Performance of RGM Medium for Isolation of Nontuberculous Mycobacteria from Respiratory Specimens from Non-Cystic Fibrosis Patients.

A new selective medium for rapidly growing mycobacteria (RGM medium) was evaluated on respiratory specimens from non-cystic fibrosis patients and compared to the mycobacterial growth indicator tube (MGIT) system and Middlebrook 7H11 agar for the isolation of all nontuberculous mycobacteria (NTM). A total of 203 mucolyzed respiratory specimens collected from 163 patients were inoculated on RGM medium and incubated at both 30°C (RGM30) and 35°C (RGM35) over a 28-day period. N-Acetyl-l-cysteine-sodium hydroxide (NALC-NaOH)-decontaminated specimens were inoculated into MGIT and Middlebrook 7H11 agar and incubated at 35°C for 42 days. NTM were identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) or gene sequencing. A total of 133 NTM isolates were recovered overall from 101 (49.8%) specimens collected from 85 (52.1%) patients by a combination of all culture methods. The sensitivity of RGM30 for the recovery of NTM was significantly higher than that of either the MGIT system (76.7% versus 59.4%; P = 0.01) or Middlebrook 7H11 agar (76.7% versus 47.4%; P = 0.0001) alone, but it was not significantly different from that of an acid-fast bacillus culture (AFC) which includes both MGIT and Middlebrook 7H11 agar (76.7% versus 63.9%; P = 0.0647). RGM35 had significantly lower sensitivity than the MGIT system (49.6% versus 59.4%; P = 0.0367) and AFC (49.6% versus 63.9%; P = 0.0023). RGM medium was highly effective at inhibiting the growth of nonmycobacterial organisms in the respiratory specimens, with breakthrough contamination rates of 5.4% and 4.4% for RGM30 and RGM35, respectively.

VT-1161 protects mice against oropharyngeal candidiasis caused by fluconazole-susceptible and -resistant Candida albicans.

BACKGROUND: Candida albicans, the most common human fungal pathogen, causes chronic mucosal infections in patients with inborn errors of IL-17 immunity that rely heavily on chronic, often lifelong, azole antifungal agents for treatment. However, a rise in azole resistance has predicated a need for developing new antifungal drugs. OBJECTIVES: To test the in vitro and in vivo efficacy of VT-1161 and VT-1129 in the treatment of oropharyngeal candidiasis with azole-susceptible or -resistant C. albicans strains. METHODS: MICs of VT-1161, VT-1129 and nine licensed antifungal drugs were determined for 31 Candida clinical isolates. The drug concentrations in mouse serum and tongues were measured following oral administration. IL-17-signalling-deficient Act1-/- mice were infected with fluconazole-susceptible or fluconazole-resistant C. albicans strains, and the amount of mucosal fungal burden was determined after fluconazole or VT-1161 treatment. RESULTS: Fourteen isolates (45%) were not fluconazole susceptible (MIC ≥4 mg/L). VT-1161 and VT-1129 showed significant in vitro activity against the majority of the 31 mucosal clinical isolates (MIC50 0.03 and 0.06 mg/L, respectively), including Candida glabrata (MIC50, 0.125 and 0.25 mg/L, respectively). After oral doses, VT-1161 and VT-1129 concentrations in mouse serum and tongues were well above their MIC50 values. VT-1161 was highly effective as treatment of both fluconazole-susceptible and -resistant oropharyngeal candidiasis in Act1-/- mice. CONCLUSIONS: VT-1129 and VT-1161 exhibit significant in vitro activity against Candida strains, including fluconazole-resistant C. albicans and C. glabrata. VT-1161 administration in mice results in significant mucosal drug accumulation and eradicates infection caused by fluconazole-susceptible and -resistant Candida strains.

VT-1598 inhibits the in vitro growth of mucosal Candida strains and protects against fluconazole-susceptible and -resistant oral candidiasis in IL-17 signalling-deficient mice.

Background: Chronic mucocutaneous candidiasis (CMC) treatment often induces drug resistance, posing long-term challenges. A novel broad-spectrum fungal CYP51 inhibitor, VT-1598, specifically targets fungal CYP51, but not human CYP enzymes. Objectives: To determine the efficacy of VT-1598 in the treatment of oral Candida infection caused by fluconazole-susceptible and -resistant clinical isolates. Methods: The MICs of VT-1598 and fluconazole for 28 Candida isolates recovered from patients with inherited CMC were determined using CLSI M27-A3 and M27-S4 guidelines. Plasma and tongue VT-1598 or fluconazole concentrations were measured in mice following oral administration to determine tissue distribution. Tongue fungal load was determined in IL-17 signalling-deficient Act1-/- mice following sublingual Candida albicans infection and oral treatment with fluconazole or VT-1598. Results: Among the 28 Candida isolates, 10 (36%) had fluconazole MICs of ≥4 mg/L, whereas VT-1598 demonstrated potent in vitro activity against all isolates (MIC90, 0.125 mg/L). After oral administration, VT-1598 levels in mouse plasma and tongue were significantly greater than those of fluconazole. In vivo, VT-1598 exhibited significant efficacy against fluconazole-susceptible and -resistant C. albicans, even at low drug doses. Furthermore, after a 10 day washout period, tongue fungal burdens in fluconazole-treated mice returned to vehicle control levels, whereas, in contrast, they were undetectable in mice treated with VT-1598. Conclusions: VT-1598 effectively controls in vitro growth of mucosally derived Candida clinical isolates, including fluconazole-resistant strains. In vivo, VT-1598 eliminates C. albicans, even after a long washout period or at low doses. Therefore, VT-1598 is a promising drug candidate that may significantly improve treatment options for CMC patients.

Multicenter Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry Study for Identification of Clinically Relevant Nocardia spp.

This multicenter study analyzed Nocardia spp., including extraction, spectral acquisition, Bruker matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) identification, and score interpretation, using three Nocardia libraries, the Bruker, National Institutes of Health (NIH), and The Ohio State University (OSU) libraries, and compared the results obtained by each center. A standardized study protocol, 150 Nocardia isolates, and NIH and OSU Nocardia MALDI-TOF MS libraries were distributed to three centers. Following standardized culture, extraction, and MALDI-TOF MS analysis, isolates were identified using score cutoffs of ≥2.0 for species/species complex-level identification and ≥1.8 for genus-level identification. Isolates yielding a score of <2.0 underwent a single repeat extraction and analysis. The overall score range for all centers was 1.3 to 2.7 (average, 2.2 ± 0.3), with common species generally producing higher average scores than less common ones. Score categorization and isolate identification demonstrated 86% agreement between centers; 118 of 150 isolates were correctly identified to the species/species complex level by all centers. Nine strains (6.0%) were not identified by any center, and six (4.0%) of these were uncommon species with limited library representation. A categorical score discrepancy among centers occurred for 21 isolates (14.0%). There was an overall benefit of 21.2% from repeat extraction of low-scoring isolates and a center-dependent benefit for duplicate spotting (range, 2 to 8.7%). Finally, supplementation of the Bruker Nocardia MALDI-TOF MS library with both the OSU and NIH libraries increased the genus-level and species-level identification by 18.2% and 36.9%, respectively. Overall, this study demonstrates the ability of diverse clinical microbiology laboratories to utilize MALDI-TOF MS for the rapid identification of clinically relevant Nocardia spp. and to implement MALDI-TOF MS libraries developed by single laboratories across institutions.

Adolescent MDMA exposure diminishes the physiological and neurotoxic consequences of an MDMA binge in female rats.

Intermittent MDMA pretreatment blocked the reductions in serotonin transporter (SERT) binding induced by an MDMA binge in a prior study in adolescent male rats. The objective of this investigation was to determine if the physiological, behavioral, and neurochemical responses to MDMA are sexually dimorphic. Female Sprague-Dawley rats received MDMA (10 mg/kg × 2) or Saline on every fifth day from postnatal day (PD) 35-60 and an MDMA binge (5 mg/kg × 4) on PD 67. The MDMA binge induced a pronounced temperature dysregulation in MDMA-naïve, but not MDMA-pretreated, groups. Similarly, MDMA-pretreated animals were resistant to the binge-induced SERT reductions, especially in the hippocampus. Motor activity at PD 68 was not reduced by the binge, unlike the responses found in males. These results show that female rats differ from males in their responses to an MDMA binge but are similar with respect to preconditioning from prior MDMA exposure.

Development of a clinically comprehensive database and a simple procedure for identification of molds from solid media by matrix-assisted laser desorption ionization-time of flight mass spectrometry.

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) is a powerful tool for the rapid and highly accurate identification of clinical pathogens but has not been utilized extensively in clinical mycology due to challenges in developing an effective protein extraction method and the limited databases available. Here, we developed an alternate extraction procedure and constructed a highly stringent database comprising 294 individual isolates representing 76 genera and 152 species. To our knowledge, this is the most comprehensive clinically relevant mold database developed to date. When challenged with 421 blinded clinical isolates from our institution, by use of the BioTyper software, accurate species-level (score of ≥ 2.0) and genus-level (score of ≥ 1.7) identifications were obtained for 370 (88.9%) and 18 (4.3%) isolates, respectively. No isolates were misidentified. Of the 33 isolates (7.8%) for which there was no identification (score of <1.7), 25 were basidiomycetes not associated with clinical disease and 8 were Penicillium species that were not represented in the database. Our library clearly outperformed the manufacturer's database that was obtained with the instrument, which identified only 3 (0.7%) and 26 (6.2%) isolates at species and genus levels, respectively. Identification was not affected by different culture conditions. Implementation into our routine workflow has revolutionized our mycology laboratory efficiency, with improved accuracy and decreased time for mold identification, eliminating reliance on traditional phenotypic features.

Aspergillus tanneri sp. nov., a new pathogen that causes invasive disease refractory to antifungal therapy.

The most common cause of invasive aspergillosis (IA) in patients with chronic granulomatous disease (CGD) is Aspergillus fumigatus followed by A. nidulans; other aspergilli rarely cause the disease. Here we review two clinical cases of fatal IA in CGD patients and describe a new etiologic agent of IA refractory to antifungal therapy. Unlike typical IA caused by A. fumigatus, the disease caused by the new species was chronic and spread from the lung to multiple adjacent organs. Mycological characteristics and the phylogenetic relationship with other aspergilli based on the sequence analysis of Mcm7, RPB2, and Tsr1 indicated that the new species, which we named as A. tanneri, belongs to Aspergillus section Circumdati. The species has a higher amphotericin B, voriconazole, and itraconazole MIC and causes more chronic infection in CGD mice than A. fumigatus. This is the first report documenting IA in CGD patients caused by a species belonging to the Aspergillus section Circumdati that is inherently resistant to azoles and amphotericin B. Unlike the results seen with many members of Aspergillus section Circumdati, ochratoxin was not detected in filtrates of cultures grown in various media. Our phenotypic and genetic characterization of the new species and the case reports will assist future diagnosis of infection caused by A. tanneri and lead to more appropriate patient management.

Safety and immunogenicity of an MF59-adjuvanted spike glycoprotein-clamp vaccine for SARS-CoV-2: a randomised, double-blind, placebo-controlled, phase 1 trial.

BACKGROUND: Given the scale of the ongoing COVID-19 pandemic, the development of vaccines based on different platforms is essential, particularly in light of emerging viral variants, the absence of information on vaccine-induced immune durability, and potential paediatric use. We aimed to assess the safety and immunogenicity of an MF59-adjuvanted subunit vaccine for COVID-19 based on recombinant SARS-CoV-2 spike glycoprotein stabilised in a pre-fusion conformation by a novel molecular clamp (spike glycoprotein-clamp [sclamp]). METHODS: We did a phase 1, double-blind, placebo-controlled, block-randomised trial of the sclamp subunit vaccine in a single clinical trial site in Brisbane, QLD, Australia. Healthy adults (aged ≥18 to ≤55 years) who had tested negative for SARS-CoV-2, reported no close contact with anyone with active or previous SARS-CoV-2 infection, and tested negative for pre-existing SARS-CoV-2 immunity were included. Participants were randomly assigned to one of five treatment groups and received two doses via intramuscular injection 28 days apart of either placebo, sclamp vaccine at 5 µg, 15 µg, or 45 µg, or one dose of sclamp vaccine at 45 µg followed by placebo. Participants and study personnel, except the dose administration personnel, were masked to treatment. The primary safety endpoints included solicited local and systemic adverse events in the 7 days after each dose and unsolicited adverse events up to 12 months after dosing. Here, data are reported up until day 57. Primary immunogenicity endpoints were antigen-specific IgG ELISA and SARS-CoV-2 microneutralisation assays assessed at 28 days after each dose. The study is ongoing and registered with ClinicalTrials.gov, NCT04495933. FINDINGS: Between June 23, 2020, and Aug 17, 2020, of 314 healthy volunteers screened, 120 were randomly assigned (n=24 per group), and 114 (95%) completed the study up to day 57 (mean age 32·5 years [SD 10·4], 65 [54%] male, 55 [46%] female). Severe solicited reactions were infrequent and occurred at similar rates in participants receiving placebo (two [8%] of 24) and the SARS-CoV-2 sclamp vaccine at any dose (three [3%] of 96). Both solicited reactions and unsolicited adverse events occurred at a similar frequency in participants receiving placebo and the SARS-CoV-2 sclamp vaccine. Solicited reactions occurred in 19 (79%) of 24 participants receiving placebo and 86 (90%) of 96 receiving the SARS-CoV-2 sclamp vaccine at any dose. Unsolicited adverse events occurred in seven (29%) of 24 participants receiving placebo and 35 (36%) of 96 participants receiving the SARS-CoV-2 sclamp vaccine at any dose. Vaccination with SARS-CoV-2 sclamp elicited a similar antigen-specific response irrespective of dose: 4 weeks after the initial dose (day 29) with 5 µg dose (geometric mean titre [GMT] 6400, 95% CI 3683-11 122), with 15 µg dose (7492, 4959-11 319), and the two 45 µg dose cohorts (8770, 5526-13 920 in the two-dose 45 µg cohort; 8793, 5570-13 881 in the single-dose 45 µg cohort); 4 weeks after the second dose (day 57) with two 5 µg doses (102 400, 64 857-161 676), with two 15 µg doses (74 725, 51 300-108 847), with two 45 µg doses (79 586, 55 430-114 268), only a single 45 µg dose (4795, 2858-8043). At day 57, 67 (99%) of 68 participants who received two doses of sclamp vaccine at any concentration produced a neutralising immune response, compared with six (25%) of 24 who received a single 45 µg dose and none of 22 who received placebo. Participants receiving two doses of sclamp vaccine elicited similar neutralisation titres, irrespective of dose: two 5 µg doses (GMT 228, 95% CI 146-356), two 15 µg doses (230, 170-312), and two 45 µg doses (239, 187-307). INTERPRETATION: This first-in-human trial shows that a subunit vaccine comprising mammalian cell culture-derived, MF59-adjuvanted, molecular clamp-stabilised recombinant spike protein elicits strong immune responses with a promising safety profile. However, the glycoprotein 41 peptide present in the clamp created HIV diagnostic assay interference, a possible barrier to widespread use highlighting the criticality of potential non-spike directed immunogenicity during vaccine development. Studies are ongoing with alternative molecular clamp trimerisation domains to ameliorate this response. FUNDING: Coalition for Epidemic Preparedness Innovations, National Health and Medical Research Council, Queensland Government, and further philanthropic sources listed in the acknowledgments.

Nitric Oxide-Releasing Emulsion with Hyaluronic Acid and Vitamin E.

S-nitrosoglutathione (GSNO) is a naturally available S-nitrosothiol that can be incorporated into non-toxic formulations intended for topical use. The value of nitric oxide (NO) delivered topically relates to its well-studied physiological functions such as vasodilation, angiogenesis, cell proliferation and broad-spectrum antibacterial activity. Previously reported topical NO-releasing substrates include polymeric materials that exhibit non-toxic behaviors on dermal tissue such as polyethylene glycol. However, they do not serve as humectants nor provide vitamins to the skin. In this study, GSNO was added to an emulsion that was fortified with α-tocopheryl acetate (vitamin E) and hyaluronic acid. The average total NO content for the NO-releasing emulsion was 58 ± 8 µmol g-1 at 150 °C and the cumulative NO release over 53 h at physiological temperature (37.4 °C) was 46 ± 4 µmol g-1. The GSNO concentration in the lotion was optimized in order to reach a pH value similar to that of human skin (pH 5.5). The viscosity was analyzed using a rotational viscometer for the S-nitrosated and the non-nitrosated emulsions to obtain a material that can be readily spread on dermal tissue. The viscosity values obtained ranged from 7.88 ± 0.99 to 8.50 ± 0.36 Pa•s. Previous studies have determined that the viscosity maximum for lotions is 100 Pa•s. A low viscosity increases the diffusion coefficient of active ingredients to the skin given that they are inversely proportional as described by the Einstein-Smoluchowski equation. The effect of the S-nitrosated and non-nitrosated emulsions on adult human dermal fibroblasts (HDFs) was assessed in comparison to untreated HDFs using Colorimetric Cell Viability Kit I - WST- 8. The findings indicate that neither the S-nitrosated nor non-nitrosated emulsions induced cytotoxicity in HDFs.

Rapid one-step protein extraction method for the identification of mycobacteria using MALDI-TOF MS.

Matrix-assisted laser desorption ionization-time-of-flight mass spectrometry is a quick and accurate method for mycobacterial identification from protein extracts. Our new one-step extraction method successfully reduced routine multistep extraction procedure time from over 60 min to under 10 min and used only 1 µL loopful of mycobacteria while providing clinically acceptable identification scores (≥1.8). Overall, 86.8% and 4.4% of mycobacteria isolates (n = 68) were identified to the species/complex and genus levels, respectively, by one-step loop extraction method, comparable to the routine extraction method. Viability studies confirmed killing of mycobacterial isolates after 5 min in the extraction solution replacing lengthy heat killing step. Retrospective 7-month data analysis showed 100% of rapidly and slowly growing mycobacterial isolates were identified to the species/complex level by rapid extraction methods. Our rapid extraction methods substantially reduced processing time and microbial biomass required for testing without sacrificing quality and accuracy of mycobacterial identification.

Multicenter Study Demonstrates Standardization Requirements for Mold Identification by MALDI-TOF MS.

OBJECTIVES: Rapid and accurate mold identification is critical for guiding therapy for mold infections. MALDI-TOF MS has been widely adopted for bacterial and yeast identification; however, few clinical laboratories have applied this technology for routine mold identification due to limited database availability and lack of standardized processes. Here, we evaluated the versatility of the NIH Mold Database in a multicenter evaluation. METHODS: The NIH Mold Database was evaluated by eight US academic centers using a solid media extraction method and a challenge set of 80 clinical mold isolates. Multiple instrument parameters important for spectra optimization were evaluated, leading to the development of two specialized acquisition programs (NIH method and the Alternate-B method). RESULTS: A wide range in performance (33-77%) was initially observed across the eight centers when routine spectral acquisition parameters were applied. Use of the NIH or the Alternate-B specialized acquisition programs, which are different than those used routinely for bacterial and yeast spectral acquisition (MBT_AutoX), in combination with optimized instrument maintenance, improved performance, illustrating that acquisition parameters may be one of the key limiting variable in achieving successful performance. CONCLUSION: Successful mold identification using the NIH Database for MALDI-TOF MS on Biotyper systems was demonstrated across multiple institutions for the first time following identification of critical program parameters combined with instrument optimization. This significantly advances our potential to implement MALDI-TOF MS for mold identification across many institutions. Because instrument variability is inevitable, development of an instrument performance standard specific for mold spectral acquisition is suggested to improve reproducibility across instruments.

Identification of Multiple Cryptococcal Fungicidal Drug Targets by Combined Gene Dosing and Drug Affinity Responsive Target Stability Screening.

UNLABELLED: Cryptococcus neoformans is a pathogenic fungus that is responsible for up to half a million cases of meningitis globally, especially in immunocompromised individuals. Common fungistatic drugs, such as fluconazole, are less toxic for patients but have low efficacy for initial therapy of the disease. Effective therapy against the disease is provided by the fungicidal drug amphotericin B; however, due to its high toxicity and the difficulty in administering its intravenous formulation, it is imperative to find new therapies targeting the fungus. The antiparasitic drug bithionol has been recently identified as having potent fungicidal activity. In this study, we used a combined gene dosing and drug affinity responsive target stability (GD-DARTS) screen as well as protein modeling to identify a common drug binding site of bithionol within multiple NAD-dependent dehydrogenase drug targets. This combination genetic and proteomic method thus provides a powerful method for identifying novel fungicidal drug targets for further development. IMPORTANCE: Cryptococcosis is a neglected fungal meningitis that causes approximately half a million deaths annually. The most effective antifungal agent, amphotericin B, was developed in the 1950s, and no effective medicine has been developed for this disease since that time. A key aspect of amphotericin B's effectiveness is thought to be because of its ability to kill the fungus (fungicidal activity), rather than just stop or slow its growth. The present study utilized a recently identified fungicidal agent, bithionol, to identify potential fungicidal drug targets that can be used in developing modern fungicidal agents. A combined protein and genetic analysis approach was used to identify a class of enzymes, dehydrogenases, that the fungus uses to maintain homeostasis with regard to sugar nutrients. Similarities in the drug target site were found that resulted in simultaneous inhibition and killing of the fungus by bithionol. These studies thus identify a common, multitarget site for antifungal development.

A high-throughput screening assay for fungicidal compounds against Cryptococcus neoformans.

Cryptococcus neoformans is a pathogenic fungus that causes meningitis worldwide, particularly in human immunodeficiency virus (HIV)-infected individuals. Although amphotericin B is the "gold standard" treatment for cryptococcal meningitis, the toxicity and inconvenience of intravenous injection emphasize a need for development of new anticryptocccal drugs. Recent data from humans and animal studies suggested that a nutrient-deprived host environment may exist in cryptococcal meningitis. Thus, a screening assay for identifying fungicidal compounds under nutrient-deprived conditions may provide an alternative strategy to develop new anticryptococcal drugs for this disease. A high-throughput fungicidal assay was developed using a profluorescent dye, alamarBlue, to detect residual metabolic activity of C. neoformans under nutrient-limiting conditions. Screening the Library of Pharmacologically Active Compounds (LOPAC) with this assay identified a potential chemical scaffold, 10058-F4, that exhibited fungicidal activity in the low micromolar range. These results thus demonstrate the feasibility of this alamarBlue-based assay for high-throughput screening of fungicidal compounds under nutrient-limiting conditions for new anticryptococcal drug development.

Reversal of disease-related pathologies in the fragile X mouse model by selective activation of GABAB receptors with arbaclofen.

Fragile X syndrome (FXS), the most common inherited cause of intellectual disability and autism, results from the transcriptional silencing of FMR1 and loss of the mRNA translational repressor protein fragile X mental retardation protein (FMRP). Patients with FXS exhibit changes in neuronal dendritic spine morphology, a pathology associated with altered synaptic function. Studies in the mouse model of fragile X have shown that loss of FMRP causes excessive synaptic protein synthesis, which results in synaptic dysfunction and altered spine morphology. We tested whether the pharmacologic activation of the γ-aminobutyric acid type B (GABA(B)) receptor could correct or reverse these phenotypes in Fmr1-knockout mice. Basal protein synthesis, which is elevated in the hippocampus of Fmr1-knockout mice, was corrected by the in vitro application of the selective GABA(B) receptor agonist STX209 (arbaclofen, R-baclofen). STX209 also reduced to wild-type values the elevated AMPA receptor internalization in Fmr1-knockout cultured neurons, a known functional consequence of increased protein synthesis. Acute administration of STX209 in vivo, at doses that modify behavior, decreased mRNA translation in the cortex of Fmr1-knockout mice. Finally, the chronic administration of STX209 in juvenile mice corrected the increased spine density in Fmr1-knockout mice without affecting spine density in wild-type mice. Thus, activation of the GABA(B) receptor with STX209 corrected synaptic abnormalities considered central to fragile X pathophysiology, a finding that suggests that STX209 may be a potentially effective therapy to treat the core symptoms of FXS.