Microevolution of serial clinical isolates of Cryptococcus neoformans var. grubii and C. gattii

This is the final version. Available from the publisher via the DOI in this record.The pathogenic species of Cryptococcus are a major cause of mortality owing to severe infections in immunocompromised as well as immunocompetent individuals. Although antifungal treatment is usually effective, many patients relapse after treatment, and in such cases, comparative analyses of the genomes of incident and relapse isolates may reveal evidence of determinative, microevolutionary changes within the host. Here, we analyzed serial isolates cultured from cerebrospinal fluid specimens of 18 South African patients with recurrent cryptococcal meningitis. The time between collection of the incident isolates and collection of the relapse isolates ranged from 124 days to 290 days, and the analyses revealed that, during this period within the patients, the isolates underwent several genetic and phenotypic changes. Considering the vast genetic diversity of cryptococcal isolates in subSaharan Africa, it was not surprising to find that the relapse isolates had acquired different genetic and correlative phenotypic changes. They exhibited various mechanisms for enhancing virulence, such as growth at 39°C, adaptation to stress, and capsule production; a remarkable amplification of ERG11 at the native and unlinked locus may provide stable resistance to fluconazole. Our data provide a deeper understanding of the microevolution of Cryptococcus species under pressure from antifungal chemotherapy and host immune responses. This investigation clearly suggests a promising strategy to identify novel targets for improved diagnosis, therapy, and prognosis.Wellcome TrustNational Institute of Allergy and Infectious Disease

A Prospective Longitudinal Study of the Clinical Outcomes from Cryptococcal Meningitis following Treatment Induction with 800 mg Oral Fluconazole in Blantyre, Malawi

Introduction: Cryptococcal meningitis is the most common neurological infection in HIV infected patients in Sub Saharan Africa, where gold standard treatment with intravenous amphotericin B and 5 flucytosine is often unavailable or difficult to administer. Fluconazole monotherapy is frequently recommended in national guidelines but is a fungistatic drug compromised by uncertainty over optimal dosing and a paucity of clinical end-point outcome data. Methods: From July 2010 until March 2011, HIV infected adults with a first episode of cryptococcal meningitis were recruited at Queen Elizabeth Central Hospital, Blantyre, Malawi. Patients were treated with oral fluconazole monotherapy 800 mg daily, as per national guidelines. ART was started at 4 weeks. Outcomes and factors associated with treatment failure were assessed 4, 10 and 52 weeks after fluconazole initiation. Results: Sixty patients were recruited. 26/60 (43%) died by 4 weeks. 35/60 (58.0%) and 43/56 (77%) died or failed treatment by 10 or 52 weeks respectively. Reduced consciousness (Glasgow Coma Score ,14 of 15), moderate/severe neurological disability (modified Rankin Score .3 of 5) and confusion (Abbreviated Mental Test Score ,8 of 10) were all common at baseline and associated with death or treatment failure. ART prior to recruitment was not associated with better outcomes. Conclusions: Mortality and treatment failure from cryptococcal meningitis following initiation of treatment with 800 mg oral fluconazole is unacceptably high. To improve outcomes, there is an urgent need for better therapeutic strategies and point-of-care diagnostics, allowing earlier diagnosis before development of neurological deficit

Revision and Update of the Consensus Definitions of Invasive Fungal Disease From the European Organization for Research and Treatment of Cancer and the Mycoses Study Group Education and Research Consortium.

BACKGROUND: Invasive fungal diseases (IFDs) remain important causes of morbidity and mortality. The consensus definitions of the Infectious Diseases Group of the European Organization for Research and Treatment of Cancer and the Mycoses Study Group have been of immense value to researchers who conduct clinical trials of antifungals, assess diagnostic tests, and undertake epidemiologic studies. However, their utility has not extended beyond patients with cancer or recipients of stem cell or solid organ transplants. With newer diagnostic techniques available, it was clear that an update of these definitions was essential. METHODS: To achieve this, 10 working groups looked closely at imaging, laboratory diagnosis, and special populations at risk of IFD. A final version of the manuscript was agreed upon after the groups' findings were presented at a scientific symposium and after a 3-month period for public comment. There were several rounds of discussion before a final version of the manuscript was approved. RESULTS: There is no change in the classifications of "proven," "probable," and "possible" IFD, although the definition of "probable" has been expanded and the scope of the category "possible" has been diminished. The category of proven IFD can apply to any patient, regardless of whether the patient is immunocompromised. The probable and possible categories are proposed for immunocompromised patients only, except for endemic mycoses. CONCLUSIONS: These updated definitions of IFDs should prove applicable in clinical, diagnostic, and epidemiologic research of a broader range of patients at high-risk

A MIQE-Compliant Real-Time PCR Assay for Aspergillus Detection

PMCID: PMC3393739This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

Hospital-acquired invasive pulmonary aspergillosis in patients with hepatic failure

<p>Abstract</p> <p>Background</p> <p>Invasive pulmonary aspergillosis (IPA) is a rapid, progressive, fatal disease that occurs mostly in immunocompromised patients. Patients with severe liver disease are at a heightened risk for infections. Little is known about the clinical presentation including predisposing factors and treatment of IPA in patients with hepatic failure.</p> <p>Methods</p> <p>Medical records of patients with hepatic failure between November 2005 and February 2007 were reviewed for lung infection. Nine medical records of definitive diagnosis of IPA and three of probable IPA were identified.</p> <p>Results</p> <p>The main predisposing factors were found to be prolonged antibiotic therapy and steroid exposure. Clinical signs and radiological findings were non-specific and atypical. Timely use of caspofungin was found to reduce the mortality due to the disease.</p> <p>Conclusion</p> <p>A high index of suspicion is required for early IPA diagnosis in patients with hepatic failure.</p

High dose fluconazole in salvage therapy for HIV-uninfected cryptococcal meningitis.

BACKGROUND: The 2010 Infectious Diseases Society of America (IDSA) guidelines for management of cryptococcal diseases recommend high dose fluconazole (≥ 800 mg/day), either alone or with other antifungal drugs, as alternative anticryptococcal choices. But evidence for its use in the treatment of HIV-uninfected cryptococcal meningitis (CM) remains sparse. METHODS: A retrospective analysis of HIV-uninfected CM patients who received fluconazole 800 mg/day for salvage therapy from January 2011 to December 2016 at Huashan Hospital, Shanghai, China was performed. Efficacy and safety were assessed, and mortality and prognostic factors evaluated. RESULTS: A total of 44 patients were studied including 19 refractory to amphotericin B induction therapy, 8 refractory to fluconazole consolidation therapy (400 mg/d), and 17 intolerant of antifungal drugs. For salvage, 11 patients received triple therapy of high dose fluconazole, amphotericin B and flucytosine, 20 received dual therapy of high dose fluconazole and flucytosine, 13 received monotherapy of high dose fluconazole. Median duration of high dose fluconazole in salvage regimens was 136.5 days (range, 1-667 days). Clinical response rates were 72.1% (31/43) and 83.7% (36/43) when assessed at 2 weeks and the end of salvage therapy, respectively. Adverse events possibly related to high dose fluconazole occurred in 54.5% (24/44) of the patients, and all were mild or moderate. From the initiation of salvage therapy, 1-year all-cause mortality was 13.6% (6 of 44 patients) among the study population with no significant difference in refractory or intolerant patients. CONCLUSIONS: Adherence to guideline recommendations of high dose fluconazole, alone or in combination with other antifungals, was safe and often effective for salvage therapy of HIV-uninfected CM patients

Deletion of Cryptococcus neoformans AIF Ortholog Promotes Chromosome Aneuploidy and Fluconazole-Resistance in a Metacaspase-Independent Manner

Apoptosis is a form of programmed cell death critical for development and homeostasis in multicellular organisms. Apoptosis-like cell death (ALCD) has been described in several fungi, including the opportunistic human pathogen Cryptococcus neoformans. In addition, capsular polysaccharides of C. neoformans are known to induce apoptosis in host immune cells, thereby contributing to its virulence. Our goals were to characterize the apoptotic signaling cascade in C. neoformans as well as its unique features compared to the host machinery to exploit the endogenous fungal apoptotic pathways as a novel antifungal strategy in the future. The dissection of apoptotic pathways revealed that apoptosis-inducing factor (Aif1) and metacaspases (Mca1 and Mca2) are independently required for ALCD in C. neoformans. We show that the apoptotic pathways are required for cell fusion and sporulation during mating, indicating that apoptosis may occur during sexual development. Previous studies showed that antifungal drugs induce ALCD in fungi and that C. neoformans adapts to high concentrations of the antifungal fluconazole (FLC) by acquisition of aneuploidy, especially duplication of chromosome 1 (Chr1). Disruption of aif1, but not the metacaspases, stimulates the emergence of aneuploid subpopulations with Chr1 disomy that are resistant to fluconazole (FLCR) in vitro and in vivo. FLCR isolates in the aif1 background are stable in the absence of the drug, while those in the wild-type background readily revert to FLC sensitivity. We propose that apoptosis orchestrated by Aif1 might eliminate aneuploid cells from the population and defects in this pathway contribute to the selection of aneuploid FLCR subpopulations during treatment. Aneuploid clinical isolates with disomies for chromosomes other than Chr1 exhibit reduced AIF1 expression, suggesting that inactivation of Aif1 might be a novel aneuploidy-tolerating mechanism in fungi that facilitates the selection of antifungal drug resistance

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 enrolment, 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

Tracing Genetic Exchange and Biogeography of Cryptococcus neoformans var. grubii at the Global Population Level.

Cryptococcus neoformans var. grubii is the causative agent of cryptococcal meningitis, a significant source of mortality in immunocompromised individuals, typically HIV/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 characterized 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 A/A) and of hybrids with C. neoformans var. neoformans (serotype A/D) 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