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 C. 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 (2.5x10-6). Scheme 2 markers did not provide further genotypic discrimination of Y132F fluconazole-resistant genotypes. CONCLUSION: Both proposed PCR formats allow 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.

Pneumonia in Children With Complex Chronic Conditions With Tracheostomy: An Emerging Challenge.

BACKGROUND: Despite respiratory infections being a leading cause of hospitalization in children with tracheostomy tubes, there are no published guidelines for their diagnosis and management. This study aims to outline the clinical, laboratory and microbiological aspects of pneumonia in these children, along with the antibiotics used and outcomes. Additionally, it seeks to determine pneumonia incidence and associated risk factors. METHODS: We conducted a retrospective study using the medical records of tracheostomized children at La Paz University Hospital in Madrid from 2010 to 2021. RESULTS: Thirty-three pneumonia cases were observed in 25 tracheostomized children. Pseudomonas aeruginosa was the predominant bacterium (52%), followed by Escherichia coli, Staphylococcus aureus and Serratia marcescens. The same microorganism isolated in the tracheal aspirate culture during pneumonia was previously isolated in 83% of cases that had a similar culture, with some growth obtained within 7-30 days prior. Multiplex respiratory PCR detected respiratory viruses in 73% of cases tested. Antibiotic treatment was administered in all cases except 1, mostly intravenously (81%), with piperacillin/tazobactam and meropenem being commonly used. Only 1 of the described episodes had a fatal outcome. CONCLUSIONS: It is advisable to include coverage for P. aeruginosa, E. coli, S. aureus, and S. marcescens in the empirical antibiotic treatment for pneumonia in tracheostomized children, along with the microorganisms identified in tracheal cultures obtained within 7-30 days prior, if available. A positive PCR for respiratory viruses is often discovered in bacterial pneumonia in tracheostomized children.

Increase in gonococcal arthritis in Madrid, Spain, 2022-2023.

In recent years, there has been an increase in Neisseria gonorrhoeae infections in Europe and Spain. Disseminated gonococcal infection is an uncommon clinical presentation that includes gonococcal arthritis. Improved antibiotic treatment has reduced the incidence of gonococcal arthritis. However, the increase in gonococcal infections may have increased the frequency of this clinical entity in recent times. We report five cases of gonococcal arthritis in patients in a tertiary-care hospital in the northern area of Madrid (Spain) from October 2022 to October 2023. Major cases occurred in male patients with unprotected sex and polyarticular symptoms requiring hospital admission and treatment with ceftriaxone and cefixime. The use of molecular techniques has allowed the detection of a greater number of culture-negative cases of gonococcal arthritis, as well as the detection of mutations associated with resistance to fluoroquinolone for switching to oral treatment.

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.

Comparison of gradient diffusion and molecular methods using Allplex™ NG&DR assay (Seegene®) for macrolide and fluoroquinolone screening resistance in Neisseria gonorrhoeae.

Antimicrobial resistance in Neisseria gonorrhoeae (NG) is increasing worldwide. Second-line treatments with macrolides or fluoroquinolones are an option for NG infections in some cases following the STI guideline recommendations. In our study, we compared the gradient diffusion test using EUCAST 2024 breakpoints with a new molecular method using the Allplex™ NG&DR assay (Seegene®) including A2059G/C2611 mutations (23S rRNA) associated with high/moderate-level macrolide resistance and S91F mutation (gyrA) relationship with fluoroquinolone resistance in NG isolates (n = 100). We calculated the sensitivity, specificity, and correlation of the molecular test for fluoroquinolone using the gradient diffusion as the reference method. In twenty-three strains was not detected any mutation associated with macrolides or fluoroquinolone resistance. No A2059G/C2611T mutations were detected, and the S91F mutations were detected in 77 out of the 100 isolates screened. Twenty-three NG isolates were reported to be resistant to azithromycin (ECOFF: >1 mg/L), and 78 NG isolates were resistant to ciprofloxacin (MIC: >0.06 mg/L). The molecular method showed a sensitivity of 96.1% and, a specificity of 90.9% for fluoroquinolone susceptibility, but the statistical analysis between the molecular test and gradient diffusion test was not statistically significant for fluoroquinolone resistance (p = 1). Statistical analysis was not performed for macrolides because of the absence of positive RT-PCR results. According to our data, Allplex™ assay cannot replace the gradient diffusion test for macrolide resistance. However, the assay could be used to test fluoroquinolone resistance in NG isolates as a replacement for phenotypic methods.

Single case of lymphogranuloma venereum on the tongue.

Traditionally, lymphogranuloma venereum (LGV) has been associated with disease of the genital area. However, atypical presentations and proctitis are increasingly observed. We report a case of LGV affecting the dorsum of the tongue, which presented as a very painful ulcer. The response to doxycycline (100 mg two times per day for 21 days) was satisfactory. This case may represent a paradigm shift in the differential diagnosis of lingual ulcers.

Trends in antifungal resistance in Candida from a multicenter study conducted in Madrid (CANDIMAD study): fluconazole-resistant C. parapsilosis spreading has gained traction in 2022.

We previously conducted a multicenter surveillance study on Candida epidemiology and antifungal resistance in Madrid (CANDIMAD study; 2019-2021), detecting an increase in fluconazole-resistant Candida parapsilosis. We here present data on isolates collected in 2022. Furthermore, we report the epidemiology and antifungal resistance trends during the entire period, including an analysis per ward of admission. Candida spp. incident isolates from blood cultures and intra-abdominal samples from patients cared for at 16 hospitals in Madrid, Spain, were tested with the EUCAST E.Def 7.3.2 method against amphotericin B, azoles, micafungin, anidulafungin, and ibrexafungerp and were molecularly characterized. In 2022, we collected 766 Candida sp. isolates (686 patients; blood cultures, 48.8%). Candida albicans was the most common species found, and Candida auris was undetected. No resistance to amphotericin B was found. Overall, resistance to echinocandins was low (0.7%), whereas fluconazole resistance was 12.0%, being higher in blood cultures (16.0%) mainly due to fluconazole-resistant C. parapsilosis clones harboring the Y132F-R398I ERG11p substitutions. Ibrexafungerp showed in vitro activity against the isolates tested. Whereas C. albicans was the dominant species in most hospital wards, we observed increasing C. parapsilosis proportions in blood. During the entire period, echinocandin resistance rates remained steadily low, while fluconazole resistance increased in blood from 6.8% (2019) to 16% (2022), mainly due to fluconazole-resistant C. parapsilosis (2.6% in 2019 to 36.6% in 2022). Up to 7 out of 16 hospitals were affected by fluconazole-resistant C. parapsilosis. In conclusion, rampant clonal spreading of C. parapsilosis fluconazole-resistant genotypes is taking place in Madrid.

Isolation of filamentous basidiomycetes from respiratory samples in a tertiary care Spanish hospital / Aislamiento de basidiomicetos filamentosos en muestras respiratorias en un hospital español de tercer nivel

Background The clinical significance of the filamentous basidiomycetes isolated from clinical samples is not always clear. Thus, these fungi have been considered environmental contaminants traditionally. Aims To review those clinical cases in which filamentous basidiomycetes from respiratory samples had been isolated. Methods The retrospective study was carried out in a single tertiary care hospital. We recovered all culture-confirmed isolations of filamentous basidiomycetes from respiratory samples (bronchial aspirate [BAS], bronchoalveolar lavage [BAL] and sputum) analyzed between the years 2020 and 2023. Isolates were identified by ITS region sequencing. Results In six patients a filamentous basidiomycete had been isolated from a respiratory sample. The species identified were all different: Fomitopsis sp. (BAS), Trametes ljubarskyi (BAL), Stereum gausapatum (BAS), Porostereum spadiaceum (BAS), Phlebia subserialis (sputum) and Inonotus levis (BAL). All the patients were immunosuppressed or had an underlying disease with pulmonary involvement. None of them received any specific antifungal treatment (in relation with the fungus isolated) and all six improved clinically and were discharged. Conclusions The isolation of filamentous basidiomycetes in these patients had uncertain clinical significance. However, the isolation of any filamentous basidiomycete in respiratory samples from immunosuppressed patients or patients with chronic pulmonary disease is an emerging situation that should be carefully assessed in the context of chronic allergic episodes or suspicion of invasive fungal infections. (AU)

Antecedentes La importancia clínica de los basidiomicetos filamentosos procedentes de muestras clínicas no siempre es clara. Por ello, estos hongos se han considerado tradicionalmente como contaminantes ambientales. Objetivos Describir las características de los pacientes con aislamientos de basidiomicetos filamentosos en muestras respiratorias para intentar aclarar el significado clínico de estos hallazgos. Métodos Se realizó un estudio retrospectivo en nuestro hospital de tercer nivel; fueron incluidos pacientes con el aislamiento de un basidiomiceto filamentoso confirmado en cultivo a partir de una muestra respiratoria (aspirados bronquiales [BAS], lavados broncoalveolares [BAL] y esputos) procesadas entre los años 2020 y 2023. Realizamos la identificación molecular del aislamiento mediante la secuenciación de la región ITS. Resultados En seis pacientes se obtuvo un aislamiento de basidiomiceto filamentoso de una muestra respiratoria, y los seis aislamientos pertenecían a diferentes géneros: Fomitopsis sp. (BAS), Trametes ljubarskyi (BAL), Stereum gausapatum (BAS), Porostereum spadiaceum (BAS), Phlebia subserialis (esputo) e Inonotus levis (BAL). Todos los pacientes sufrían inmunosupresión o alguna enfermedad de base con afectación pulmonar. Ninguno de ellos recibió tratamiento antifúngico específico (en relación con el aislamiento recuperado de la muestra) y finalmente fueron dados de alta tras la mejoría clínica. Conclusiones Los aislamientos de basidiomicetos filamentosos en nuestros pacientes tuvieron un significado clínico incierto. Sin embargo, en los pacientes inmunodeprimidos o con enfermedades pulmonares crónicas, el aislamiento de un basidiomiceto filamentoso en el tracto respiratorio inferior representa una situación emergente que debe evaluarse cuidadosamente en el contexto de un episodio alérgico crónico o ante la sospecha de una infección fúngica invasiva. (AU)

Prevalence of mutations associated with macrolide and fluoroquinolone resistance in Neisseria gonorrhoeae with Allplex™ NG&DR Assay (Seegene®) in a tertiary hospital from Madrid, Spaina / Prevalencia de mutaciones asociadas a resistencia a macrólidos y fluoroquinolonas en Neisseria gonorrhoeae con el kit Allplex™ NG&DR Assay (Seegene®) en un hospital terciario de la Comunidad de Madrid, España

Background. The prevalence of drug-resistant Neisseria gonorrhoeae (NG) infections is increasing. Studies report the prevalence of NG strains presenting A2059G/C2611T (rRNA 23S) and S91F (parC) mutations conferring resistance to azith romycin and ciprofloxacin. Material and methods. We conducted a prospective cohort study evaluating first void-urine urines, rectal, and oropharyngeal swabs collected from a cohort of patients in a tertiary hospital in Madrid between October 2022 and January 2023. Samples were screened by Allplex™ 7-STI Essential As say (Seegene®). Drug resistances were performed by Allplex™ NG&DR Assay (Seegene®). Results. A total of 1,415 patients were included, of which 112 had a positive sample for NG infection. One patient had a C2611T mutation (0.9%) and neither patient showed A2059G mutation. We found 67 (59.8%) S91F-positive patients. For ty-four patients (39.3%) not had any mutations. Conclusions. We report a low-prevalence of mutations A2059G/C2611T to macrolides and a high-prevalence to S91F in NG infections. Molecular methods for the detection of NG resistance could be useful in direct non-culturable samples (AU)

Introducción. La infección por Neisseria gonorrhoeae (NG) resistente está aumentando. Se ha descrito la prevalencia de cepas de NG con mutaciones A2059G/C2611T (rRNA 23S) y S91F (parC) que confieren resistencia a azitromicina y cipro floxacino. Material y métodos. Realizamos un estudio prospecti vo evaluando orinas de primera micción, hisopos anales y fa ríngeos recogidos de una cohorte de pacientes en un hospital terciario de Madrid entre octubre de 2022 y enero de 2023. El cribado de las muestras se realizó mediante Allplex™ 7-STI Es sential Assay (Seegene®). Las resistencias a macrólidos y fluo roquinolonas se realizaron mediante Allplex™ NG&DR Assay (Seegene®). Resultados. Se incluyeron 1.415 pacientes, de los cua les 112 fueron positivos para NG. Un paciente presentaba una mutación C2611T (0,9%) y en ningún paciente se detec tó A2059G. Encontramos 67 pacientes (59,8%) positivos pa ra S91F. Cuarenta y cuatro pacientes (39,3%) no presentaban mutaciones. Conclusiones. Reportamos una baja prevalencia de mu taciones A2059G/C2611T a macrólidos y una alta prevalencia de S91F en NG. Los métodos moleculares para la detección de resistencias en NG podrían ser útiles en muestras directas no cultivables (AU)

Antifungal resistance in Candida spp within the intra-abdominal cavity: study of resistance acquisition in patients with serial isolates.

OBJECTIVES: Antifungal susceptibility testing is mostly conducted on blood-cultured Candida spp isolates. Because the intra-abdominal cavity has been highlighted as a hidden echinocandin-resistant C. glabrata reservoir, we assessed whether testing sequential isolates from a given patient might increase the chances of detecting antifungal resistance. METHODS: Intra-abdominal initial and sequential isolates from the same species from patients included in the CANDIdaemia in MADrid study (January 2019 to June 2022) were studied. We assessed antifungal susceptibility to amphotericin B, azoles, anidulafungin, micafungin, and ibrexafungerp using European Committee on Antimicrobial Susceptibility Testing (EUCAST) methodology and molecularly characterized resistant isolates. RESULTS: We collected 308 isolates (C. albicans [n = 179/308; 58.1%], C. glabrata [n = 101/308; 32.8%], C. tropicalis [n = 17/308; 5.5%], and C. parapsilosis [n = 11/308; 3.6%]) from 112 patients distributed as incident (n = 125/308) and sequential (n = 183/308). Per patient resistance rates of fluconazole (13.4% [15/112] vs. 8% [9/112]); 5.4% proportions difference (95% CI, -2.7% to 13.5%, p 0.09) and echinocandins (8.9% [10/112] vs. 1.8% [2/112]); 7.1% proportions difference (95% CI; 1.2-12.9%; p 0.01) were higher when considering all available isolates than only incident isolates. Resistance was detected in 18 of 112 patients and would have been overlooked in 11 of 18 (61.1%) patients if only incident isolates had been studied. Of the patients who harboured fluconazole or echinocandin-resistant isolates, 14 of 15 and 8 of 10 had received or were receiving fluconazole or echinocandins, respectively. DISCUSSION: Testing sequential Candida isolates from intra-abdominal samples is required to detect antifungal resistance, particularly to echinocandins, in patients whose incident isolates turned out to be susceptible. Furthermore, patients with echinocandin-resistant infections had frequently used echinocandins and had common secondary resistance acquisition.

Epidemiology and prevalence of mutations associated with resistance to macrolides and fluoroquinolones in Mycoplasma genitalium in a tertiary hospital from Madrid, Spain / Epidemiología y prevalencia de mutaciones asociadas a la resistencia a macrólidos y fluoroquinolonas en Mycoplasma genitalium en un hospital terciario de Madrid, España

Objectives: Mycoplasma genitalium causes persistent sexually transmitted infections. The aims of this study were to estimate the prevalence of resistances to macrolides and fluoroquinolones in M. genitalium and the sexually transmitted coinfections in patients at Hospital Universitario La Paz (Madrid, Spain). Material and methods: Patients attended between January and October 2021 were studied. Screening for sexually transmitted pathogens and detection of 23S rRNA and parC genes mutations were performed by real-time PCR (Allplex,SeegeneTM). Results: A total of 1,518 females and 1,136 males were studied. The prevalence of M. genitalium was 2.1%. The macrolides resistance rate was 51.8%. The mutations found were A2059G, A2058T and A2058G. The rate of resistance to fluoroquinolones was 17.8% being the G248T mutation (S83I) the most frequent. Seven males had some sexual transmitted coinfection. Conclusions: Although the percentage of M. genitalium infections is low, the high rate of resistance to macrolides makes it necessary to revise the protocols for diagnosis and empirical treatment of sexually transmitted infections. The use of fluoroquinolones is appropriate after screening of macrolide resistance profile. (AU)

Objetivos: Mycoplasma genitalium causa infecciones de transmisión sexual persistentes. Los objetivos de este trabajo fueron estimar la prevalencia de resistencias a macrólidos y fluoroquinolonas en M. genitalium así como las coinfecciones de transmisión sexual en pacientes del Hospital Universitario La Paz (Madrid, España). Material y métodos: Se estudiaron pacientes atendidos entre enero y octubre de 2021. El cribado de patógenos de transmisión sexual y la detección de mutaciones de los genes ARNr 23S y parC se realizaron por PCR en tiempo real (Allplex, SeegeneTM). Resultados: Se estudiaron 1.518 mujeres y 1.136 hombres. La prevalencia de M. genitalium fue del 2,1%. La tasa de resistencia a macrólidos fue del 51.8%. Las mutaciones encontradas fueron A2059G, A2058T y A2058G. La tasa de resistencias a fluoroquinolonas fue del 17.8% siendo la mutación G248T (S83I) la más frecuente. Siete hombres presentaron alguna coinfección de transmisión sexual. Conclusiones: Aunque el porcentaje de infecciones por M. genitalium es bajo, la elevada tasa de resistencias frente a macrólidos hace necesario modificar los protocolos de diagnóstico y tratamiento empírico de las infecciones de transmisión sexual. El uso de fluoroquinolonas es adecuado tras testar previamente el perfil de resistencia a macrólidos. (AU)

Correction: Díaz-García et al. Candida Genotyping of Blood Culture Isolates from Patients Admitted to 16 Hospitals in Madrid: Genotype Spreading during the COVID-19 Pandemic Driven by Fluconazole-Resistant C. parapsilosis. J. Fungi 2022, 8, 1228.

In the original publication [...].

Sexually transmitted infections and clinical features in monkeypox (mpox) patients in Madrid, Spain.

Since the beginning of the monkeypox (mpox) virus outbreak in May 2022, there has been an increase in the number of cases worldwide in the setting of sexual transmission. We have tested by real-time PCR 187 mpox patients, of which 157 patients were screened for sexually transmitted infections (STI) in 245 samples. Thirty-six pathogens were detected in 30 patients: herpes simplex virus (HSV-I/II, 12/36, 33.3%), Neisseria gonorrhoeae (NG, 9/36, 25%), Chlamydia trachomatis (CT, 5/36, 13.8%), Chlamydia trachomatis-lymphogranuloma venereum (CT-LGV, 3/36, 8.3%), Treponema pallidum (TP, 4/36, 11.1%) and Mycoplasma genitalium (MG, 3/36, 8.3%). Screening of STI is recommended in mpox patients for the differential diagnosis of the main infections of sexual tract especially in patients with rectal involvement.

Isolation of filamentous basidiomycetes from respiratory samples in a tertiary care Spanish hospital.

BACKGROUND: The clinical significance of the filamentous basidiomycetes isolated from clinical samples is not always clear. Thus, these fungi have been considered environmental contaminants traditionally. AIMS: To review those clinical cases in which filamentous basidiomycetes from respiratory samples had been isolated. METHODS: The retrospective study was carried out in a single tertiary care hospital. We recovered all culture-confirmed isolations of filamentous basidiomycetes from respiratory samples (bronchial aspirate [BAS], bronchoalveolar lavage [BAL] and sputum) analyzed between the years 2020 and 2023. Isolates were identified by ITS region sequencing. RESULTS: In six patients a filamentous basidiomycete had been isolated from a respiratory sample. The species identified were all different: Fomitopsis sp. (BAS), Trametes ljubarskyi (BAL), Stereum gausapatum (BAS), Porostereum spadiaceum (BAS), Phlebia subserialis (sputum) and Inonotus levis (BAL). All the patients were immunosuppressed or had an underlying disease with pulmonary involvement. None of them received any specific antifungal treatment (in relation with the fungus isolated) and all six improved clinically and were discharged. CONCLUSIONS: The isolation of filamentous basidiomycetes in these patients had uncertain clinical significance. However, the isolation of any filamentous basidiomycete in respiratory samples from immunosuppressed patients or patients with chronic pulmonary disease is an emerging situation that should be carefully assessed in the context of chronic allergic episodes or suspicion of invasive fungal infections.