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.

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.

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.

Blood and intra-abdominal Candida spp. from a multicentre study conducted in Madrid using EUCAST: emergence of fluconazole resistance in Candida parapsilosis, low echinocandin resistance and absence of Candida auris.

OBJECTIVES: We prospectively monitored the epidemiology and antifungal susceptibility of Candida spp. from blood cultures and intra-abdominal samples in patients admitted to hospitals in the Madrid area. METHODS: Between 2019 and 2021, we prospectively collected incident isolates [one per species, patient and compartment (blood cultures versus intra-abdominal samples)] from patients admitted to any of 16 hospitals located in Madrid. We studied the antifungal susceptibilities to amphotericin B, triazoles, micafungin, anidulafungin and ibrexafungerp following the EUCAST E.Def 7.3.2 procedure. RESULTS: A total of 2107 Candida spp. isolates (1895 patients) from blood cultures (51.7%) and intra-abdominal samples were collected. Candida albicans, the Candida glabrata complex, the Candida parapsilosis complex, Candida tropicalis and Candida krusei accounted for 96.9% of the isolates; in contrast, Candida auris was undetected. Fluconazole resistance in Candida spp. was higher in blood cultures than in intra-abdominal samples (9.1% versus 8.2%; P > 0.05), especially for the C. parapsilosis complex (16.6% versus 3.6%, P < 0.05), whereas echinocandin resistance tended to be lower in blood cultures (0.5% versus 1.0%; P > 0.05). Resistance rates have risen, particularly for fluconazole in blood culture isolates, which increased sharply in 2021. Ibrexafungerp showed in vitro activity against most isolates. Species distributions and resistance rates varied among hospitals. CONCLUSIONS: Whereas no C. auris isolates were detected, fluconazole-resistant C. parapsilosis isolates have been spreading across the region and this has pulled up the rate of fluconazole resistance. In contrast, the rate of echinocandin resistance continues to be low.

A Phase I/IIa Prospective, Randomized, Open-Label Study on the Safety and Efficacy of Nebulized Liposomal Amphotericin for Invasive Pulmonary Aspergillosis.

Although nebulized liposomal amphotericin B (NLAB) is being used in invasive pulmonary aspergillosis (IPA) prophylaxis, no clinical trial has shown its efficacy as a therapeutic strategy. NAIFI is the inaugural randomized, controlled clinical trial designed to examine the safety and effectiveness of NLAB (dosage: 25 mg in 6 mL, three times per week for 6 weeks) against a placebo, in the auxiliary treatment of IPA. Throughout the three-year clinical trial, thirteen patients (six NLAB, seven placebo) were included, with 61% being onco-hematological with less than 100 neutrophils/µL. There were no significant differences noted in their pre- and post-nebulization results of forced vital capacity (FVC), forced expiratory volume in 1 s (FEV1), and oxygen saturation between the groups. Neither bronchospasm nor serum amphotericin B levels were reported in any patients given NLAB. 18F-Fluorodeoxyglucose positron emission tomography (FDG-PET-TC) was carried out at the baseline and after 6 weeks. A notable decrease in median SUV (standardized uptake value) was observed in NLAB patients after 6 weeks (-3.6 vs. -0.95, p: 0.039, one tail). Furthermore, a reduction in serum substance galactomannan and beta-D-Glucan was identified within NLAB recipients. NLAB is well tolerated and safe for patients with IPA. Encouraging indirect efficacy data have been derived from image monitoring or biomarkers. However, further studies involving more patients are necessary.

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.

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.

Pharmacokinetics of echinocandins in suspected candida peritonitis: A potential risk for resistance.

INTRODUCTION: A possible increase in Candida resistance, especially in Candida glabrata, has been speculated according to poor diffusion of echinocandins to peritoneal fluid. MATERIALS/METHODS: Peritoneal and serum concentrations of caspofungin, micafungin and anidulafungin were analysed in surgical patients with suspected candida peritonitis. After 4 days of starting therapy, serum and peritoneal samples (through peritoneal drainage) were obtained at baseline, 1, 6, 12 and 24 h of drug administration. Micafungin and anidulafungin concentrations were determined using high-performance liquid chromatography (HPLC/F), whereas caspofungin concentrations were established by bioassay. RESULTS: Twenty-three critically ill patients with suspected abdominal fungal infection who were receiving an echinocandin were prospectively recruited. No specific criteria were applied to prescribe one specific echinocandin. No special clinical differences were observed among the three groups of patients. All were receiving antibiotic therapy, 80% required inotropic drugs, and fungal peritonitis was confirmed in 74% of them. The AUC0_24h (mg × h/L) obtained in serum and peritoneal fluid were: 126.84 and 34.38, 98.52 and 18.83, and 66.9 and 8.78 for anidulafungin, micafungin and caspofungin, respectively. The median concentration in peritoneal fluid ranged from 0.66 to 1.82 µg/mL for anidulafungin, 0.68-0.88 µg/mL for micafungin and 0.21-0.46 µg/mL for caspofungin. CONCLUSION: The results showed moderate penetration of echinocandins into the peritoneal fluid of these patients. These levels are below the threshold of resistance mutant selection published by other authors. This could justify a potential risk of resistance in patients with prolonged treatment with echinocandins and suboptimal control of abdominal infection.

First national survey of the diagnosis of Helicobacter pylori infection in Clinical Microbiology Laboratories in Spain. / Primera encuesta nacional sobre el diagnóstico de la infección por Helicobacter pylori en los laboratorios de microbiología clínica en España.

INTRODUCTION: The aim of this study was to know, through a national survey, the methods and techniques used for the diagnosis of Helicobacter pylori (Hp) in the different Clinical Microbiology Services/Laboratories in Spain, as well as antibiotic resistance data. METHODS: The survey requested information about the diagnostic methods performed for Hp detection in Clinical Microbiology laboratories, including serology, stool antigen, culture from gastric biopsies, and PCR. In addition, the performance of antibiotic susceptibility was collected. Data on the number of samples processed in 2016, positivity of each technique and resistance data were requested. The survey was sent by email (October-December 2017) to the heads of 198 Clinical Microbiology Laboratories in Spain. RESULTS: Overall, 51 centers from 29 regions answered the survey and 48/51 provided Hp microbiological diagnostic testing. Concerning the microbiological methods used to diagnose Hp infection, the culture of gastric biopsies was the most frequent (37/48), followed by stool antigen detection (35/48), serology (19/48) and biopsy PCR (5/48). Regarding antibiotic resistance, high resistance rates were observed, especially in metronidazole and clarithromycin (over 33%). CONCLUSION: Culture of gastric biopsies was the most frequent method for detection of Hp, but the immunochromatographic stool antigen test was the one with which the largest number of samples were analyzed. Nowadays, in Spain, it concerns the problem of increased antibiotic resistance to 'first-line' antibiotics.

T2 magnetic resonance for the diagnosis of deep-seated invasive candidiasis in a liver recipient without candidemia.

BACKGROUND: T2 magnetic resonance imaging (T2MR) is a new method for the diagnosis of invasive candidiasis, although most studies have analyzed its role in patients with candidemia or not infection. CASE REPORT: We present the case of a patient with arteritis and thrombosis of the hepatic graft resulted from an undocumented fungal infection in the explanted liver.T2MR in serum was a suitable diagnostic tool for the diagnosis of the deep-seated invasive candidiasis in the absence of candidemia or the isolation of the yeast in culture. CONCLUSIONS: T2MR allowed the diagnosis of deep-seated invasive candidiasis in an immunodepressed patient without candidemia, even before the onset of symptoms.