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 October 5, 2020, to September 25, 2021, 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 for donor culture was 75% (6/8), compared with 33% (5/15) for PCR. Donor screening via culture predicted all serious recipient mollicute infections and had better positive predictive value than PCR; however, neither screening test predicted all mollicute infections. Independent of screening results, clinicians should remain suspicious for posttransplant mollicute infection.

Structures of trehalose-6-phosphate synthase, Tps1, from the fungal pathogen Cryptococcus neoformans: A target for antifungals.

Invasive fungal diseases are a major threat to human health, resulting in more than 1.5 million annual deaths worldwide. The arsenal of antifungal therapeutics remains limited and is in dire need of drugs that target additional biosynthetic pathways that are absent from humans. One such pathway involves the biosynthesis of trehalose. Trehalose is a disaccharide that is required for pathogenic fungi to survive in their human hosts. In the first step of trehalose biosynthesis, trehalose-6-phosphate synthase (Tps1) converts UDP-glucose and glucose-6-phosphate to trehalose-6-phosphate. Here, we report the structures of full-length Cryptococcus neoformans Tps1 (CnTps1) in unliganded form and in complex with uridine diphosphate and glucose-6-phosphate. Comparison of these two structures reveals significant movement toward the catalytic pocket by the N terminus upon ligand binding and identifies residues required for substrate binding, as well as residues that stabilize the tetramer. Intriguingly, an intrinsically disordered domain (IDD), which is conserved among Cryptococcal species and closely related basidiomycetes, extends from each subunit of the tetramer into the "solvent" but is not visible in density maps. We determined that the IDD is not required for C. neoformans Tps1-dependent thermotolerance and osmotic stress survival. Studies with UDP-galactose highlight the exquisite substrate specificity of CnTps1. In toto, these studies expand our knowledge of trehalose biosynthesis in Cryptococcus and highlight the potential of developing antifungal therapeutics that disrupt the synthesis of this disaccharide or the formation of a functional tetramer and the use of cryo-EM in the structural characterization of CnTps1-ligand/drug complexes.

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.

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.

Biolistic Transformation of Cryptococcus neoformans.

Biolistic transformation of Cryptococcus neoformans is used as a molecular tool to genetically alter or delete targeted genes. The DNA is introduced into the yeast on DNA-coated gold beads by a helium shock wave produced using a biolistic particle system. The procedure often involves insertion of a dominant selectable marker into the desired site by homologous recombination. To increase the likelihood of homologous recombination, large fragments of overlapping DNA are used. The two most used dominant selectable markers are nourseothricin and Geneticin. With the need to generate multiple gene deletions in the same strain, there are recyclable marker systems, such as the bacteriophage P1 Cre-loxP system or CRISPR that provide additional useful molecular tools. While newer strategies exist to generate deletions and introduce markers and other gene modifications, biolistic transformation has remained a viable tool to facilitate the construction of genetically modified yeast strains. This chapter provides a working protocol on how to delete and restore a gene in C. neoformans.

The Rabbit Model of Cryptococcal Meningitis.

Cryptococcal meningitis (CM) is a fungal disease caused by the invasion of Cryptococcus yeast cells into the central nervous system. The organism is thought to enter the body through the lungs and then escape due to dysregulation of the immune response. Multiple animal species have been used to model the infection and characterize CM including mice, rats, dogs, guinea pigs, and rabbits. The rabbit model has over 40 years of data and has been used to study host-pathogen interactions and the efficacy of antifungal therapeutics. The model begins with immune suppression to eliminate the lymphocytic cell population followed by direct infection of the central nervous system via an injection of a suspension of yeast cells into the cisterna magna. The organism remains in the CNS during the course of infection, and cerebrospinal fluid can be repeatedly sampled to quantify the burden of organism, measure drug levels in the CSF, profile the immune response in the CSF, and/or characterize the yeast cells. The rabbit model of infection is a robust experimental model for better understanding CM and Cryptococcus cellular behavior.

A ketogenic diet enhances fluconazole efficacy in murine models of systemic fungal infection.

Invasive fungal infections are a significant public health concern, with mortality rates ranging from 20% to 85% despite current treatments. Therefore, we examined whether a ketogenic diet could serve as a successful treatment intervention in murine models of Cryptococcus neoformans and Candida albicans infection in combination with fluconazole-a low-cost, readily available antifungal therapy. The ketogenic diet is a high-fat, low-carbohydrate diet that promotes fatty acid oxidation as an alternative to glycolysis through the production of ketone bodies. In this series of experiments, mice fed a ketogenic diet prior to infection with C. neoformans and treated with fluconazole had a significant decrease in fungal burden in both the brain (mean 2.66 ± 0.289 log10 reduction) and lung (mean 1.72 ± 0.399 log10 reduction) compared to fluconazole treatment on a conventional diet. During C. albicans infection, kidney fungal burden of mice in the keto-fluconazole combination group was significantly decreased compared to fluconazole alone (2.37 ± 0.770 log10-reduction). Along with higher concentrations of fluconazole in the plasma and brain tissue, fluconazole efficacy was maximized at a significantly lower concentration on a keto diet compared to a conventional diet, indicating a dramatic effect on fluconazole pharmacodynamics. Our findings indicate that a ketogenic diet potentiates the effect of fluconazole at multiple body sites during both C. neoformans and C. albicans infection and could have practical and promising treatment implications.IMPORTANCEInvasive fungal infections cause over 2.5 million deaths per year around the world. Treatments for fungal infections are limited, and there is a significant need to develop strategies to enhance antifungal efficacy, combat antifungal resistance, and mitigate treatment side effects. We determined that a high-fat, low-carbohydrate ketogenic diet significantly potentiated the therapeutic effect of fluconazole, which resulted in a substantial decrease in tissue fungal burden of both C. neoformans and C. albicans in experimental animal models. We believe this work is the first of its kind to demonstrate that diet can dramatically influence the treatment of fungal infections. These results highlight a novel strategy of antifungal drug enhancement and emphasize the need for future investigation into dietary effects on antifungal drug activity.

Hormonal steroids induce multidrug resistance and stress response genes in Neisseria gonorrhoeae by binding to MtrR.

Transcriptional regulator MtrR inhibits the expression of the multidrug efflux pump operon mtrCDE in the pathogenic bacterium Neisseria gonorrhoeae. Here, we show that MtrR binds the hormonal steroids progesterone, ß-estradiol, and testosterone, which are present at urogenital infection sites, as well as ethinyl estrogen, a component of some hormonal contraceptives. Steroid binding leads to the decreased affinity of MtrR for cognate DNA, increased mtrCDE expression, and enhanced antimicrobial resistance. Furthermore, we solve crystal structures of MtrR bound to each steroid, thus revealing their binding mechanisms and the conformational changes that induce MtrR.

The war on cryptococcosis: A Review of the antifungal arsenal

Cryptococcal meningitis is the most common central nervous system infection in the world today. It occurs primarily, but not exclusively, in immunocompromised individuals and despite substantial improvement in management of clinical events like AIDS, the numbers of cases of cryptococcosis remain very high. Unfortunately, despite several antifungal agents available for treatment, morbidity and mortality rates remain high with this fungal infection. In this Review, we will describe the treatments and strategies for success, identify the failures, and provide insights into the future developments / improvements for management. This sugar-coated yeast can play havoc within the human brain. Our goals must be to either prevent or diagnose disease early and treat aggressively with all our clinical tools when disease is detected.