Fluconazole-resistant Candida parapsilosis genotypes from hospitals located in five Spanish cities and one in Italy: Description of azole-resistance profiles associated with the Y132F ERG11p substitution.

BACKGROUND: Fluconazole-resistant Candida parapsilosis is a matter of concern. OBJECTIVES: To describe fluconazole-resistant C. parapsilosis genotypes circulating across hospitals in Spain and Rome and to study their azole-resistance profile associated with ERG11p substitutions. PATIENTS/METHODS: We selected fluconazole-resistant C. parapsilosis isolates (n = 528 from 2019 to 2023; MIC ≥8 mg/L according to EUCAST) from patients admitted to 13 hospitals located in five Spanish cities and Rome. Additionally, we tested voriconazole, posaconazole, isavuconazole, amphotericin B, micafungin, anidulafungin and ibrexafungerp susceptibility. RESULTS: Of the 53 genotypes found, 49 harboured the Y132F substitution, five of which were dominating city-specific genotypes involving almost half the isolates. Another genotype involved isolates harbouring the G458S substitution. Finally, we found two genotypes with the wild-type ERG11 gene sequence and one with the R398I substitution. All isolates were fully susceptible/wild-type to amphotericin B, anidulafungin, micafungin and ibrexafungerp. The azole-resistance patterns found were: voriconazole-resistant (74.1%) or voriconazole-intermediate (25.2%), posaconazole-resistant (10%) and isavuconazole non-wild-type (47.5%). Fluconazole-resistant and voriconazole non-wild-type isolates were likely to harbour substitution Y132F if posaconazole was wild type; however, if posaconazole was non-wild type, substitution G458S was indicated if isavuconazole MIC was >0.125 mg/L or substitution Y132F if isavuconazole MIC was ≤0.125 mg/L. CONCLUSIONS: We detected a recent clonal spread of fluconazole-resistant C. parapsilosis across some cities in Spain, mostly driven by dominating city-specific genotypes, which involved a large number of isolates harbouring the Y132F ERG11p substitution. Isolates harbouring substitution Y132F can be suspected because they are non-susceptible to voriconazole and rarely posaconazole-resistant.

Evidence of Fluconazole-Resistant Candida parapsilosis Genotypes Spreading across Hospitals Located in Madrid, Spain and Harboring the Y132F ERG11p Substitution.

We have been monitoring the antifungal resistance in Candida parapsilosis isolates collected from inpatients at Madrid metropolitan area hospitals for the last 3 years. The study aimed to elucidate the presence of fluconazole-resistant C. parapsilosis genotypes in Madrid. From January 2019 to December 2021, a total of 354 C. parapsilosis isolates (n = 346 patients) from blood (76.6%) or intraabdominal samples were collected and genotyped using species-specific microsatellite markers. Antifungal susceptibilities to amphotericin B, the triazoles, micafungin, anidulafungin, and ibrexafungerp were performed according to EUCAST E.Def 7.3.2; the ERG11 gene was sequenced in fluconazole-resistant isolates. A total of 13.6% (n = 48/354) isolates (one per patient) were found to be resistant to fluconazole and non-wild-type to voriconazole but fully susceptible to ibrexafungerp. Resistant isolates were mostly sourced from blood (n = 45/48, 93.8%) and were detected in five hospitals. Two hospitals accounted for a high proportion of resistant isolates (n = 41/48). Resistant isolates harbored either the Y132F ERG11p amino acid substitution (n = 43) or the G458S substitution (n = 5). Isolates harboring the Y132F substitution clustered into a clonal complex involving three genotypes (one genotype accounted for n = 39/43 isolates) that were found in four hospitals. Isolates harboring the G458S substitution clustered into another genotype found in a fifth hospital. C. parapsilosis genotypes demonstrating resistance to fluconazole have been spreading across hospitals in Madrid, Spain. Over the last 3 years, the frequency of isolation of such isolates and the number of hospitals affected is on the rise.

Neisseria gonorrhoeae antimicrobial resistance in Spain: a prospective multicentre study.

OBJECTIVES: Gonococcal infection is one of the most reported sexually transmitted infections and antimicrobial resistance in Neisseria gonorrhoeae (NG) is challenging for the treatment of this infection. This observational study aimed to describe antimicrobial resistance of NG and epidemiological data from patients with gonococcal infection in eight regions of Spain, for updating the local therapeutic guidelines. METHODS: MICs of penicillin, cefixime, ceftriaxone, azithromycin, ciprofloxacin, fosfomycin and gentamicin were determined by Etest for all NG isolates recovered from 1 April 2018 to 30 September 2019 from 10 hospitals in Spain. Resistance determinants were identified using logistic regression analysis. Differences with a P value <0.05 were considered statistically significant. RESULTS: Antimicrobial susceptibility testing was performed for 2571 gonococci isolated from 2429 patients. 44.5% (945/2124) of patients were MSM. The resistance rate to extended-spectrum cephalosporins was low, with 0.2% (6/2561) of isolates resistant to ceftriaxone and 1.7% (44/2517) of isolates resistant to cefixime. The overall azithromycin resistance rate was 12.1% (310/2560), but differed greatly depending on the area. 56.2% (1366/2429) of the strains studied were ciprofloxacin resistant. MIC50 and MIC90 values of gentamicin and fosfomycin were 4 and 8 mg/L and 24 and 48 mg/L, respectively. CONCLUSIONS: Our study shows that NG susceptibility to extended-spectrum cephalosporins remains high in Spain. The azithromycin resistance rate questions the suitability of dual therapy. This study provides data of interest for updating the national treatment guidelines and highlights the need to develop and implement a national sentinel gonococcal antimicrobial susceptibility programme.

Fluconazole-resistant Candida parapsilosis: fast detection of the Y132F ERG11p substitution, and a proposed microsatellite genotyping scheme.

OBJECTIVES: We propose fast and accurate molecular detection of the Y132F ERG11p substitution directly on pure-cultured Candida parapsilosis isolates. We also assessed a discriminative genotyping scheme to track circulating genotypes. METHODS: A total of 223 C. parapsilosis isolates (one patient each) from 20 hospitals, located in Spain and Italy were selected. Isolates were fluconazole-resistant (n = 94; harbouring the Y132F ERG11p substitution [n = 85], the G458S substitution [n = 6], the R398I substitution [n = 2], or the wild-type ERG11 gene sequence) or fluconazole-susceptible (n = 129). Two targeted-A395T-mutation PCR formats (conventional and real-time) were engineered and optimized on fluconazole-susceptible and fluconazole-resistant pure-cultured isolates, thus skipping DNA extraction. Two genotyping schemes were compared: Scheme 1 (CP1, CP4a, CP6, and B markers), and Scheme 2 (6A, 6B, 6C, CP1, CP4a, and CP6 markers). RESULTS: The screening performed using both PCR formats showed 100% specificity (fluconazole-susceptible isolates; n = 129/129) and sensitivity (Y132F isolates; n = 85/85) values; however, results were available in 3 and 1.5 hours with the conventional and real-time PCR formats, respectively. Overall, Scheme 1 showed higher genetic diversity than Scheme 2, as shown by the number of alleles detected (n = 98; mean 23, range 13-38), the significantly higher observed and expected heterozygosity, and the probability of identity index (2.5 × 10-6). Scheme 2 markers did not provide further genotypic discrimination of Y132F fluconazole-resistant genotypes. CONCLUSION: Both proposed PCR formats allow us to speed up the accurate detection of substitution Y132F ERG11p in C. parapsilosis isolates with 100% specificity and sensitivity. In addition, we recommend CP1, CP4a, CP6, and B microsatellite markers for genotyping fluconazole-resistant isolates.

Fluconazole-resistant Candida parapsilosis clonally related genotypes: first report proving the presence of endemic isolates harbouring the Y132F ERG11 gene substitution in Spain.

OBJECTIVES: We report here for the first time the presence of Candida parapsilosis isolates harbouring the Y132F ERG11 gene substitution in patients admitted to a Spanish hospital. METHODS: We studied the available (n = 104) C parapsilosis isolates from patients admitted to the Son Espases reference hospital in the Balearic Islands from 1 April 2019 to 30 November 2020. Isolates were sourced from 70 patients: catheter (n = 41), blood cultures (n = 37), lower respiratory tract (n = 15), intra-abdominal (n = 8), and other samples (n = 3). Isolates were genotyped and tested for antifungal susceptibilities to amphotericin B, triazoles, anidulafungin, and micafungin using EUCAST E.Def 7.3.2. The ERG11 gene was sequenced in fluconazole-resistant isolates. RESULTS: Among the 104 isolates, fluconazole and voriconazole resistance was found in 87 (84%) and 30 (29%) isolates, respectively; all isolates were fully susceptible to echinocandins and amphotericin B. All fluconazole-resistant isolates harboured the Y132F substitution in the ERG11 gene and were grouped into 11 clonally related genotypes. A genotype accounted for 70% (61/87) of fluconazole-resistant isolates. Genotypes involving the fluconazole-resistant isolates were different from those found in the remaining fluconazole-susceptible genotypes. Fifty-six patients harboured fluconazole-resistant genotypes, and 35 of the 56 had candidaemia (48%), abdominal candidiasis (17%), or other forms of candidiasis (35%). Only 20% of the study patients infected by fluconazole-resistant genotypes had a history of azole use. DISCUSSION: Fluconazole resistance in C parapsilosis isolates from patients admitted to this reference hospital is not attributable to prior azole use, but rather to the presence of a group of fluconazole-resistant C parapsilosis genotypes that have become endemic.

Global Emergence of Resistance to Fluconazole and Voriconazole in Candida parapsilosis in Tertiary Hospitals in Spain During the COVID-19 Pandemic.

Background: Candida parapsilosis is a frequent cause of candidemia worldwide. Its incidence is associated with the use of medical implants, such as central venous catheters or parenteral nutrition. This species has reduced susceptibility to echinocandins, and it is susceptible to polyenes and azoles. Multiple outbreaks caused by fluconazole-nonsusceptible strains have been reported recently. A similar trend has been observed among the C. parapsilosis isolates received in the last 2 years at the Spanish Mycology Reference Laboratory. Methods: Yeast were identified by molecular biology, and antifungal susceptibility testing was performed using the European Committee on Antimicrobial Susceptibility Testing protocol. The ERG11 gene was sequenced to identify resistance mechanisms, and strain typing was carried out by microsatellite analysis. Results: We examined the susceptibility profile of 1315 C. parapsilosis isolates available at our reference laboratory between 2000 and 2021, noticing an increase in the number of isolates with acquired resistance to fluconazole, and voriconazole has increased in at least 8 different Spanish hospitals in 2020-2021. From 121 recorded clones, 3 were identified as the most prevalent in Spain (clone 10 in Catalonia and clone 96 in Castilla-Leon and Madrid, whereas clone 67 was found in 2 geographically unrelated regions, Cantabria and the Balearic Islands). Conclusions: Our data suggest that concurrently with the coronavirus disease 2019 pandemic, a selection of fluconazole-resistant C. parapsilosis isolates has occurred in Spain, and the expansion of specific clones has been noted across centers. Further research is needed to determine the factors that underlie the successful expansion of these clones and their potential genetic relatedness.

A Case of Aeromonas trota in an Immunocompromised Patient with Diarrhea.

According to recent literature, 95.4% of the Aeromonas strains associated with human clinical cases correspond to four species: Aeromonas caviae, Aeromonas dhakensis, Aeromonas veronii and Aeromonas hydrophila. However, other less prevalent species such as Aeromonas trota, are also described from clinical samples. Based on its low incidence, the latter species can be regarded as rare and it is the only Aeromonas species susceptible to ampicillin. From the taxonomic point of view, A. trota is considered a synonym of the species Aeromonas enteropelogenes. The objective of this study is to present a new clinical case associated with A. trota in order to increase the knowledge about this species. The strain was recovered from the feces of a 69-year-old patient with a diarrheal syndrome and peritoneal psammocarcinoma. The preliminary identification as Aeromonas sp. was obtained with the API 20E, but it was characterized as Aeromonas jandei and also as Aeromonas enteropelogenes with different scores with the matrix-assisted laser desorption ionization time of flight (MALDI-TOF). Based on the sequence of the rpoD gene, it was confirmed to be A. trota. The antimicrobial resistance pattern showed that the strain was susceptible to ampicillin, penicillins in combination with beta-lactamase inhibitors, quinolones, carbapenems, aminoglycosides and cephalosporins, except cephalothin. In conclusion, the recognition of an Aeromonas strain susceptible to ampicillin should alert the clinical microbiologist of the possible involvement of this rare species. Furthermore, the MALDI-TOF database should be updated indicating that the species A. enteropelogenes, is a synonym of A. trota.

Comparison of different cell lines and incubation times in the isolation by the shell vial culture of human metapneumovirus from pediatric respiratory samples.

We report a prospective study concerning the efficacy of LLC-MK2 (continuous monkey kidney cell), Hep-2, MDCK (Madin-Darby Canine Kidney), Vero and MRC-5 cell lines, by shell vial assay, and incubation time in the isolation of hMPV from pediatric respiratory samples. The overall sensitivity of the cell lines studied were: 100% for the LLC-MK2, 68.7% for the Hep-2, 28.1% for the Vero, 3.1% for the MDCK and 0% for the MRC-5. Only one strain (3.1%) showed growth in the four cell lines studied and 10 (31.2%) strains only grew in the LLC-MK2 cell line. The analysis of incubation times showed that only 14 strains (43.7%) were able to grow after 3 days of incubation, while all strains (100%) showed growth after 5 days. The use of shell vials with commercial LLC-MK2 cells could be a method for isolating hMPV from respiratory samples in the pediatric population.

[Clinical and epidemiological characteristics of respiratory infections caused by human metapneumovirus in pediatric patients]. / Características clínicas y epidemiológicas de las infecciones respiratorias causadas por el metapneumovirus humano en pacientes pediátricos.

INTRODUCTION: Human Metapneumovirus (hMPV) was first identified in 2001 in respiratory samples from children and adults with acute respiratory tract infection. The aim of this prospective study was to determine the clinical and epidemiological characteristics of pediatric patients with an acute respiratory tract infection and exclusive isolation of hMPV in respiratory samples (December 2005-January 2007). MATERIAL AND METHODS: All respiratory tract samples were submitted to rapid antigen detection against respiratory syncytial virus (RSV) and influenza A and B viruses. To isolate respiratory viruses, samples were inoculated in various cell lines using the shell vial culture assay. To isolate hMPV, the LLC-MK2 cell line was used. Only antigen-negative samples were studied for the presence of hMPV. RESULTS: Over the study period, 32 hMPV were isolated from different patients, accounting for 1.7% of all samples studied for this virus (only RSV-negative samples, 1,791) and 1.5% of all samples studied. Peak incidence was found between December and March of the two years studied. Of the 32 patients in whom hMPV was detected, 17 (53.2%) were female and 15 (46.8%) male. Mean age was 12.5 months (range, 1 month-4 years). The most frequent clinical symptoms were fever (90.6%), cough (87.5%), rhinorrhea and bronchiolitis (46.8%). Three patients (9.4%) were hospitalized. CONCLUSIONS: Respiratory infection caused by hMPV is considered an emergent disease in pediatric patients. The clinical manifestations are very similar to those of RSV infection; hence, only virological study can establish the definite etiological diagnosis.