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.
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.
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.
This study proposes an algorithm for microbiological diagnosis of urinary tract infections based on screening by luminometry and Gram-stain, followed by identification by matrix-assisted laser ...desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Positive urine samples detected with the luminometry screening Coral UTI Screen
TM
system underwent Gram staining and identification of the causative organism was performed by MALDI-TOF Microflex LT mass spectrometer (Bruker Daltonics, Germany). Subsequently, the results were compared with those of conventional culture identification using WIDER MIC/id system (Francisco Soria Melguizo SA, Spain). Considering the conventional approach as the gold standard, the proposed algorithm presented both a high specificity (98.1%) and a positive likelihood ratio of 37.42. The implementation of this algorithm would allow diagnosis of urinary tract infection in less than an hour in 92.4% of positive samples. This combination of techniques would be useful particularly for patients with severe UTI, pyelonephritis or urinary sepsis.
This study proposes an algorithm for microbiological diagnosis of urinary tract infections based on screening by luminometry and Gram-stain, followed by identification by matrix-assisted laser ...desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Positive urine samples detected with the luminometry screening Coral UTI Screen
system underwent Gram staining and identification of the causative organism was performed by MALDI-TOF Microflex LT mass spectrometer (Bruker Daltonics, Germany). Subsequently, the results were compared with those of conventional culture identification using WIDER MIC/id system (Francisco Soria Melguizo SA, Spain). Considering the conventional approach as the gold standard, the proposed algorithm presented both a high specificity (98.1%) and a positive likelihood ratio of 37.42. The implementation of this algorithm would allow diagnosis of urinary tract infection in less than an hour in 92.4% of positive samples. This combination of techniques would be useful particularly for patients with severe UTI, pyelonephritis or urinary sepsis.
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.
To include a specific antibiotic in the empiric therapy, it is necessary to predict when a nosocomial pneumonia (NP) is caused by methicillin-resistant Staphylococcus aureus (MRSA). We have developed ...a model for the prediction of the probability of a NP being caused by MRSA, when the carrier status and the microbiological diagnosis are unknown.
A retrospective case-control study (1999-2005) was designed. A univariate and multivariate logistic regression was performed to identify the risk factors for suffering a NP due to MRSA. Demographic factors, related to hospitalization, immunosuppression or neutropenia, to medication and severity were included.
Three hundred and sixty three patients (121 cases and 242 controls) were studied. The final model of multivariate logistic regression included an age>14 years (OR 7.4, CI 95% 1.5-37.4, P<.015), NP appearance>6 days after admittance (OR 4.1, CI 95% 2.4-7,1, P<.001), NP development excluding summers (OR 2.5, CI 95% 1.2-5.2, P<.015), respiratory diseases (OR 4.9, CI 95% 1.5-15.8, P<.007) and multilobar involvement (OR 4, CI 95% 2.3-7.2, P<.001).The probability of developing a pneumonia due to MRSA was studied for each of the possible combinations and subsequently classified in minor and major criteria.
MRSA coverage should be included in the empirical treatment of NP when: a) an adult patient (>14 years old) presents, at least, 2 major criteria or 1 major criterion together with 2 minor criteria, and b) a patient <14 years-old has 2 major criteria as well as 2 minor criteria.
The objective of this study is to characterize Streptococcus pyogenes isolates with a mucoid phenotype and to compare them with non-mucoid isolates obtained between April and August 2016.
...Identification and antimicrobial susceptibility were performed in all isolates. The emm type and exotoxin genes speA, speB, speC, speF, speG, speH, speJ, speZ and ssa were analyzed. Clinical and demographic data were collected.
From 96 isolates analyzed, 47% had a mucoid phenotype and 95.5% of them presented speA-speB-speF-speG-ssa genes and emm3 genotype. The main clinical manifestation was pharyngotonsillitis (77.1%) evolving to scarlet fever in 67.5% of the cases.
This study describes the circulation of a mucoid phenotype strain with a speA-speB-speF-speG-ssa toxin profile and emm3.1 genotype considered one of the most frequent and virulent of SGA.
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