Olorofim Susceptibility Testing of 1,423 Danish Mold Isolates Obtained in 2018-2019 Confirms Uniform and Broad-Spectrum Activity.

Olorofim is a novel antifungal drug in phase 2 trials. It has shown promising in vitro activity against various molds, except for Mucorales. Initially, we observed a broad range of EUCAST MICs for Aspergillus fumigatus Here, we explored the MIC variability in more detail and prospectively investigated the susceptibility of contemporary clinical mold isolates, as population data are needed for future epidemiological cutoff (ECOFF) settings. Fifteen A. fumigatus isolates previously found with low/medium/high MICs (≤0.002 to 0.25 mg/liter) were tested repeatedly and EUCAST MICs read in a blinded fashion by three observers. pyrE, encoding the olorofim target enzyme dihydroorotate dehydrogenase (DHODH), was sequenced. A total of 1,423 mold isolates (10 Aspergillus species complexes [including 1,032 A. fumigatus isolates] and 105 other mold/dermatophyte isolates) were examined. Olorofim susceptibility (modal MIC, MIC50, MIC90, and wild-type upper limits [WT-ULs] [species complexes with ≥15 isolates]) was determined and compared to that of four comparators. MICs (mg/liter) were within two 2-fold dilutions (0.016 to 0.03) for 473/476 determinations. The MIC range spanned four dilutions (0.008 to 0.06). No significant pyrE mutations were found. Modal MIC/WT-UL97.5 (mg/liter) values were 0.03/0.06 (A. terreus and A. flavus), 0.06/0.125 (A. fumigatus and Trichophyton rubrum), and 0.06/0.25 (A. niger and A. nidulans). The MIC range for Scedosporium spp. was 0.008 to 0.25. Olorofim susceptibility was similar for azole-resistant and -susceptible isolates of A. fumigatus but reduced for A. montevidensis and A. chevalieri (MICs of >1). With experience, olorofim susceptibility testing is robust. The testing of isolates from our center showed uniform and broad-spectrum activity. Single-center WT-ULs are suggested.

Rezafungin In Vitro Activity against Contemporary Nordic Clinical Candida Isolates and Candida auris Determined by the EUCAST Reference Method.

Rezafungin (formerly CD101) is a novel echinocandin in clinical development. EUCAST epidemiological cutoff values (ECOFFs) have not yet been established. We determined the in vitro activity of rezafungin and comparators against 1,293 Nordic yeast isolates and 122 Indian Candida auris isolates and established single-center wild-type upper limits (WT-UL). The isolates (19 Candida spp. and 13 other yeast species) were identified using Chromagar; matrix-assisted laser desorption ionization-time of flight (MALDI-TOF); and, when needed, internal transcribed spacer sequencing. EUCAST E.Def 7.3.1 susceptibility testing included rezafungin, anidulafungin, micafungin, amphotericin B, and fluconazole. WT-UL were established following EUCAST principles for visual and statistical ECOFF setting. fks target genes were sequenced for rezafungin non-wild-type isolates. EUCAST clinical breakpoints for fungi version 9.0 were adopted for susceptibility classification. Rezafungin had species-specific activity similar to that of anidulafungin and micafungin. On a milligram-per-liter basis, rezafungin was overall less active than anidulafungin and micafungin but equally or more active than fluconazole and amphotericin B against the most common Candida species, except C. parapsilosis We identified 37 (3.1%) rezafungin non-wild-type isolates of C. albicans (1.9%), C. glabrata (3.0%), C. tropicalis (2.7%), C. dubliniensis (2.9%), C. krusei (1.2%), and C. auris (14.8%). Alterations in Fks hot spots were found in 26/26 Nordic and 8/18 non-wild-type C. auris isolates. Rezafungin displayed broad in vitro activity against Candida spp., including C. auris Adopting WT-UL established here, few Nordic strains, but a significant proportion of C. auris isolates, had elevated MICs with mutations in fks target genes that conferred echinocandin cross-resistance. fks1 mutations raised rezafungin MICs notably less than anidulafungin and micafungin MICs in C. auris.

Bacterial vaginosis diagnosed by analysis of first-void-urine specimens.

Bacterial vaginosis (BV) is traditionally diagnosed using vaginal samples. The aim of this study was to investigate whether BV can be diagnosed from first-void urine (FVU). Self-collected vaginal smears, vaginal swabs, and FVU were obtained from 176 women. BV was diagnosed by Nugent's criteria. The FVU and vaginal swabs were analyzed by quantitative PCRs (qPCRs) for selected vaginal bacteria (Atopobium vaginae, Prevotella spp., Gardnerella vaginalis, bacterial vaginosis-associated bacterium 2, Eggerthella-like bacterium, "Leptotrichia amnionii," Megasphaera type 1), and all had an area under the receiver operating characteristic (ROC) curve of >85%, suggesting good prediction of BV according to the Nugent score. All seven bacteria in FVU were significantly associated with BV in univariate analysis. An accurate diagnosis of BV from urine was obtained in this population by a combination of qPCRs for Megasphaera type 1 and Prevotella spp. The same two bacteria remained significantly associated with BV in a multivariate model after adjusting for the other five species. There was no statistically significant difference between the sensitivities and specificities of BV diagnosis by molecular methods performed on swabs and FVU samples. A linear regression analysis showed good agreement between bacterial loads from swabs and FVU, but Prevotella spp. could be detected in high numbers in a few FVU samples without being present in swabs. This method will allow diagnosis of BV in studies where only urine has been collected and where detection of BV is considered relevant.

Vaginal microbiome in women from Greenland assessed by microscopy and quantitative PCR.

BACKGROUND: Bacterial vaginosis (BV) is a common condition, although its aetiology remains unexplained. The aim of this study was to analyse the composition of vaginal microbiota in women from Greenland to provide a quantitative description and improve the understanding of BV. METHODS: Self-collected vaginal smears and swabs were obtained from 177 women. The vaginal smears were graded for BV according to Nugent's criteria. The vaginal swab samples were analysed by 19 quantitative PCRs (qPCRs) for selected vaginal bacteria and by PCR for four sexually transmitted infections (STIs). RESULTS: STIs were common: Mycoplasma genitalium 12%, Chlamydia trachomatis 7%, Neisseria gonorrhoeae 1%, and Trichomonas vaginalis 0.5%. BV was found in 45% of women, but was not associated with individual STIs. Seven of the 19 vaginal bacteria (Atopobium vaginae, Prevotella spp., Gardnerella vaginalis, BVAB2, Eggerthella-like bacterium, Leptotrichia amnionii, and Megasphaera type 1) had areas under the receiver operating characteristic (ROC) curve > 85%, suggesting they are good predictors of BV according to Nugent. Prevotella spp. had the highest odds ratio for BV (OR 437; 95% CI 82-2779) in univariate analysis considering only specimens with a bacterial load above the threshold determined by ROC curve analysis as positive, as well as the highest adjusted odds ratio in multivariate logistic regression analysis (OR 4.4; 95% CI 1.4-13.5). BV could be subdivided into clusters dominated by a single or a few species together. CONCLUSIONS: BV by Nugent score was highly prevalent. Two of seven key species (Prevotella spp. and A. vaginae) remained significantly associated with BV in a multivariate model after adjusting for other bacterial species. G. vaginalis and Prevotella spp. defined the majority of BV clusters.

Targeted AntiBiotics for Chronic pulmonary diseases (TARGET ABC): can targeted antibiotic therapy improve the prognosis of Pseudomonas aeruginosa-infected patients with chronic pulmonary obstructive disease, non-cystic fibrosis bronchiectasis, and asthma? A multicenter, randomized, controlled, open-label trial.

BACKGROUND: Pseudomonas aeruginosa infection is seen in chronic pulmonary disease and is associated with exacerbations and poor long-term prognosis. However, evidence-based guidelines for the management and treatment of P. aeruginosa infection in chronic, non-cystic fibrosis (CF) pulmonary disease are lacking. The aim of this study is to investigate whether targeted antibiotic treatment against P. aeruginosa can reduce exacerbations and mortality in patients with chronic obstructive pulmonary disease (COPD), non-CF bronchiectasis, and asthma. METHODS: This study is an ongoing multicenter, randomized, controlled, open-label trial. A total of 150 patients with COPD, non-CF bronchiectasis or asthma, and P. aeruginosa-positive lower respiratory tract samples will be randomly assigned with a 1:1 ratio to either no antibiotic treatment or anti-pseudomonal antibiotic treatment with intravenous beta-lactam and oral ciprofloxacin for 14 days. The primary outcome, analyzed with two co-primary endpoints, is (i) time to prednisolone and/or antibiotic requiring exacerbation or death, in the primary or secondary health sector, within days 20-365 from study allocation and (ii) days alive and without exacerbation within days 20-365 from the study allocation. DISCUSSION: This trial will determine whether targeted antibiotics can benefit future patients with chronic, non-CF pulmonary disease and P. aeruginosa infection in terms of reduced morbidity and mortality, thus optimizing therapeutic approaches in this large group of chronic patients. TRIAL REGISTRATION: ClinicalTrials.gov NCT03262142 . Registered on August 25, 2017.

Update 2016-2018 of the Nationwide Danish Fungaemia Surveillance Study: Epidemiologic Changes in a 15-Year Perspective.

As part of a national surveillance programme initiated in 2004, fungal blood isolates from 2016-2018 underwent species identification and EUCAST susceptibility testing. The epidemiology was described and compared to data from previous years. In 2016-2018, 1454 unique isolates were included. The fungaemia rate was 8.13/100,000 inhabitants compared to 8.64, 9.03, and 8.38 in 2004-2007, 2008-2011, and 2012-2015, respectively. Half of the cases (52.8%) involved patients 60-79 years old and the incidence was highest in males ≥70 years old. Candida albicans accounted for 42.1% of all isolates and Candida glabrata for 32.1%. C. albicans was more frequent in males (p = 0.03) and C. glabrata in females (p = 0.03). During the four periods, the proportion of C. albicans decreased (p < 0.001), and C. glabrata increased (p < 0.001). Consequently, fluconazole susceptibility gradually decreased from 68.5% to 59.0% (p < 0.001). Acquired fluconazole resistance was found in 4.6% Candida isolates in 2016-2018. Acquired echinocandin resistance increased during the four periods 0.0%, 0.6%, 1.7% to 1.5% (p < 0.0001). Sixteen echinocandin-resistant isolates from 2016-2018 harboured well-known FKS resistance-mutations and one echinocandin-resistant C. albicans had an FKS mutation outside the hotspot (P1354P/S) of unknown importance. In C. glabrata specifically, echinocandin resistance was detected in 12/460 (2.6%) in 2016-2018 whereas multidrug-class resistance was rare (1/460 isolates (0.2%)). Since the increase in incidence during 2004-2011, the incidence has stabilised. In contrast, the species distribution has changed gradually over the 15 years, with increased C. glabrata at the expense of C. albicans. The consequent decreased fluconazole susceptibility and the emergence of acquired echinocandin resistance complicates the management of fungaemia and calls for antifungal drug development.

Characterization of the vaginal microflora in health and disease.

BACKGROUND: Bacterial vaginosis (BV) is an imbalance of the vaginal bacterial microbiota and its aetiology is still unknown. Our aims were to investigate the diagnostic potential of species/genus specific quantitative PCR (qPCR) for bacteria present in swabs and first-void urine (FVU) samples using Nugent's and Claeys' criteria and 454 sequencing of the vaginal microbiome as reference. METHODS: Self-collected swabs, vaginal smears and FVU were obtained from 177 women from Greenland (Study I and III) and physician-collected vaginal swabs and smears were obtained from 163 Swedish women (Study II). BV was diagnosed by Nugent's criteria in Study I and III and by Amsel's criteria in Study II. The vaginal swabs and FVU samples were analysed by qPCR for selected vaginal bacteria in all three studies and for four sexually transmitted infections (STIs) in Study I. RESULTS: Study I: STIs were common in women from Greenland and BV was found in 45% of these women but was not associated with individual STIs. In multivariate logistic analysis, Atopobium vaginae and Prevotella spp. were both independently associated with BV in swabs. BV could be subdivided into clusters dominated by a single or a few species together. Seven vaginal bacteria (A. vaginae, Prevotella spp. Gardnerella vaginalis, Bacterial vaginosis associated bacterium (BVAB) 2, Eggerthella-like bacterium, Leptotrichia amnionii and Megasphaera type 1) had areas under the receiver operating characteristic (ROC) curve > 85% in swabs, suggesting that they were good predictors of BV according to Nugent. Study II: For the majority of species/genera, the kappa values indicated fair to good agreement when their presence was determined by 454 pyrosequencing versus real-time PCR. The same seven vaginal bacteria as found in Study I, had areas under the ROC-curve > 85% in swabs from Swedish women, demonstrating a good diagnostic accuracy for BV according to Amsel. Study III: In a multivariate model, Megasphaera type 1 and Prevotella spp. remained significantly associated with BV in FVU samples. A linear regression analysis showed good agreement between bacterial load from swabs and FVU, but Prevotella spp. could be detected in high numbers in a few FVU samples without being present in swabs. After applying ROC curve analysis, the same seven vaginal bacteria as previously mentioned showed good prediction for BV according to Nugent in FVU. BV could be detected with comparable sensitivity in FVU and vaginal swabs. CONCLUSION: BV can be diagnosed by molecular methods performed either on swabs or urine but it is important to apply thresholds in order to improve the accuracy of the diagnosis. Furthers it was possible to identify clusters of BV dominated by single or paired bacteria, and these clusters could classify BV into subgroups, providing a more detailed understanding of the condition. Seven vaginal bacteria were highly accurate for BV diagnosis both in swabs and FVU. Finally a good agreement between Nugent and Claeys was found.

Composition of the vaginal microbiota in women of reproductive age--sensitive and specific molecular diagnosis of bacterial vaginosis is possible?

BACKGROUND AND OBJECTIVE: Bacterial vaginosis (BV) is the most common vaginal disorder, characterized by depletion of the normal lactobacillus-dominant microbiota and overgrowth of commensal anaerobic bacteria. This study aimed to investigate the composition of the vaginal microbiota in women of reproductive age (healthy women and women with BV), with the view of developing molecular criteria for BV diagnosis. MATERIALS AND METHODS: Vaginal samples from 163 women (79 control, 73 BV and 11 intermediate (Lactobacillary grade II flora) cases) were analyzed using 454 pyrosequencing of the hypervariable regions V3-V4 of the 16S rRNA gene and 16 quantitative bacterial species/genus-specific real-time PCR assays. Sensitivities and specificities of potential BV markers were computed using the Amsel criteria as reference standard for BV. The use of quantitative thresholds for prediction of BV, determined for both relative abundance measured with 454 pyrosequencing and bacterial load measured with qPCR, was evaluated. RESULTS: Relative to the healthy women, the BV patients had in their vaginal microbiota significantly higher prevalence, loads and relative abundances of the majority of BV associated bacteria. However, only Gardnerella vaginalis, Atopobium vaginae, Eggerthella, Prevotella, BVAB2 and Megasphaera type 1 detected at or above optimal thresholds were highly predictable for BV, with the best diagnostic accuracy shown for A. vaginae. The depletion of Lactobacillus species combined with the presence of either G. vaginalis or A. vaginae at diagnostic levels was a highly accurate BV predictor. CONCLUSIONS: Quantitative determination of the presence of G. vaginalis, A. vaginae, Eggerthella, Prevotella, BVAB2 and Megasphaera type 1 as well as the depletion of Lactobacillus was highly accurate for BV diagnosis. Measurements of abundance of normal and BV microbiota relative to total bacteria in vaginal fluid may provide more accurate BV diagnosis, and be used for test-of-cure, rather than qualitative detection or absolute counts of BV related microorganisms.

[Septic shock caused by Gardnerella vaginalis and Peptostreptococcus species after Cesarean section]. / Septisk shock forårsaget af Gardnerella vaginalis og peptostreptokokker efter sectio.

A 41-year old multisectio patient got a life-threatening postoperative infection with Gardnerella vaginalis and Peptostreptococcus spp. Perioperative treatment with cefuroxime and metronidazol is recommended.