pathogens. Based on limited data, these fungi are emerging. Invasive fusariosis increased from 0.08 cases/100 000 admissions to 0.22 cases/100 000 admissions over the time periods of 2000-2009 and 2010-2015, respectively, and in lung transplant recipients, Scedosporium spp. and L. prolificans were only detected from 2014 onwards. Global surveillance to better delineate antifungal susceptibility, risk factors, sequelae, and outcomes is required.

Susceptibility to Cryptococcus neoformans Infection with Bruton's Tyrosine Kinase Inhibition.

Patients receiving the Bruton's tyrosine kinase (BTK) inhibitor ibrutinib have an increased likelihood of fungal infections. The objectives of this study were to determine if Cryptococcus neoformans infection severity was isolate dependent with BTK inhibition and whether blocking BTK impacted infection severity in a mouse model. We compared four clinical isolates from patients on ibrutinib to virulent (H99) and avirulent (A1-35-8) reference strains. BTK knockout (KO) and wild-type (WT) C57 mice and WT CD1 mice were infected by intranasal (i.n.), oropharyngeal aspiration (OPA), and intravenous (i.v.) routes. Infection severity was assessed by survival and fungal burden (CFU per gram of tissue). Ibrutinib (25 mg/kg) or vehicle was administered daily through intraperitoneal injections. In the BTK KO model, no isolate-dependent effect on fungal burden was observed, and infection severity was not significantly different from that of the WT with i.n., OPA, and i.v. routes. Ibrutinib treatment did not impact infection severity. However, when the four clinical isolates were compared to H99, two of these isolates were less virulent, with significantly longer survival and reduced rates of brain infection. In conclusion, C. neoformans infection severity in the BTK KO model does not appear to be isolate dependent. BTK KO and ibrutinib treatment did not result in significantly different infection severities. However, based on repeated clinical observations of increased susceptibility to fungal infections with BTK inhibitor therapy, further work is needed to optimize a mouse model with BTK inhibition to better understand the role that this pathway plays in susceptibility to C. neoformans infection.

Pharmacodynamics of ATI-2307 in a rabbit model of cryptococcal meningoencephalitis.

Cryptococcal meningoencephalitis (CM) is a devastating fungal disease with high morbidity and mortality. The current regimen that is standard-of-care involves a combination of three different drugs administered for up to one year. There is a critical need for new therapies due to both toxicity and inadequate fungicidal activity of the currently available antifungal drugs. ATI-2307 is a novel aryl amidine that disrupts the mitochondrial membrane potential and inhibits the respiratory chain complexes of fungi-it thus represents a new mechanism for direct antifungal action. Furthermore, ATI-2307 selectively targets fungal mitochondria via a fungal-specific transporter that is not present in mammalian cells. It has very potent in vitro anticryptococcal activity. In this study, the efficacy of ATI-2307 was tested in a rabbit model of CM. ATI-2307 demonstrated significant fungicidal activity at dosages between 1 and 2 mg/kg/d, and these results were superior to fluconazole and similar to amphotericin B treatment. When ATI-2307 was combined with fluconazole, the antifungal effect was greater than either therapy alone. While ATI-2307 has potent anticryptococcal activity in the subarachnoid space, its ability to reduce yeasts in the brain parenchyma was relatively less over the same study period. This new drug, with its unique mechanism of fungicidal action and ability to positively interact with an azole, has demonstrated sufficient anticryptococcal potential in this experimental setting to be further evaluated in clinical studies.

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.

Development and validation of a colorimetric antifungal susceptibility testing method for the dimorphic fungus Talaromyces marneffei.

Antifungal drug resistance is an emerging cause of treatment failure in invasive fungal infections, and antifungal susceptibility testing (AFST) may inform treatment decisions. Currently, there are no established AFST guidelines for Talaromyces marneffei (Tm) or other dimorphic fungi. We developed a colorimetric AFST method using a fluorescent redox indicator alamarBlue, which changes from blue to pink in proportion to cellular metabolic activity. We determined the optimal time for alamarBlue addition to be 24 h post-inoculation and for MIC reading to be 72 h post-inoculation. Our method allows three ways to determine minimum inhibitory concentration (MIC): visual inspection of color change, optical density, and fluorescence intensity. We validated the assay by determining the MICs for seven antifungals against 32 Tm clinical isolates and assessed the essential agreement (EA) and inter-rater reliability between our alamarBlue and the Clinical Laboratory Standard Institute (CLSI) broth microdilution methods. The MIC ranges (from low to high) were: 0.008-0.025 µg/ml for itraconazole, 0.004-0.13 µg/ml for voriconazole, 0.03-0.13 µg/ml for posaconazole, 0.06-0.5 µg/ml for flucytosine, 0.5-1 µg/ml for amphotericin B, 0.5-4 µg/ml for caspofungin, and 0.5-16 µg/ml for fluconazole. The EAs were 100% between all three MIC readouts of the alamarBlue method, and 94%-100% between the alamarBlue and CLSI methods. Our alamarBlue method had substantially higher inter-rater agreement and offers a more reliable method that can be standardized across laboratories in both high- and low-resource settings compared to the established CLSI methodology.

We developed a colorimetric alamarBlue method to determine the susceptibility of antifungal drugs against Talaromyces marneffei. We observed excellent agreement between the alamarBlue method and the Clinical Laboratory Standard Institute broth microdilution method, and the alamarBlue method had substantially higher inter-rater agreement.

The current state of Clinical Mycology in Eastern and South-Eastern Europe.

The ability of medical centers in Eastern and South-Eastern Europe to diagnose and treat fungal infections remains unknown. In order to investigate that, here we conducted a cross-sectional online survey, released at both The International Society for Human & Animal Mycology (ISHAM) and European Confederation of Medical Mycology (ECMM) websites. A total of 31 institutions responded to the questionnaire. Most centers (87.1%, n = 27) had access to Aspergillus spp. ELISA galactomannan testing as well as to Cryptococcus spp. antigen testing (83.9%, n = 26). Serological tests were mostly available for Aspergillus species (80.6%, n = 25); and most institutions reported access to mold-active antifungal drugs (83.9%; n = 26), but 5-flucytosine was available to only 29% (n = 9) of the participant centers. In conclusion, this study represents the first attempt to document the strengths and limitations of the Eastern and South-Eastern European region for diagnosing and treating fungal diseases. LAY SUMMARY: Our article is about the availability of diagnostic and treatments tools related to fungal infections in the countries of Eastern and South-Eastern region. Surveys like these are important to understand the gaps and point towards the fungal infections as a global health issue.

Cryptococcal Antigen in Serum and Cerebrospinal Fluid for Detecting Cryptococcal Meningitis in Adults Living With Human Immunodeficiency Virus: Systematic Review and Meta-Analysis of Diagnostic Test Accuracy Studies.

Cryptococcal antigen (CrAg) detection could direct the timely initiation of antifungal therapy. We searched MEDLINE and Embase for studies where CrAg detection in serum/cerebrospinal fluid (CSF) and CSF fungal culture were done on adults living with human immunodeficiency virus (HIV) who had suspected cryptococcal meningitis (CM). With Quality Assessment of Diagnostic Accuracy Studies 2 (QUADAS-2), we evaluated the risk of bias in 11 included studies with 3600 participants, and used a random-effects meta-analysis to obtain summary sensitivity and specificity of serum and CSF CrAg, as well as agreement between CSF CrAg and CSF culture. Summary sensitivity and specificity of serum CrAg were 99.7% (97.4-100) and 94.1% (88.3-98.1), respectively, and summary sensitivity and specificity of CSF CrAg were 98.8% (96.2-99.6) and 99.3% (96.7-99.9), respectively. Agreement between CSF CrAg and CSF culture was 98% (97-99). In adults living with HIV who have CM symptoms, serum CrAg negativity may rule out CM, while positivity should prompt induction antifungal therapy if lumbar puncture is not feasible. In a first episode of CM, CSF CrAg positivity is diagnostic.

Superiority of a Novel Mp1p Antigen Detection Enzyme Immunoassay Compared to Standard BACTEC Blood Culture in the Diagnosis of Talaromycosis.

BACKGROUND: Talaromycosis is an invasive mycosis endemic in Southeast Asia and causes substantial morbidity and mortality in individuals with advanced human immunodeficiency virus (HIV) disease. Current diagnosis relies on isolating Talaromyces marneffei in cultures, which takes up to 14 days and is detectable only during late-stage infection, leading to high mortality. METHODS: In this retrospective case-control study, we assessed the accuracy of a novel Mp1p antigen-detecting enzyme immunoassay (EIA) in stored plasma samples of 372 patients who had culture-proven talaromycosis from blood or sterile body fluids (reference standard) and 517 individuals without talaromycosis (338 healthy volunteers; 179 with other infections). All participants were recruited between 2011 and 2017 in Vietnam. RESULTS: Of cases and controls, 66.1% and 75.4%, respectively, were male; the median age was 33 and 37, respectively. All cases were HIV infected; median CD4 count was 10 cells/µL. At an optical density cutoff of 0.5, the specificity was 98.1% (95% CI, 96.3%-99.0%); the sensitivity was superior to blood culture (86.3% [95% CI, 82.3%-89.5%] vs 72.8% [95% CI, 68.0%-77.2%]) (P < .001, McNemar test). The time to diagnosis was 6 hours vs 6.6 ± 3.0 days for blood culture. Paired plasma and urine testing in the same patients (n = 269) significantly increased sensitivity compared to testing plasma alone or testing urine alone (P < .001 and P = .02, respectively, McNemar test). CONCLUSIONS: The Mp1p EIA is highly specific and is superior in sensitivity and time to diagnosis compared to blood culture for the diagnosis of talaromycosis. Paired plasma and urine testing further increases sensitivity, introducing a new tool for rapid diagnosis, enabling early treatment and potentially reducing mortality.

MSG07: An International Cohort Study Comparing Epidemiology and Outcomes of Patients With Cryptococcus neoformans or Cryptococcus gattii Infections.

BACKGROUND: Cryptococcosis due to Cryptococcus neoformans and Cryptococcus gattii varies with geographic region, populations affected, disease manifestations, and severity of infection, which impact treatment. METHODS: We developed a retrospective cohort of patients diagnosed with culture-proven cryptococcosis during 1995-2013 from 5 centers in North America and Australia. We compared underlying diseases, clinical manifestations, treatment, and outcomes in patients with C. gattii or C. neoformans infection. RESULTS: A total of 709 patients (452 C. neoformans; 257 C. gattii) were identified. Mean age was 50.2 years; 61.4% were male; and 52.3% were white. Time to diagnosis was prolonged in C. gattii patients compared with C. neoformans (mean, 52.2 vs 36.0 days; P < .003), and there was a higher proportion of C. gattii patients without underlying disease (40.5% vs 10.2%; P < .0001). Overall, 59% had central nervous system (CNS) infection, with lung (42.5%) and blood (24.5%) being common sites. Pulmonary infection was more common in patients with C. gattii than in those with C. neoformans (60.7% vs 32.1%; P < .0001). CNS or blood infections were more common in C. neoformans-infected patients (P ≤ .0001 for both). Treatment of CNS disease with induction therapy of amphotericin B and flucytosine occurred in 76.4% of patients. Crude 12-month mortality was higher in patients with C. neoformans (28.4% vs 20.2%; odds ratio, 1.56 [95% confidence interval, 1.08-2.26]). CONCLUSIONS: This study emphasizes differences in species-specific epidemiology and outcomes of patients with cryptococcosis, including underlying diseases, site of infection, and mortality. Species identification in patients with cryptococcosis is necessary to discern epidemiologic patterns, guide treatment regimens, and predict clinical progression and outcomes.

Correction: Cryptococcus neoformans resists to drastic conditions by switching to viable but non-culturable cell phenotype.

[This corrects the article DOI: 10.1371/journal.ppat.1007945.].

Cryptococcus neoformans resists to drastic conditions by switching to viable but non-culturable cell phenotype.

Metabolically quiescent pathogens can persist in a viable non-replicating state for months or even years. For certain infectious diseases, such as tuberculosis, cryptococcosis, histoplasmosis, latent infection is a corollary of this dormant state, which has the risk for reactivation and clinical disease. During murine cryptococcosis and macrophage uptake, stress and host immunity induce Cryptococcus neoformans heterogeneity with the generation of a sub-population of yeasts that manifests a phenotype compatible with dormancy (low stress response, latency of growth). In this subpopulation, mitochondrial transcriptional activity is regulated and this phenotype has been considered as a hallmark of quiescence in stem cells. Based on these findings, we worked to reproduce this phenotype in vitro and then standardize the experimental conditions to consistently generate this dormancy in C. neoformans. We found that incubation of stationary phase yeasts (STAT) in nutriment limited conditions and hypoxia for 8 days (8D-HYPOx) was able to produced cells that mimic the phenotype obtained in vivo. In these conditions, mortality and/or apoptosis occurred in less than 5% of the yeasts compared to 30-40% of apoptotic or dead yeasts upon incubation in normoxia (8D-NORMOx). Yeasts in 8D-HYPOx harbored a lower stress response, delayed growth and less that 1% of culturability on agar plates, suggesting that these yeasts are viable but non culturable cells (VBNC). These VBNC were able to reactivate in the presence of pantothenic acid, a vitamin that is known to be involved in quorum sensing and a precursor of acetyl-CoA. Global metabolism of 8D-HYPOx cells showed some specific requirements and was globally shut down compared to 8D-NORMOx and STAT conditions. Mitochondrial analyses showed that the mitochondrial mass increased with mitochondria mostly depolarized in 8D-HYPOx compared to 8D-NORMox, with increased expression of mitochondrial genes. Proteomic and transcriptomic analyses of 8D-HYPOx revealed that the number of secreted proteins and transcripts detected also decreased compared to 8D-NORMOx and STAT, and the proteome, secretome and transcriptome harbored specific profiles that are engaged as soon as four days of incubation. Importantly, acetyl-CoA and the fatty acid pathway involving mitochondria are required for the generation and viability maintenance of VBNC. Altogether, these data show that we were able to generate for the first time VBNC phenotype in C. neoformans. This VBNC state is associated with a specific metabolism that should be further studied to understand dormancy/quiescence in this yeast.