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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 ¼ 828) and cryptic species (n ¼ 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 TR 34 -L98H substitutions (n ¼ 24 out of 63). Out of the 725 patients, 48 (6.6%) carried either cryptic species (n ¼ 14) or A. fumigatus sensu stricto (n ¼ 34; 4.7%) resistant isolates. Aspergillus fumigatus sensu stricto harbouring either the TR 34 -L98H (n ¼ 19) or TR 46 /Y121F/T289A (n ¼ 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. TR 34 -L98H is the dominant cyp51A gene substitutions, although its presence is not widespread.

A voriconazole-resistant isolate of Aspergillus fumigatus was recovered from an immunocompetent patient receiving long-term antifungal therapy for cerebral aspergillosis. A G448S amino acid substitution in the azole target (Cyp51A) was identified as the cause of the resistance phenotype. This article describes the first isolation of a voriconazole-resistant A. fumigatus isolate from an immunocompetent patient in Spain. CASE REPORT

Laboratory cross-contamination by Mycobacterium tuberculosis is known to be responsible for the misdiagnosis of tuberculosis, but its impact on other contexts has not been analyzed. We present the findings of a molecular epidemiology analysis in which the recent transmission events identified by a genotyping reference center were overestimated as a result of unnoticed laboratory cross-contamination in the original diagnostic laboratories.The phenomenon of misdiagnosing tuberculosis by laboratory cross-contamination when Mycobacterium tuberculosis is cultured has been widely reported (3, 4-8, 10, 11). The production of aerosolized particles after the processing of smearpositive specimens, cultures positive for M. tuberculosis, or positive control strains may be responsible for the inoculation of other specimens processed on the same day or of reagents used for the decontamination of specimens (5). False positivity is suspected (i) if M. tuberculosis is cultured from a sample processed together with a smear-positive specimen, (ii) if M. tuberculosis is cultured from only one of the cultures in the set (usually with a low yield of bacteria), and (iii) if the clinician is considering an alternative diagnosis, that is, a diagnosis other than tuberculosis (TB). Suspicion of false positivity is increased when two or more of these conditions are met. Finally, if molecular analysis is available, cross-contamination is confirmed when the strains cultured from both truly infected and contaminated specimens share the same genotypic pattern and no epidemiological links can be found between the cases. Several studies, some of which are based on molecular analysis, have estimated that the rate of laboratory cross-contamination for M. tuberculosis ranges from 0.1% to 3%, although massive contamination has caused up to 65% of false-positive cases (11).False-positive results for tuberculosis have been a matter of concern because of the clinical, therapeutic, and social impacts of the misdiagnosis of tuberculosis. The economic load associated with each misdiagnosed case of tuberculosis has been estimated to be $32,618 (9). However, another area on which false positivity has an impact but which has received little attention is the misidentification of recent transmission events by molecular epidemiology studies. Molecular epidemiology is based on the analysis of the genotypes of cultured M. tuberculosis isolates to identify cases infected by the same M. tuberculosis strain. These cases are defined as clustered and are considered to be caused by recent transmission events and to belong to the same transmission chain. If an analysis to determine the existence of potential false-positive cases is not performed before molecular analysis, as a quality control of microbiological procedures, there is a risk of misassigning clustered cases. This refined preanalysis is not usually performed because molecular epidemiology studies are generally run by laboratories which are different from those which culture M. tuberculosis from clinical spec...

Background Carbapenem-resistant Gram-negative bacilli (CR-GNB) are among the most threatening microorganisms worldwide and carbapenem use facilitates their spread. Antimicrobial stewardship programmes (ASPs) can help to optimize the use of antibiotics. This study evaluates the impact of a multifaceted educational ASP on carbapenem use and on the epidemiology of CR-GNB. Methods We conducted a quasi-experimental, time-series study in seven hospitals, from January 2014 to September 2018. The key intervention was composed of educational interviews promoting the appropriate use of carbapenems. The primary endpoints were carbapenem consumption and incidence density (ID) of CR-GNB. All non-duplicated CR-GNB clinical isolates were tested using phenotypic assays and PCR for the presence of carbapenemases. Joinpoint regression and interrupted time-series analyses were used to determine trends. Results A decrease in carbapenem consumption throughout the study period [average quarterly percentage change (AQPC) −1.5%, P < 0.001] and a −8.170 (−16.064 to −0.277) level change following the intervention were observed. The ID of CR-Acinetobacter baumannii decreased (AQPC −3.5%, P = 0.02) and the overall ID of CR-GNB remained stable (AQPC −0.4%, P = 0.52). CR-GNB, CR-Pseudomonas aeruginosa and CR-A. baumannii IDs per hospital correlated with the local consumption of carbapenems. The most prevalent carbapenem resistance mechanisms were OXA-23 for CR-A. baumannii (76.1%), OXA-48 for CR-Klebsiella pneumoniae (66%) and no carbapenemases for CR-P. aeruginosa (91.7%). The epidemiology of carbapenemases was heterogeneous throughout the study, especially for carbapenemase-producing Enterobacteriaceae. Conclusions In conclusion, a multifaceted, educational interview-based ASP targeting carbapenem prescribing reduced carbapenem use and the ID of CR-A. baumannii.

The EUCAST 9.3.2 procedure recommends visual readings of azole and amphotericin B MICs against Aspergillus spp. Visual determination of MICs may be challenging. In this work, we aim to obtain and compare visual and spectrophotometric MICs readings of azoles and amphotericin B against A. fumigatus sensu lato isolates. Eight hundred and forty-seven A. fumigatus sensu lato isolates (A. fumigatus sensu stricto [n=828] and cryptic species [n=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 >95% compared to control; read at 540 nm) MICs were compared. Essential (±1 twofold dilutions) 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 misclassifications for voriconazole and isavuconazole were found to be associated with MIC results falling either in the area of technical uncertainty or in one two-fold dilutions above the breakpoint. Resistance rate was slightly lower when the MICs were obtained by spectrophotometric readings. However, all relevant cyp51A mutants were correctly classified as resistant. Spectrophotometric determination of azole and amphotericin B MICs against A. fumigatus sensu lato isolates may be a convenient alternative to visual endpoint readings.

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Respiratory syncytial virus (RSV) is the viral agent which is more frequently involved in lower respiratory tract infections (LRTIs) in infants under 1 year of age in developed countries. A new oligochromatographic assay, Speed-Oligo® RSV, was designed and optimized for the specific detection and identification of RSV subtypes A and B. The test was evaluated in 289 clinical samples from 169 hospitalized children using an immunochromatography (IC) test, virus isolation by culture, and an in-house real-time polymerase chain reaction (RT-PCR). Other viruses causing LRTIs were investigated by cell culture or PCR-based tests. Sixty-two patients were infected by RSV (36.7%). In addition, adenovirus, influenza B, parainfluenza 2, and human metapneumovirus were detected in rates ranging from 5 to 8%. A proportion of 10.1% of the patients had mixed infections. The sensitivity, specificity, and positive and negative predictive values were, respectively, 94.9, 99.4, 98.9, and 97.4% for Speed-Oligo® RSV, 92.9, 96.3, 92.9, and 96.3% for RT-PCR/RSV, and 58.4, 98.1, 93.3, and 82.6% for IC. Our rates of viral detection and co-infection were similar to those of previously reported series. Finally, we find that Speed-Oligo® RSV is a rapid and easy-to-perform technique for the detection of RSV and the identification of subtypes A and B.