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PURPOSE OF REVIEW: Environmental fungi such as Cryptococcus neoformans and Aspergillus fumigatus must survive many different and changing environments as they transition from their environmental niches to human lungs and other organs. Fungi alter their cell surfaces and secreted macromolecules to respond to and manipulate their surroundings. RECENT FINDINGS: This review focuses on exo-polysaccharides, chains of sugars that transported out of the cell and spread to the local environment. Major exo-polysaccharides for C. neoformans and A. fumigatus are glucuronylxylomannan (GXM) and galactosaminogalactan (GAG), respectively, which accumulate at high concentrations in growth medium and infected patients SUMMARY: Here we discuss GXM and GAG synthesis and export, their immunomodulatory properties, and their roles in biofilm formation. We also propose areas of future research to address outstanding questions in the field that could facilitate development of new disease treatments.

Hospitals are increasingly filled with people admitted for medical and surgical complications of substance use disorder (SUD). Hospitalization can be a reachable moment to engage and initiate SUD care. Yet most hospitals do not have systems in place to adequately address addiction, and most providers have little to no addiction training. There is widespread need for protocols and tools to implement hospital-based SUD care. We share best practices from our hospital-based Improving Addiction Care Team (IMPACT). We include a description of interprofessional roles (medical providers, social workers, peers with lived experience in recovery) and include detailed appendices of practical tools such as medication protocols (eg, buprenorphine induction), risk assessments (eg, outpatient parenteral antibiotic therapy) and treatment tools (eg, a patient safety care plan to manage patient and staff expectations surrounding risks for in hospital drug use). A case example illustrates how IMPACT works and how tools can be applied. We hope other hospitals can adapt and integrate these tools to support widespread implementation of hospital-based SUD care.

SUMMARYEnvironmental pathogens, which move from ecological niches to mammalian hosts, must adapt to dramatically different environments. Microbes that disseminate farther, including the fungal meningitis pathogen Cryptococcus neoformans, require additional adaptation to diverse tissues. When C. neoformans enters the lungs, infecting cells (30 μm diameter), then form a heterogeneous population. The brain contains uniformly small cells (∼7 μm). We demonstrate that formation of a small C. neoformans morphotype – called “seed” cells due to their disseminating ability – is critical for extrapulmonary organ entry. Seed cell formation is triggered by environmental factors, including C. neoformans’ environmental niche, pigeon guano. The underlying trigger, phosphate, can be released by tissue damage, potentially establishing a feed-forward loop of seed cell formation and dissemination. We demonstrate that C. neoformans’ size variation is not just a continuum but inducible subpopulations that change host interactions to facilitate microbe survival and spread.

Invasive fungal infections cause 1.6 million deaths annually, primarily in immunocompromised individuals. Mortality rates are as high as 90% due to limited treatments. The azole class antifungal, fluconazole, is widely available and has multi-species activity but only inhibits growth instead of killing fungal cells, necessitating long treatments. To improve treatment, we used our novel high-throughput method, the overlap2 method (O2M) to identify drugs that interact with fluconazole, either increasing or decreasing efficacy. We identified 40 molecules that act synergistically (amplify activity) and 19 molecules that act antagonistically (decrease efficacy) when combined with fluconazole. We found that critical frontline beta-lactam antibiotics antagonize fluconazole activity. A promising fluconazole-synergizing anticholinergic drug, dicyclomine, increases fungal cell permeability and inhibits nutrient intake when combined with fluconazole. In vivo, this combination doubled the time-to-endpoint of mice with Cryptococcus neoformans meningitis. Thus, our ability to rapidly identify synergistic and antagonistic drug interactions can potentially alter the patient outcomes., eLife digest Individuals with weakened immune systems – such as recipients of organ transplants – can fall prey to illnesses caused by fungi that are harmless to most people. These infections are difficult to manage because few treatments exist to fight fungi, and many have severe side effects. Antifungal drugs usually slow the growth of fungi cells rather than kill them, which means that patients must remain under treatment for a long time, or even for life. One way to boost efficiency and combat resistant infections is to combine antifungal treatments with drugs that work in complementary ways: the drugs strengthen each other’s actions, and together they can potentially kill the fungus rather than slow its progression. However, not all drug combinations are helpful. In fact, certain drugs may interact in ways that make treatment less effective. This is particularly concerning because people with weakened immune systems often take many types of medications. Here, Wambaugh et al. harnessed a new high-throughput system to screen how 2,000 drugs (many of which already approved to treat other conditions) affected the efficiency of a common antifungal called fluconazole. This highlighted 19 drugs that made fluconazole less effective, some being antibiotics routinely used to treat patients with weakened immune systems. On the other hand, 40 drugs boosted the efficiency of fluconazole, including dicyclomine, a compound currently used to treat inflammatory bowel syndrome. In fact, pairing dicyclomine and fluconazole more than doubled the survival rate of mice with severe fungal infections. The combined treatment could target many species of harmful fungi, even those that had become resistant to fluconazole alone. The results by Wambaugh et al. point towards better treatments for individuals with serious fungal infections. Drugs already in circulation for other conditions could be used to boost the efficiency of fluconazole, while antibiotics that do not decrease the efficiency of this medication should be selected to treat at-risk patients.

SummaryInvasive fungal infections cause 1.6 million deaths annually, primarily in immunocompromised individuals. Mortality rates are as high as 90% due to limited number of efficacious drugs and poor drug availability. The azole class antifungal, fluconazole, is widely available and has multi-species activity but only inhibits fungal cell growth instead of killing fungal cells, necessitating long treatments. To improve fluconazole treatments, we used our novel high-throughput method, the overlap2method (O2M), to identify drugs that interact with fluconazole, either increasing or decreasing efficacy. Although serendipitous identification of these interactions is rare, O2M allows us to screen molecules five times faster than testing combinations individually and greatly enriches for interactors. We identified 40 molecules that act synergistically (amplify activity) and 19 molecules that act antagonistically (decrease efficacy) when combined with fluconazole. We found that critical frontline beta-lactam antibiotics antagonize fluconazole activity. A promising fluconazole-synergizing anticholinergic drug, dicyclomine, increases fungal cell permeability and inhibits nutrient intake when combined with fluconazole.In vivo, this combination doubled the time-to-endpoint of mice with disseminatedCryptococcus neoformansinfections. Thus, our ability to rapidly identify synergistic and antagonistic drug interactions can potentially alter the patient outcomes.

Although antimicrobial drugs have dramatically increased the lifespan and quality of life in the 20th century, antimicrobial resistance threatens our entire society's ability to treat systemic infections. In the United States alone, antibiotic-resistant infections kill approximately 23,000 people a year and cost around 20 billion USD in additional healthcare. One approach to combat antimicrobial resistance is combination therapy, which is particularly useful in the critical early stage of infection, before the infecting organism and its drug resistance profile have been identified. Many antimicrobial treatments use combination therapies. However, most of these combinations are additive, meaning that the combined efficacy is the same as the sum of the individual antibiotic efficacy. Some combination therapies are synergistic: the combined efficacy is much greater than additive. Synergistic combinations are particularly useful because they can inhibit the growth of antimicrobial drug resistant strains. However, these combinations are rare and difficult to identify. This is due to the sheer number of molecules needed to be tested in a pairwise manner: a library of 1,000 molecules has 1 million potential combinations. Thus, efforts have been made to predict molecules for synergy. This article describes our high-throughput method for predicting synergistic small molecule pairs known as the Overlap2 Method (O2M). O2M uses patterns from chemical-genetic datasets to identify mutants that are hypersensitive to each molecule in a synergistic pair but not to other molecules. The Brown lab exploits this growth difference by performing a high-throughput screen for molecules that inhibit the growth of mutant but not wild-type cells. The lab's work previously identified molecules that synergize with the antibiotic trimethoprim and the antifungal drug fluconazole using this strategy. Here, the authors present a method to screen for novel synergistic combinations, which can be altered for multiple microorganisms.

Cryptococcus neoformansis a common environmental yeast and opportunistic pathogen responsible for 15% of AIDS-related deaths worldwide. Mortality primarily results from meningoencephalitis, which occurs when fungal cells disseminate from the initial pulmonary infection site and spread to the brain. A keyC. neoformansvirulence trait is the polysaccharide capsule. Capsule shields C. neoformans from immune-mediated recognition and destruction. The main capsule component, glucuronoxylomannan (GXM), is found both attached to the cell surface and free in the extracellular space (as exo-GXM). Exo-GXM accumulates in patient serum and cerebrospinal fluid at μg/mL concentrations, has well-documented immunosuppressive properties, and correlates with poor patient outcomes. However, it is poorly understood whether exo-GXM release is regulated or the result of shedding during normal capsule turnover. We demonstrate that exo-GXM release is regulated by environmental cues and inversely correlates with surface capsule levels. We identified genes specifically involved in exo-GXM release that do not alter surface capsule thickness. The first mutant,liv7∆, released less GXM than wild-type cells when capsule is not induced. The second mutant,cnag_00658∆, released more exo-GXM under capsule-inducing conditions. Exo-GXM release observedin vitrocorrelated with polystyrene adherence, virulence, and fungal burden during murine infection. Additionally, we find that exo-GXM reduces cell size and capsule thickness in capsule-inducing conditions, potentially influencing dissemination. Finally, we demonstrated that exo-GXM prevents immune cell infiltration into the brain during disseminated infection and highly inflammatory intracranial infection. Our data suggest that exo-GXM performs a different role from capsule GXM during infection, altering cell size and suppressing inflammation.ImportanceCryptococcus neoformansis a leading cause of life-threatening meningoencephalitis in humans.C. neoformanscells produce an immunosuppressive polysaccharide, glucuronoxylomannan (GXM), that is the main component of a protective surface capsule. GXM is also released free into extracellular space as exo-GXM, although the distinction between cell-attached GXM and exo-GXM has been unclear. Exo-GXM influences the outcome of infection, is the basis for current diagnostic tools, and has potential therapeutic applications. This study increases our basic understanding of the fungal biology that regulates polysaccharide release, suggesting that the release of cell-attached GXM and exo-GXM are distinctly regulated. We also introduce a new concept that exo-GXM may alter cell body and capsule size, thereby influencing dissemination in the host. Finally, we provide experimental evidence to confirm clinical observations that exo-GXM influences inflammation during brain infection.

Although a surgical removal of tunneled central venous catheter is usually simple, it can become complicated when the catheter is found to be stuck because of its adherence within a central vein. If a catheter is pulled too hard, it may fracture within a central vein in two pieces. In this report, we describe the mechanics of central venous catheter breakage and provide a solution that minimizes the possibility of catheter from breaking as it is pulled out from its stuck position within a central vein.

Cryptococcus neoformans is a common environmental saprophyte and human fungal pathogen that primarily causes disease in immunocompromised individuals. Similar to many environmentally acquired human fungal pathogens, C. neoformans initiates infection in the lungs. However, the main driver of mortality is invasive cryptococcosis leading to fungal meningitis. After C. neoformans gains a foothold in the lungs, a critical early step in invasion is transversal of the respiratory epithelium. In this review, we summarize current knowledge relating to pulmonary escape. We focus on fungal factors that allow C. neoformans to disseminate from the lungs via intracellular and extracellular routes.

The commensal microbiota has an important impact on host health, which is only beginning to be elucidated. Despite the presence of fungal, archaeal, and viral members, most studies have focused solely on the bacterial microbiota. Antibodies against the yeast Saccharomyces cerevisiae are found in some patients with Crohn’s disease (CD), suggesting that the mycobiota may contribute to disease severity. We report that S. cerevisiae exacerbated intestinal disease in a mouse model of colitis and increased gut barrier permeability. Transcriptome analysis of colon tissue from germ-free mice inoculated with S. cerevisiae or another fungus, Rhodotorula aurantiaca, revealed that S. cerevisiae colonization affected the intestinal barrier and host metabolism. A fecal metabolomics screen of germ-free animals demonstrated that S. cerevisiae colonization enhanced host purine metabolism, leading to an increase in uric acid production. Treatment with uric acid alone worsened disease and increased gut permeability. Allopurinol, a clinical drug used to reduce uric acid, ameliorated colitis induced by S. cerevisiae in mice. In addition, we found a positive correlation between elevated uric acid and anti-yeast antibodies in human sera. Thus, yeast in the gut may be able to potentiate metabolite production that negatively affects the course of inflammatory bowel disease.

Summary The fungal meningitis pathogen Cryptococcus neoformans is a central driver of mortality in HIV/AIDS. We report a genome-scale chemical genetic data map for this pathogen that quantifies the impact of 439 small-molecule challenges on 1,448 gene knockouts. We identified chemical phenotypes for 83% of mutants screened and at least one genetic response for each compound. C. neoformans chemical-genetic responses are largely distinct from orthologous published profiles of Saccharomyces cerevisiae , demonstrating the importance of pathogen-centered studies. We used the chemical-genetic matrix to predict novel pathogenicity genes, infer compound mode of action, and to develop an algorithm, O2M, that predicts antifungal synergies. These predictions were experimentally validated, thereby identifying virulence genes, a molecule that triggers G2/M arrest and inhibits the Cdc25 phosphatase, and many compounds that synergize with the antifungal drug fluconazole. Our work establishes a chemical-genetic foundation for approaching an infection responsible for greater than one-third of AIDS-related deaths. PaperClip

Antibiotic-resistant infections kill approximately 23,000 people and cost $20,000,000,000 each year in the United States alone despite the widespread use of small-molecule antimicrobial combination therapy. Antibiotic combinations typically have an additive effect: the efficacy of the combination matches the sum of the efficacies of each antibiotic when used alone. Small molecules can also act synergistically when the efficacy of the combination is greater than the additive efficacy. However, synergistic combinations are rare and have been historically difficult to identify. High-throughput identification of synergistic pairs is limited by the scale of potential combinations: a modest collection of 1,000 small molecules involves 1 million pairwise combinations. Here, we describe a high-throughput method for rapid identification of synergistic small-molecule pairs, the overlap2 method (O2M). O2M extracts patterns from chemical-genetic datasets, which are created when a collection of mutants is grown in the presence of hundreds of different small molecules, producing a precise set of phenotypes induced by each small molecule across the mutant set. The identification of mutants that show the same phenotype when treated with known synergistic molecules allows us to pinpoint additional molecule combinations that also act synergistically. As a proof of concept, we focus on combinations with the antibiotics trimethoprim and sulfamethizole, which had been standard treatment against urinary tract infections until widespread resistance decreased efficacy. Using O2M, we screened a library of 2,000 small molecules and identified several that synergize with the antibiotic trimethoprim and/or sulfamethizole. The most potent of these synergistic interactions is with the antiviral drug azidothymidine (AZT). We then demonstrate that understanding the molecular mechanism underlying small-molecule synergistic interactions allows the rational design of additional combinations that bypass drug resistance. Trimethoprim and sulfamethizole are both folate biosynthesis inhibitors. We find that this activity disrupts nucleotide homeostasis, which blocks DNA replication in the presence of AZT. Building on these data, we show that other small molecules that disrupt nucleotide homeostasis through other mechanisms (hydroxyurea and floxuridine) also act synergistically with AZT. These novel combinations inhibit the growth and virulence of trimethoprim-resistant clinical Escherichia coli and Klebsiella pneumoniae isolates, suggesting that they may be able to be rapidly advanced into clinical use. In sum, we present a generalizable method to screen for novel synergistic combinations, to identify particular mechanisms resulting in synergy, and to use the mechanistic knowledge to rationally design new combinations that bypass drug resistance., Author summary Antibiotic resistance is a growing problem that threatens our ability to treat systemic bacterial infections. One strategy to combat antibiotic resistance is the use of synergistic antibiotic pairs that, when combined, have activity that is considerably greater than the sum of each individual drug’s activity on its own. Synergistic combinations can even inhibit the growth of bacteria that are resistant to the individual treatment drugs. However, synergistic pairs are rare and difficult to identify. High-throughput identification of synergistic pairs is challenging due to scale: 1 million different pairs are possible for a relatively small collection of 1,000 small molecules. Here, we describe a high-throughput method for rapid identification of synergistic small-molecule pairs, termed the overlap2 method (O2M), that dramatically speeds up the screening process. First, we identify mutants that show the same phenotype when treated with each individual molecule in a synergistic pair, then use this information to guide screens for additional synergistic pairs. As a proof of concept, we studied the synergistic antibiotic pair trimethoprim and sulfamethizole, and we identified several additional synergistic molecules. Among these is the antiviral drug azidothymidine (AZT), which blocks bacterial DNA replication. Trimethoprim and sulfamethizole both inhibit folate biosynthesis, which is necessary for the proper synthesis of nucleotides for DNA replication and repair. We found that reduced nucleotide levels sensitize E. coli cells to AZT. When we substitute trimethoprim with other small molecules that also reduce nucleotide levels, we find that these small molecules also act synergistically with AZT. Indeed, AZT in combination with trimethoprim substitutes inhibits the growth of trimethoprim-resistant clinical isolates more potently than trimethoprim and AZT or trimethoprim and sulfamethizole. This work demonstrates that when we resolve the pathways that underlie synergistic interactions, we can then identify additional small molecules that act by similar mechanisms, providing a means to bypass antibiotic resistance.

Between 2005 and 2011, the WHO Regional Office for Europe assisted the member states of the WHO European Region to prepare and respond to outbreaks of avian influenza H5N1, the 2009 pandemic, and to enhance their capacities for the prevention and control of seasonal influenza. It did this through conducting a combination of regional and subregional meetings and trainings, establishing a regional network for influenza surveillance, providing operational guidance for implementing influenza surveillance and strengthening the capacities of National Influenza Centers, and through assistance at the country-level where needed. In all, close to 60 country-missions or country-level activities were conducted. These activities were conducted in close coordination with WHO headquarters, WHO European Region Country Offices, the European Commission, the European Centre for Disease Prevention and Control, and with other partner organizations, and were in line with the implementation of the International Health Regulations (2005). The results of activities as well as guidance documents were disseminated to a wide audience through publication on the WHO Regional Office for Europe Influenza website, on the EuroFlu website, and through peer-reviewed publications.

An international collaborative study was conducted at ten sites to examine the performance of enzyme immunoassays (EIAs) for the quantitation of IgG1, IgG2, IgG3, IgG4 and total IgG anti-Haemophilus influenzae type b (Hib) capsular polysaccharide in human serum. All groups used the same reagents: microtiter plates coated with polyribosylribitol phosphate (PRP) conjugated to poly-L-lysine (PLL), reference, control and test human sera, biotin-conjugated International Union of Immunological Societies (IUIS)-documented monoclonal anti-human IgG1-4 and IgG Pan detection antibodies, avidin-peroxidase and TMB substrate. Initial mixing of soluble PRP antigen or an equal volume of buffer with the 20 test sera prior to analysis confirmed PRP antigen specificity in all five EIAs with greater than 80% competitive inhibition at most sites. Positive correlation between the total IgG anti-Hib and sum of IgG1-4 anti-Hib was demonstrated (r2 = 0.99, Y = 1.13X -0.15). Good agreement was shown between the total IgG anti-Hib as measured by EIA and the total Hib-specific antibodies measured by the current radiolabeled antigen binding assay (r2 = 0.97, Y = 4.6X -5.8). Assay parallelism was demonstrated with an average interdilutional %CV of 22% and parallel dose-response curve slopes. The interdilutional %CVs were calculated as an average per sample of the variation of microgram/ml (corrected for dilution) at different dilutions per laboratory for all participating sites. The interlaboratory variation was the only performance parameter studied that exceeded the target level of 35% CV in all IgG1-4 and total IgG anti-Hib assays. IgG subclass distributions in the test sera demonstrated a predominance of IgG1 anti-Hib in the pediatric serum pools and IgG2 anti-Hib in the adult sera, with low but detectable levels of IgG3 and IgG4 anti-Hib in each group.

Cisplatin (CDDP) has been used as a DNA cross-linking agent to evaluate whether there is a specific cell cycle checkpoint response to such damage in Saccharomyces cerevisiae (S. cerevisiae). Fluorescent-activated cell sorting (FACS) analysis showed only a G2/M checkpoint, normal exit from G1 and progression through S-phase following alpha-factor arrest and CDDP treatment. Of the checkpoint mutants tested, rad9, rad17 and rad24, did not show increased sensitivity to CDDP compared to isogenic wild-type cells. However, other checkpoint mutants tested (mec1, mec3 and rad53) showed increased sensitivity to CDDP, as did controls with a defect in excision repair (rad1 and rad14) or a defect in recombination (rad51 and rad52). Thus, by survival and cell cycle kinetics, it appears that DNA cross-links do not inhibit entry into S-phase or slow DNA replication and that replication continues after cisplatin treatment in yeast.

An immunodominant region on baculovirus-produced parvovirus B19 VP2 capsids was localized between amino acids 259 and 426 by mapping the binding sites of a panel of monoclonal antibodies which recognize determinants on the particles. The binding sites of three monoclonal antibodies were fine-mapped within this antigenic domain. Six VP2-specific monoclonal antibodies recognized determinants common to both the empty capsids and native parvovirus. The defined antigenic region is most probably exposed on the native B19 virion and corresponds to part of the threefold spike on the surface of canine parvovirus particles.

B. Dresser’,‘, E. Pope’,*, N. Chin3, J. Liu*, N. Loskutoff4, E. Behnke3, C. Brown4, M. McRae’, C. Sinoway3, M. Campbell’, K. Cameron’, R. Evans’, 0. 0wens3, C. Johnson’, M. Cedars* ‘Cincinnati Zoo Center for Reproduction of Endangered Wildlife (CREW), 3400 Vine Street, Cincinnati, OH 45220 USA; *University of Cincinnati College of Medicine, Department of Obstetrics and Gynecology, Cincinnati, OH; 3The Christ Hospital, Cincinnati, OH; 40maha’s Henry Doorly Zoo, Omaha, NE; 5Bethesda Hospital, Cincinnati, OH.