Background
Studies comparing gradient diffusion strips (GDSs) and the EUCAST E.Def 9.4 microdilution method are scarce, thwarted by a low number of isolates, and restricted to selected antifungal ...agents.
Objectives
We evaluated the performance of GDSs to detect azole resistance in A. fumigatus, including cryptic species.
Patients/Methods
A. fumigatus sensu stricto (n = 89) and cryptic species (n = 52) were classified as susceptible or resistant to itraconazole, voriconazole, posaconazole and isavuconazole (EUCAST E.Def 9.4; clinical breakpoints v10). A. fumigatus sensu stricto azole‐resistant isolates had the following cyp51A gene mutations: TR34‐L98H (n = 24), G54R (n = 5), TR46‐Y121F‐T289A (n = 1), F46Y‐M172V‐N248T‐D255E‐E427K (n = 1), F165L (n = 1) and cyp51A gene wild type (n = 3). GDSs (ETEST®, bioMèrieux, Marcy‐l'Etoile, France and Liofilchem®, Roseto degli Abruzzi, Italy) MICs were obtained by following the manufacturer's guidelines.
Results
For A. fumigatus sensu stricto, itraconazole MICs >1.5 mg/L, voriconazole >0.38 mg/L, posaconazole >0.75 mg/L, and isavuconazole >0.5 mg/L correctly separated resistant from susceptible isolates with two exceptions. Considering the aforementioned cut‐off MICs, sensitivity/specificity values of GDSs to detect azole resistance were: itraconazole (97%/100%), voriconazole (97%/100%), posaconazole (97%/100%) and isavuconazole (93.3%/100%). For cryptic species isolates, voriconazole MICs >1 mg/L and isavuconazole >0.75 mg/L separated resistant isolates from susceptible isolates with 15 and 27 exceptions, respectively. Considering the aforementioned cut‐off MICs, sensitivity/specificity values were as follows: voriconazole (68.1%/100%) and isavuconazole (25%/100%). For itraconazole and posaconazole, it was not possible to establish cut‐off values.
Conclusions
We set tentative cut‐off MIC values to correctly spot resistant Aspergillus fumigatus sensu stricto isolates using GDSs. The performance against cryptic species was poor.
We aimed to assess the percentage of azole resistance in Aspergillus fumigatus in Spain.
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.
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.
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.
The main goal of this study was to accurately detect azole resistance in species of the Aspergillus fumigatus complex by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry ...(MALDI-TOF MS).
Identification of isolates (n = 868) was done with MALDI-TOF MS using both commercial and in-house libraries. To determine azole susceptibility, the EUCAST E.Def. 9.3.2 method was applied as the reference standard. Identification of resistant isolates was confirmed by DNA sequence analysis. Protein spectra obtained by MALDI-TOF MS were analysed to differentiate species within the A. fumigatus complex and to detect azole-resistant A. fumigatus sensu stricto isolates.
Correct discrimination of A. fumigatus sensu stricto from cryptic species was accomplished in 100% of the cases applying principal component analysis (PCA) to protein spectra generated by MALDI-TOF MS. Furthermore, a specific peak (4586 m/z) was found to be present only in cryptic species. The application of partial least squares (PLS) discriminant analysis allowed 98.43% (±0.038) discrimination between susceptible and azole-resistant A. fumigatus sensu stricto isolates. Finally, based on PLS and SVM, A. fumigatus sensu stricto isolates with different cyp51A gene mutations were correctly clustered in 91.5% of the cases.
MALDI-TOF MS combined with peak analysis is a novel tool that allows the differentiation of A. fumigatus sensu stricto from other species within the A. fumigatus complex, as well as the detection of azole-resistant A. fumigatus sensu stricto. Although further studies are still needed, the results reported here show the great potential of MALDI-TOF and machine learning for the rapid detection of azole-resistant Aspergillus fumigatus isolates from clinical origins.
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The EUCAST EDef 9.3.2 procedure recommends visual readings of azole and amphotericin B MICs against
spp. Visual determination of MICs may be challenging. In this work, we aim to obtain and compare ...visual and spectrophotometric MIC readings of azoles and amphotericin B against
isolates. A total of 847
isolates (
= 828 and cryptic species
= 19) were tested against amphotericin B, itraconazole, voriconazole, posaconazole, and isavuconazole using the EUCAST EDef 9.3.2 procedure. Isolates were classified as susceptible or resistant/non-wild type according to the 2020 updated breakpoints. The area of technical uncertainty for the azoles was defined in the updated breakpoints. Visual and spectrophotometric (fungal growth reduction of >95% compared to the control, read at 540 nm) MICs were compared. Essential (±1 2-fold dilution) and categorical agreements were calculated. Overall, high essential (97.1%) and categorical (99.6%) agreements were found. We obtained 100% categorical agreements for amphotericin B, itraconazole, and posaconazole, and consequently, no errors were found. Categorical agreements were 98.7 and 99.3% for voriconazole and isavuconazole, respectively. Most of the misclassifications for voriconazole and isavuconazole were found to be associated with MIC results falling either in the area of technical uncertainty or within one 2-fold dilution above the breakpoint. The resistance rate was slightly lower when the MICs were obtained by spectrophotometric readings. However, all relevant
mutants were correctly classified as resistant. Spectrophotometric determination of azole and amphotericin B MICs against
isolates may be a convenient alternative to visual endpoint readings.
Background
Azole resistance screening in Aspergillus fumigatus isolates can be routinely carried out by using azole‐containing plates (E.Def 10.2 method), that requires filtering conidial suspensions ...prior inoculum adjustment.
Objectives
We evaluated whether skipping the filtration step of conidial suspensions negatively influences the performance of the E.Def 10.2.
Patients/Methods
A. fumigatus sensu stricto isolates (n = 92), classified as azole‐susceptible or azole‐resistant according to the EUCAST microdilution E.Def 9.4 method, were studied. Azole‐resistant isolates had either wild type cyp51A gene sequence (n = 3) or the TR34‐L98H (n = 26), G54R (n = 5), TR46‐Y121F‐T289A (n = 1), F46Y‐M172V‐N248T‐D255E‐E427K (n = 1), F165L (n = 1) or G448S (n = 1) cyp51A gene substitutions. In‐house azole‐containing agar plates were prepared according to the EUCAST E.Def 10.2 procedure. Conidial suspensions were obtained by adding distilled water (Tween 20 0.1%). Subsequently, the suspensions were either filtered or left unfiltered prior to inoculum adjustment to 0.5 McFarland. Using microdilution as the gold standard, agreement, sensitivity and specificity of the agar plates inoculated with two inoculums were assessed.
Results
Agreements for the agar screening method with either unfiltered or filtered conidial suspensions were high for itraconazole (100%), voriconazole (100%) and posaconazole (97.8%). Sensitivity (100%) and specificity (98.2%) of the procedure to rule in or out resistance when unfiltered suspensions were used were also high. Isolates harbouring the TR34‐L98H, G54R and TR46‐Y121F‐T289A substitutions were detected with the modified method.
Conclusions
Unfiltered conidial suspensions does not negatively influence the performance of the E.Def 10.2 method when screening for A. fumigatus sensu stricto.
Aim: The aim of the present study was to explore the significance of the mealtime experience among residents of nursing homes in Spain.
Methods: A qualitative phenomenological approach was ...followed. An initial purposeful sampling of Spanish residents in for‐profit nursing homes in the southern area of Madrid was carried out. A theoretical sampling was also implemented in order to gain a more in‐depth understanding of dependence. Inclusion criteria for nursing home residents were: age (60 years or older) and lack of any cognitive impairment. Data were collected using unstructured and semistructured interviews. Data collection was concluded once theoretical saturation was reached, and the data were analysed using the Giorgi proposal.
Results: A total of 26 residents with a mean age of 83 years were included. Three main themes that describe the significance of meals in nursing homes emerged from the data: (i) timing of the meals – mealtimes serve as a point of reference for organizing activities in the nursing home and orient the residents during the day; (ii) table allocation – table allocation depends on the judgment of the personnel, the behavior of each resident and on the input from the residents that use a table; and (iii) the meals themselves – food is experienced as a privilege, as a sign of autonomy and normality, and as an indicator of personal identity.
Conclusion: Understanding the social significance of meals for residents in nursing homes would provide deeper insight into resident expectations. This will in turn help to improve service and quality of life for residents. Geriatr Gerontol Int 2013; 13: 482–489.
BACKGROUNDPassive immunotherapy with convalescent plasma (CP) is a potential treatment for COVID-19. Evidence from controlled clinical trials is inconclusive.METHODSWe conducted a randomized, ...open-label, controlled clinical trial at 27 hospitals in Spain. Patients had to be admitted for COVID-19 pneumonia within 7 days from symptom onset and not on mechanical ventilation or high-flow oxygen devices. Patients were randomized 1:1 to treatment with CP in addition to standard of care (SOC) or to the control arm receiving only SOC. The primary endpoint was the proportion of patients in categories 5 (noninvasive ventilation or high-flow oxygen), 6 (invasive mechanical ventilation or extracorporeal membrane oxygenation ECMO), or 7 (death) at 14 days. Primary analysis was performed in the intention-to-treat population.RESULTSBetween April 4, 2020, and February 5, 2021, 350 patients were randomly assigned to either CP (n = 179) or SOC (n = 171). At 14 days, proportion of patients in categories 5, 6, or 7 was 11.7% in the CP group versus 16.4% in the control group (P = 0.205). The difference was greater at 28 days, with 8.4% of patients in categories 5-7 in the CP group versus 17.0% in the control group (P = 0.021). The difference in overall survival did not reach statistical significance (HR 0.46, 95% CI 0.19-1.14, log-rank P = 0.087).CONCLUSIONCP showed a significant benefit in preventing progression to noninvasive ventilation or high-flow oxygen, invasive mechanical ventilation or ECMO, or death at 28 days. The effect on the predefined primary endpoint at 14 days and the effect on overall survival were not statistically significant.TRIAL REGISTRATIONClinicaltrials.gov, NCT04345523.FUNDINGGovernment of Spain, Instituto de Salud Carlos III.
Cocaine addiction disorder is notably aggravated by concomitant cognitive and emotional pathology that impedes recovery. We studied whether a persistent cognitive/emotional dysregulation in mice ...withdrawn from cocaine holds a neurobiological correlate within the hippocampus, a limbic region with a key role in anxiety and memory but that has been scarcely investigated in cocaine addiction research. Mice were submitted to a chronic cocaine (20 mg/kg/day for 12 days) or vehicle treatment followed by 44 drug-free days. Some mice were then assessed on a battery of emotional (elevated plus-maze, light/dark box, open field, forced swimming) and cognitive (object and place recognition memory, cocaine-induced conditioned place preference, continuous spontaneous alternation) behavioral tests, while other mice remained in their home cage. Relevant hippocampal features basal c-Fos activity, GABA
, parvalbumin (PV)
and neuropeptide Y (NPY)
interneurons and adult neurogenesis (cell proliferation and immature neurons) were immunohistochemically assessed 73 days after the chronic cocaine or vehicle protocol. The cocaine-withdrawn mice showed no remarkable exploratory or emotional alterations but were consistently impaired in all the cognitive tasks. All the cocaine-withdrawn groups, independent of whether they were submitted to behavioral assessment or not, showed enhanced basal c-Fos expression and an increased number of GABA
cells in the dentate gyrus. Moreover, the cocaine-withdrawn mice previously submitted to behavioral training displayed a blunted experience-dependent regulation of PV
and NPY
neurons in the dentate gyrus, and neurogenesis in the hippocampus. Results highlight the importance of hippocampal neuroplasticity for the ingrained cognitive deficits present during chronic cocaine withdrawal.
Mitochondrial energetic deficit is one of the hallmarks of neurodegenerative disorders, e.g. Alzheimer´s disease (AD). Adherence to a Mediterranean diet is associated with lower incidence of ...cognitive decline and AD and extra virgin olive oil's (poly)phenols such as oleuropein and hydroxytyrosol (HT) are being actively studied in this respect. In this study, we assessed the effects of HT on mitochondrial energetic dysfunction in the 7PA2 cells cellular model, i.e. one of the best cellular models of Aβ toxicity with a well-characterized mitochondrial dysfunction typically observed in AD. We report an increase of new mitochondria at 8 h post HT-treatment, which was followed by higher mitochondrial fusion. Further, ATP concentrations were significantly increased after 24 h of treatment with HT as compared with controls. Our data suggest that HT may revert the energetic deficit of a cellular model of AD by potentiating mitochondrial activity. Because HT is being proposed as dietary supplement or component of functional foods, future studies in appropriate animal models and - eventually - humans are warranted to further investigate its potential neuroprotective actions in AD.
Efficiency of expanded genomic profiling (EGP) programmes in terms of final inclusion of patients in genomically matched therapies is still unknown. Fit patients with advanced and refractory ...colorectal cancer (CRC) were selected for an EGP programme. Next-generation sequencing (NGS) analysis from formalin-fixed paraffin-embedded tumour samples was performed. The purpose was to describe the prevalence of genomic alterations defined by the ESMO Scale for Clinical Actionability of Molecular Targets (ESCAT), as well as the percentage of patients finally included in genomically guided clinical trials. In total, 187 patients were recruited. Mutational profile was obtained in 177 patients (10 patients were failure due to insufficient tumour sample), copy number alterations in 41 patients and fusions in 31 patients. ESCAT-defined alterations were detected in 28.8% of the intention-to-analyse population. BRAF V600E was clustered in ESCAT I, with a prevalence of 3.7%, KRAS G12C and ERBB2 amplification were clustered in ESCAT II, whose prevalence was 4.2% and 1.6%, respectively. Most alterations were classified in ESCAT III (mutations in ERBB2, PIK3CA or FGFR genes and MET amplification) and IV (mutations in BRAF non-V600E, ERBB3, FBXW7, NOTCH, RNF43), with a single prevalence under 5%, except for PIK3CA mutation (9%). The final rate of inclusion into genomically guided clinical trials was 2.7%, including therapies targeting BRAF V600E or RNF43 mutations in two patients each, and ERBB2 mutation in one patient. In conclusion, EGP programmes in patients with advanced CRC are feasible and identify a subset of patients with potentially druggable genomic alterations. However, further efforts must be made to increase the rate of patients treated with genomically guided therapies.
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