Essential oils of Pinus sylvestris, Citrus limon and Origanum vulgare exhibit high bactericidal and anti-biofilm activities against Neisseria gonorrhoeae and Streptococcus suis.

Antimicrobial resistance is a worldwide problem that urges novel alternatives to treat infections. In attempts to find novel molecules, we assess the antimicrobial potential of seven essential oils (EO) of different plants (Pinus sylvestris, Citrus limon, Origanum vulgare, Cymbopogon martini, Cinnamomum cassia, Melaleuca alternifolia and Eucalyptus globulus) against two multidrug-resistant bacteria species, i.e. Neisseria gonorrhoeae and Streptococcus suis. EOs of P. sylvestris and C. limon revealed higher bactericidal activity (MIC ≤ 0.5 mg/mL) and capacity to rapidly disperse biofilms of several N. gonorrhoeae clinical isolates than other EOs. Examination of biofilms exposed to both EO by electron microscopy revealed a reduction of bacterial aggregates, high production of extracellular vesicles, and alteration of cell integrity. This activity was dose-dependent and was enhanced in DNase I-treated biofilms. Antibiotic susceptibility studies confirmed that both EOs affected the outer membrane permeability, and analysis of EO- susceptibility of an LPS-deficient mutant suggested that both EO target the LPS bilayer. Further analysis revealed that α- and ß-pinene and d-limonene, components of both EO, contribute to such activity. EO of C. martini, C. cassia, and O. vulgare exhibited promising antimicrobial activity (MIC ≤ 0.5 mg/mL) against S. suis, but only EO of O. vulgare exhibited a high biofilm dispersal activity, which was also confirmed by electron microscopy studies. To conclude, the EO of P. sylvestris, C. limon and O. vulgare studied in this work exhibit bactericidal and anti-biofilm activities against gonococcus and streptococcus, respectively.

In vitro synergy screens of FDA-approved drugs reveal novel zidovudine- and azithromycin-based combinations with last-line antibiotics against Klebsiella pneumoniae.

Treatment of infections caused by multi-drug resistant (MDR) enterobacteria remains challenging due to the limited therapeutic options available. Drug repurposing could accelerate the development of new urgently needed successful interventions. This work aimed to identify and characterise novel drug combinations against Klebsiella pneumoniae based on the concepts of synergy and drug repurposing. We first performed a semi-qualitative high-throughput synergy screen (sHTSS) with tigecycline, colistin and fosfomycin (last-line antibiotics against MDR Enterobacteriaceae) against a FDA-library containing 1430 clinically approved drugs; a total of 109 compounds potentiated any of the last-line antibiotics. Selected hits were further validated by secondary checkerboard (CBA) and time-kill (TKA) assays, obtaining 15.09% and 65.85% confirmation rates, respectively. Accordingly, TKA were used for synergy classification based on determination of bactericidal activities at 8, 24 and 48 h, selecting 27 combinations against K. pneumoniae. Among them, zidovudine or azithromycin combinations with last-line antibiotics were further evaluated by TKA against a panel of 12 MDR/XDR K. pneumoniae strains, and their activities confronted with those clinical combinations currently used for MDR enterobacteria treatment; these combinations showed better bactericidal activities than usual treatments without added cytotoxicity. Our studies show that sHTSS paired to TKA are powerful tools for the identification and characterisation of novel synergistic drug combinations against K. pneumoniae. Further pre-clinical studies might support the translational potential of zidovudine- and azithromycin-based combinations for the treatment of these infections.

Clinical Performance Evaluation of a Rapid Real-Time PCR Assay for Monkeypox Diagnosis: a Retrospective and Comparative Study.

In an increasingly globalized and interconnected world, the outbreak of an infectious disease in one country can become a worrying health emergency for the whole world. A current example is the 2022 monkeypox virus (mpox) outbreak affecting multiple areas across the world. In this context, strategies to interrupt transmission as soon as possible by identifying cases, clusters, and sources of infection should be developed around the world to prevent these crises. The aim of this retrospective and collaborative study was to perform external clinical validation of the VIASURE monkeypox virus real-time PCR detection kit (CerTest Biotec, Spain) with ready-to-use reagents designed for the rapid detection of mpox. A total of 165 samples with suspected infection were used for this analysis. The standard procedures of the clinical microbiology laboratory of the Miguel Servet University Hospital, using the RealStar Orthopoxvirus PCR kit v1.0 (Altona Diagnostics) and bidirectional Sanger sequencing (STAB VIDA, Caparica, Portugal), were considered reference techniques. Furthermore, a subset of 67 mpox-negative samples and 13 mpox-positive samples were routinely tested for clinical diagnosis of other rash/ulcerative pathologies. Accuracy testing resulted in appropriate clinical validation values, as follows: sensitivity, 1 (95% confidence interval [CI], 0.97 to 1); specificity, 1 (95% CI, 0.98 to 1); positive predictive value, 1 (95% CI, 0.93 to 1); negative predictive value, 1 (95% CI, 0.95 to 1). The strength of agreement between assays was almost perfect. The added value is the useful support for the specific diagnosis of mpox infections due to the diagnostic specificity data obtained. IMPORTANCE Given that a large number of mpox outbreaks have been reported worldwide since 2022 in countries in which the disease is not endemic, the main concern for clinicians and global health systems should be to develop effective, available, and easy-to-implement diagnostic strategies to interrupt mpox transmission as soon as possible. This retrospective study demonstrates the satisfactory clinical parameters of a commercially available molecular diagnostic kit for routine testing for mpox in clinical diagnostic laboratories.

Retrospective and Comparative Study of Three Molecular Assays for the Macrolide Resistance Detection in Mycoplasma genitalium Positive Urogenital Specimens.

The capacity of Mycoplasma genitalium to develop resistance to macrolides makes detection of macrolide resistance genes by rapid real-time PCR assays increasingly necessary in clinical diagnostic laboratories so as to initiate appropriate treatment as rapidly as possible. The aim of this retrospective and comparative study was to conduct the clinical evaluation of three commercially available kits for macrolide resistance detection. A total of 111 M. genitalium positive samples analyzed in the Clinical Microbiology Laboratory of the Miguel Servet University Hospital, Zaragoza (Spain) were used. After M. genitalium molecular confirmation, the three assays under study were evaluated and discrepant results were resolved via sequencing. The clinical sensitivity for resistance detection was 83% (95% confidence interval, 69% to 93%) for the ResistancePlus® MG panel kit (SpeeDx Pty Ltd., Sydney, Australia), 95% (84% to 99%) for AllplexTM MG & AziR Assay (Seegene®, Seoul, Korea), and 97% (88% to 99%) for the VIASURE macrolide resistance-associated mutations (23SrRNA) Real time PCR detection kit (Certest Biotec, Zaragoza, Spain). The clinical specificity was 100% (94% to 100%) for Allplex and VIASURE assays and 95% (86% to 99%) for SpeeDx assay. The results arising from this study are cause for strong consideration for the implementation of rapid real-time PCR assays in clinical diagnosis laboratories to eliminate treatment failure and transmission as soon as possible.

Survival of Patient With Hemorrhagic Meningitis Associated With Inhalation Anthrax.

This report describes a 49-year-old male construction worker who acquired a Bacillus anthracis infection after working on a sheep farm. He experienced a severe respiratory infection, septic shock, and hemorrhagic meningoencephalitis with severe intracranial hypertension. After several weeks with multiple organ dysfunction syndrome, he responded favorably to antibiotic treatment. Three weeks into his hospitalization, an intracranial hemorrhage and cerebral edema led to an abrupt deterioration in his neurological status. A single dose of raxibacumab was added to his antimicrobial regimen on hospital day 27. His overall status, both clinical and radiographic, improved within a few days. He was discharged 2 months after admission and appears to have fully recovered.

Retrospective Study for the Clinical Evaluation of a Real-Time PCR Assay with Lyophilized and Ready-to-Use Reagents for Streptococcus agalactiae Detection in Prenatal Screening Specimens.

Streptococcus agalactiae is a leading cause of sepsis and meningitis in newborns and young infants. Screening programs and intrapartum antibiotic prophylaxis have reduced early neonatal onset of disease. The aim of this study was to evaluate a molecular assay with lyophilized and ready-to-use reagents: VIASURE® Streptococcus B Real Time PCR detection kit (CerTest Biotec) (Viasure qPCR assay) compared to both the GBS culture and a molecular assay with separated and frozen reagents: Strep B Real-TM Quant (Sacace Biotecnologies®) (Sacace qPCR assay). A total of 413 vaginal−rectal swabs from women between the 35th and 37th weeks of pregnancy were processed. GBS culture was firstly achieved through Granada medium and Columbia CNA agar at 35 °C in aerobic conditions. Then, nucleic acid extraction was performed for subsequent molecular analysis using both commercial assays. Discordant results were resolved via bidirectional Sanger sequencing. Viasure qPCR assay clinical sensitivity was 0.97 (0.92−0.99) and specificity 1 (0.98−1). This retrospective study demonstrated the good clinical parameters and the strong overall agreement (99.3%) between the Viasure qPCR assay and both reference assays. Finally, the added value observed of the assay under study was the stabilized and ready-to-use format, reducing the number of time-consuming steps, permitting the storage at room temperature, facilitating transport, being environmentally respectful, and reducing additional costs.

In Vitro Antifungal Activity of Ibrexafungerp (SCY-078) Against Contemporary Blood Isolates From Medically Relevant Species of Candida: A European Study.

Background: Ibrexafungerp (SCY-078) is the newest oral and intravenous antifungal drug with broad activity, currently undergoing clinical trials for invasive candidiasis. Objective: The aim of this study was to assess the in vitro activity of ibrexafungerp and comparators against a collection of 434 European blood isolates of Candida. Methods: Ibrexafungerp, caspofungin, fluconazole, and micafungin minimum inhibitory concentrations (MICs) were collected from 12 European laboratories for 434 blood isolates, including 163 Candida albicans, 108 Candida parapsilosis, 60 Candida glabrata, 40 Candida tropicalis, 29 Candida krusei, 20 Candida orthopsilosis, 6 Candida guilliermondii, 2 Candida famata, 2 Candida lusitaniae, and 1 isolate each of Candida bracarensis, Candida catenulata, Candida dubliniensis, and Candida kefyr. MICs were determined by the EUCAST broth microdilution method, and isolates were classified according to recommended clinical breakpoints and epidemiological cutoffs. Additionally, 22 Candida auris from different clinical specimens were evaluated. Results: Ibrexafungerp MICs ranged from 0.016 to ≥8 mg/L. The lowest ibrexafungerp MICs were observed for C. albicans (geometric MIC 0.062 mg/L, MIC range 0.016-0.5 mg/L) and the highest ibrexafungerp MICs were observed for C. tropicalis (geometric MIC 0.517 mg/L, MIC range 0.06-≥8 mg/L). Modal MICs/MIC50s (mg/L) against Candida spp. were 0.125/0.06 for C. albicans, 0.5/0.5 for C. parapsilosis, 0.25/0.25 for C. glabrata, 0.5/0.5 for C. tropicalis, 1/1 for C. krusei, 4/2 for C. orthopsilosis, and 0.5/0.5 for C. auris. Ibrexafungerp showed activity against fluconazole- and echinocandin-resistant isolates. If adopting wild-type upper limits, a non-wild-type phenotype for ibrexafungerp was only observed for 16/434 (3.7%) isolates: 11 (4.6%) C. parapsilosis, 4 (5%) C. glabrata, and 1 (2.5%) C. tropicalis. Conclusion: Ibrexafungerp showed a potent in vitro activity against Candida.

Successful control of Serratia marcescens outbreak in a neonatal unit of a tertiary-care hospital in Spain.

OBJECTIVE: Serratia marcescens is a Gram-negative bacterium that is found in hospital environments and commonly associated with outbreaks in neonatal units. One S. marcescens isolate was detected from a bloodstream culture from a neonate in our hospital that was followed by an outbreak. The aim of this study was to describe the molecular epidemiology of a S. marcescens outbreak in the neonatal unit. METHODS: In order to investigate the outbreak, weekly surveillance rectal swabs were submitted for culture from all patients admitted in this unit from August to September 2018. Environmental samples were obtained from potential sources in September 2018. Typing of isolates was performed by pulsed field gel electrophoresis (PFGE). In addition, we studied the in vitro activity of chlorhexidine against S. marcescens. RESULTS: During this period, 146 infants were hospitalised in our neonatal unit, of which 16 patients had a S. marcescens-positive sample. A total of 36 environmental surveillance samples were collected, and one sample from a stethoscope from an incubator of a colonized baby was positive for S. marcescens. All the 18 isolates, including the isolate from the stethoscope, belonged to a single PFGE cluster. We found that very low concentrations of chlorhexidine, even with application times close to 0 achieved significant reductions in the amount of S. marcescens. CONCLUSION: A unique clone of S. marcescens caused this outbreak, including isolates from patients and from one stethoscope. The outbreak was controlled with the early implementation of specific control measures.

Integration of In Silico and In Vitro Analysis of Gliotoxin Production Reveals a Narrow Range of Producing Fungal Species.

Gliotoxin is a fungal secondary metabolite with impact on health and agriculture since it might act as virulence factor and contaminate human and animal food. Homologous gliotoxin (GT) gene clusters are spread across a number of fungal species although if they produce GT or other related epipolythiodioxopiperazines (ETPs) remains obscure. Using bioinformatic tools, we have identified homologous gli gene clusters similar to the A. fumigatus GT gene cluster in several fungal species. In silico study led to in vitro confirmation of GT and Bisdethiobis(methylthio)gliotoxin (bmGT) production in fungal strain cultures by HPLC detection. Despite we selected most similar homologous gli gene cluster in 20 different species, GT and bmGT were only detected in section Fumigati species and in a Trichoderma virens Q strain. Our results suggest that in silico gli homology analyses in different fungal strains to predict GT production might be only informative when accompanied by analysis about mycotoxin production in cell cultures.

In Vitro Effect of Photodynamic Therapy with Different Lights and Combined or Uncombined with Chlorhexidine on Candida spp.

Candidiasis is very common and complicated to treat in some cases due to increased resistance to antifungals. Antimicrobial photodynamic therapy (aPDT) is a promising alternative treatment. It is based on the principle that light of a specific wavelength activates a photosensitizer molecule resulting in the generation of reactive oxygen species that are able to kill pathogens. The aim here is the in vitro photoinactivation of three strains of Candida spp., Candida albicans ATCC 10231, Candida parapsilosis ATCC 22019 and Candida krusei ATCC 6258, using aPDT with different sources of irradiation and the photosensitizer methylene blue (MB), alone or in combination with chlorhexidine (CHX). Irradiation was carried out at a fluence of 18 J/cm2 with a light-emitting diode (LED) lamp emitting in red (625 nm) or a white metal halide lamp (WMH) that emits at broad-spectrum white light (420-700 nm). After the photodynamic treatment, the antimicrobial effect is evaluated by counting colony forming units (CFU). MB-aPDT produces a 6 log10 reduction in the number of CFU/100 µL of Candida spp., and the combination with CHX enhances the effect of photoinactivation (effect achieved with lower concentration of MB). Both lamps have similar efficiencies, but the WMH lamp is slightly more efficient. This work opens the doors to a possible clinical application of the combination for resistant or persistent forms of Candida infections.

Photodynamic Therapy Combined with Antibiotics or Antifungals against Microorganisms That Cause Skin and Soft Tissue Infections: A Planktonic and Biofilm Approach to Overcome Resistances.

The present review covers combination approaches of antimicrobial photodynamic therapy (aPDT) plus antibiotics or antifungals to attack bacteria and fungi in vitro (both planktonic and biofilm forms) focused on those microorganisms that cause infections in skin and soft tissues. The combination can prevent failure in the fight against these microorganisms: antimicrobial drugs can increase the susceptibility of microorganisms to aPDT and prevent the possibility of regrowth of those that were not inactivated during the irradiation; meanwhile, aPDT is effective regardless of the resistance pattern of the strain and their use does not contribute to the selection of antimicrobial resistance. Additive or synergistic antimicrobial effects in vitro are evaluated and the best combinations are presented. The use of combined treatment of aPDT with antimicrobials could help overcome the difficulty of fighting high level of resistance microorganisms and, as it is a multi-target approach, it could make the selection of resistant microorganisms more difficult.

Chalcogenide nanoparticles and organic photosensitizers for synergetic antimicrobial photodynamic therapy.

Synergistic antimicrobial effects were observed for copper sulfide (CuS) nanoparticles together with indocyanine green (ICG) in the elimination of wild type pathogenic bacteria (Staphylococcus aureus ATCC 29213 and Pseudomonas aeruginosa ATCC 27853) and also opportunistic fungal infective yeast (Candida albicans ATCC 10231). Furthermore, large antibacterial effects were observed for clinical isolates of Methicillin-resistant S. aureus (MRSA) PFGE strain-type USA300. This efficient antimicrobial action was attributed to the combined extra- and intracellular generation of reactive oxygen species upon light irradiation. Instead of the use of visible-light for the activation of common photosensitizers, both ICG and CuS nanoparticles can be activated in the near infrared (NIR)-region of the electromagnetic spectrum and therefore, superior tissue penetration would be expected in a potential elimination of pathogenic microorganisms not only on the skin but also in the soft tissue. In the different bacteria studied a 3-log reduction in the bacterial counts was achieved after only 6 min of NIR irradiation and treatment with ICG or CuS alone at concentrations of 40 and 160 µg mL-1, respectively. A maximum bactericidal effect against S. aureus and USA300 strains was obtained for the combination of both photosensitizers at the same concentration. Regarding P. aeruginosa, a 4-log reduction in the CFU was observed for the combination of CuS and ICG at various concentrations. In Candida albicans the combination of both ICG and CuS and light irradiation showed an antimicrobial dose-dependent effect with the reduction of at least 3-log in the cell counts for the combination of ICG + CuS at reduced concentrations. The observed antimicrobial effect was solely attributed to a photodynamic effect and any photothermal effect was avoided to discard any potential thermal injury in a potential clinical application. The generation of reactive oxygen species upon near infrared-light irradiation for those photosensitizers used was measured either alone or in combination. The cytocompatibility of the proposed materials at the doses used in photodynamic therapy was also demonstrated in human dermal fibroblasts and keratinocytes by cell culturing and flow cytometry studies.

Successful control of Serratia marcescens outbreak in a neonatal unit of a tertiary-care hospital in Spain.

OBJECTIVE: Serratia marcescens is a Gram-negative bacterium that is found in hospital environments and commonly associated with outbreaks in neonatal units. One S. marcescens isolate was detected from a bloodstream culture from a neonate in our hospital that was followed by an outbreak. The aim of this study was to describe the molecular epidemiology of a S. marcescens outbreak in the neonatal unit. METHODS: In order to investigate the outbreak, weekly surveillance rectal swabs were submitted for culture from all patients admitted in this unit from August to September 2018. Environmental samples were obtained from potential sources in September 2018. Typing of isolates was performed by pulsed field gel electrophoresis (PFGE). In addition, we studied the in vitro activity of chlorhexidine against S. marcescens. RESULTS: During this period, 146 infants were hospitalised in our neonatal unit, of which 16 patients had a S. marcescens-positive sample. A total of 36 environmental surveillance samples were collected, and one sample from a stethoscope from an incubator of a colonized baby was positive for S. marcescens. All the 18 isolates, including the isolate from the stethoscope, belonged to a single PFGE cluster. We found that very low concentrations of chlorhexidine, even with application times close to 0 achieved significant reductions in the amount of S. marcescens. CONCLUSION: A unique clone of S. marcescens caused this outbreak, including isolates from patients and from one stethoscope. The outbreak was controlled with the early implementation of specific control measures.

Comparison of Antibacterial Activity and Wound Healing in a Superficial Abrasion Mouse Model of Staphylococcus aureus Skin Infection Using Photodynamic Therapy Based on Methylene Blue or Mupirocin or Both.

Background: Antibiotic resistance and impaired wound healing are major concerns in S. aureus superficial skin infections, and new therapies are needed. Antimicrobial photodynamic therapy (aPDT) is a new therapeutic approach for infections, but it also improves healing in many wound models. Objective: To compare the antimicrobial activity and the effects on wound healing of aPDT based on Methylene Blue (MB-aPDT) with mupirocin treatment, either alone or in combination, in superficial skin wounds of S. aureus-infected mice. Additionally, to evaluate the clinical, microbiological, and cosmetic effects on wound healing. Materials and Methods: A superficial skin infection model of S. aureus was established in SKH-1 mice. Infected wounds were treated with MB-aPDT, MB-aPDT with a daily topical mupirocin or only with mupirocin. No treatment was carried out in control animals. Daily clinical and microbiological examinations were performed until complete clinical wound healing. Histopathological studies and statistical analysis were performed at the end of the study. Results: MB-aPDT treatment induced the best wound healing compared to mupirocin alone or to mupirocin plus MB-aPDT. Superficial contraction at 24 h and a greater reduction in size at 48 h, quicker detachment of the crust, less scaling, and absence of scars were observed. Histopathological studies correlated with clinical and gross findings. By contrast, mupirocin showed the highest logaritmic reduction of S. aureus. Conclusions: MB-aPDT and mupirocin treatments are effective in a murine superficial skin infection model of S. aureus. One session of MB-aPDT was the best option for clinical wound healing and cosmetic results. The addition of mupirocin to MB-aPDT treatment improved antimicrobial activity; however, it did not enhance wound healing. No synergistic antibacterial effects were detected.

Broad-Spectrum Photo-Antimicrobial Polymers Based on Cationic Polystyrene and Rose Bengal.

New strategies to fight bacteria and fungi are necessary in view of the problem of iatrogenic and nosocomial infections combined with the growing threat of increased antimicrobial resistance. Recently, our group has prepared and described two new readily available materials based on the combination of Rose Bengal (singlet oxygen photosensitizer) and commercially available cationic polystyrene (macroporous resin Amberlite® IRA 900 or gel-type resin IRA 400). These materials showed high efficacy in the antimicrobial photodynamic inactivation (aPDI) of Pseudomonas aeruginosa. Here, we present the photobactericidal effect of these polymers against an extended group of pathogens like Escherichia coli, Enterococcus faecalis, Staphylococcus aureus, and the opportunistic yeast Candida albicans using green light. The most interesting finding is that the studied materials are able to reduce the population of both Gram-positive and Gram-negative bacteria with good activity, although, for C. albicans, in a moderate manner. In view of the results achieved and especially considering the inexpensiveness of these two types of photoactive polymers, we believe that they could be used as the starting point for the development of coatings for self-disinfecting surfaces.

In Vitro and In Vivo Antibacterial Activity of Gliotoxin Alone and in Combination with Antibiotics against Staphylococcus aureus.

Multidrug-resistant bacteria such as methicillin-resistant Staphylococcus aureus (MRSA) is one of the major causes of hospital-acquired and community infections and pose a challenge to the human health care system. Therefore, it is important to find new drugs that show activity against these bacteria, both in monotherapy and in combination with other antimicrobial drugs. Gliotoxin (GT) is a mycotoxin produced by Aspergillus fumigatus and other fungi of the Aspergillus genus. Some evidence suggests that GT shows antimicrobial activity against S. aureus in vitro, albeit its efficacy against multidrug-resistant strains such asMRSA or vancomycin-intermediate S. aureus (VISA) strainsis not known. This work aimedto evaluate the antibiotic efficacy of GT as monotherapy or in combination with other therapeutics against MRSA in vitro and in vivo using a Caenorhabditis elegans infection model.

Core Microbiota in Central Lung Cancer With Streptococcal Enrichment as a Possible Diagnostic Marker.

BACKGROUND: Dysbiosis in lung cancer has been underexplored. The aim of this study was to define the bacterial and fungal microbiota of the bronchi in central lung cancer and to compare it with that of the oral and intestinal compartments. METHODS: Twenty-five patients with central lung cancer and sixteen controls without antimicrobial intake during the previous month were recruited. Bacterial and fungal distribution was determined by massive sequencing of bronchial biopsies and saliva and faecal samples. Complex computational analysis was performed to define the core lung microbiota. RESULTS: Affected and contralateral bronchi of patients have almost identical microbiota dominated by Streptococcus, whereas Pseudomonas was the dominant genera in controls. Oral and pulmonary ecosystems were significantly more similar in patients, probably due to microaspirations. Streptococcal abundance in the bronchi differentiated patients from controls according to a ROC curve analysis (90.9% sensitivity, 83.3% specificity, AUC=0.897). The saliva of patients characteristically showed a greater abundance of Streptococcus, Rothia, Gemella and Lactobacillus. The mycobiome of controls (Candida) was significantly different from that of patients (Malassezia). Cancer patients' bronchial mycobiome was similar to their saliva, but different from their contralateral bronchi. CONCLUSIONS: The central lung cancer microbiome shows high levels of Streptococcus, and differs significantly in its composition from that of control subjects. Changes are not restricted to tumour tissue, and seem to be the consequence of microaspirations from the oral cavity. These findings could be useful in the screening and even diagnosis of this disease.

Invasive Fusariosis in Nonneutropenic Patients, Spain, 2000-2015.

Invasive fusariosis (IF) is associated with severe neutropenia in patients with concurrent hematologic conditions. We conducted a retrospective observational study to characterize the epidemiology of IF in 18 Spanish hospitals during 2000-2015. In that time, the frequency of IF in nonneutropenic patients increased from 0.08 cases per 100,000 admissions in 2000-2009 to 0.22 cases per 100,000 admissions in 2010-2015. Nonneutropenic IF patients often had nonhematologic conditions, such as chronic cardiac or lung disease, rheumatoid arthritis, history of solid organ transplantation, or localized fusariosis. The 90-day death rate among nonneutropenic patients (28.6%) and patients with resolved neutropenia (38.1%) was similar. However, the death rate among patients with persistent neutropenia (91.3%) was significantly higher. We used a multivariate Cox regression analysis to characterize risk factors for death: persistent neutropenia was the only risk factor for death, regardless of antifungal therapy.

Azole resistance survey on clinical Aspergillus fumigatus isolates in Spain.

OBJECTIVES: We aimed to assess the percentage of azole resistance in Aspergillus fumigatus in Spain. METHODS: Thirty participating Spanish hospitals stored all morphologically identified A. fumigatus sensu lato clinical isolates-regardless their clinical significance-from 15 February to 14 May 2019. Isolates showing azole resistance according to the EUCAST 9.3.2 methodology were molecularly identified and the cyp51A gene was studied in A. fumigatus sensu stricto isolates. RESULTS: Eight hundred and forty-seven isolates from 725 patients were collected in 29 hospitals (A. fumigatus sensu stricto (n = 828) and cryptic species (n = 19)). Isolates were mostly from the lower respiratory tract (94.0%; 797/847). Only cryptic species were amphotericin B resistant. Sixty-three (7.4%) out of the 847 isolates were resistant to ≥1 azole(s). Azole resistance was higher in cryptic species than in A. fumigatus sensu stricto (95%, 18/19 vs. 5.5%, 45/828); isavuconazole was associated to the lowest number of non-wild type isolates. The dominant mechanism of resistance was the presence of TR34-L98H substitutions (n = 24 out of 63). Out of the 725 patients, 48 (6.6%) carried either cryptic species (n = 14) or A. fumigatus sensu stricto (n = 34; 4.7%) resistant isolates. Aspergillus fumigatus sensu stricto harbouring either the TR34-L98H (n = 19) or TR46/Y121F/T289A (n = 1) mutations were detected in patients in hospitals located at 7/24 studied cities. DISCUSSION: Of the patients, 6.6% carry azole-resistant A. fumigatus sensu lato isolates in Spain. TR34-L98H is the dominant cyp51A gene substitutions, although its presence is not widespread.