Candida spp.: the burden of a microorganism in a microbiology department.

There is no precise information available on the entire workload of isolating a specific microorganism in a clinical microbiology laboratory, and the costs associated with it have not been specifically estimated. In this descriptive retrospective study conducted at the microbiology department of a general teaching hospital from January 2021 to December 2022, we assessed the workload associated with identifying Candida species in all types of clinical samples and patients. Costs were estimated from data obtained from the hospital's finance department and microbiology laboratory cost records. In 2 years, 1,008,231 samples were processed at our microbiology department, of which 8,775 had one or more Candida spp. isolates (9,683 total isolates). Overall, 5,151 samples with Candida spp. were identified from 2,383 inpatients. We isolated Candida spp. from 515.3 samples/100,000 population/year and from 92 samples/1,000 hospital admissions/year. By sample type, 90.8% were superficial, mainly mucosal. Only 9.1% Candida spp. were isolated from deep, usually sterile, samples, being mostly from ordinarily sterile fluids. Candida albicans was the main species (58.5%) identified, followed by C. parapsilosis complex, C. glabrata, C. tropicalis, and C. krusei. In admitted patients, the incidences of samples with Candida spp. isolates were 302.7 samples/100,000 population/year and 54 samples/1,000 admissions/year. The average cost of isolating and identifying Candida spp. was estimated at 25€ per culture-positive sample. To our knowledge, this is the first attempt to gage the workload and costs of Candida spp. isolation at a hospital microbiology department. These data can help assess the burden and significance of Candida isolation at other institutions and also help design measures for streamlining. IMPORTANCE: We believe that this work is of interest because at present, there is no really accurate information available on the total workload involved in isolating a specific microorganism in a clinical microbiology laboratory. The costs related to this have also not been described. We have described the unrestricted workload of Candida spp. in all types of samples for all types of species and patients. We believe that this information would be necessary to collect and share this information as well as to collect it in a standardized way to know the current situation of Candida spp. workload in all clinical microbiology laboratories.

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.

The Tropical Andes Biodiversity Hotspot: A Comprehensive Dataset for the Mira-Mataje Binational Basins.

We present a flora and fauna dataset for the Mira-Mataje binational basins. This is an area shared between southwestern Colombia and northwestern Ecuador, where both the Chocó and Tropical Andes biodiversity hotspots converge. We systematized data from 120 sources in the Darwin Core Archive (DwC-A) standard and geospatial vector data format for geographic information systems (GIS) (shapefiles). Sources included natural history museums, published literature, and citizen science repositories across 13 countries. The resulting database has 33,460 records from 6,821 species, of which 540 have been recorded as endemic, and 612 as threatened. The diversity represented in the dataset is equivalent to 10% of the total plant species and 26% of the total terrestrial vertebrate species in both hotspots. The dataset can be used to estimate and compare biodiversity patterns with environmental parameters and provide value to ecosystems, ecoregions, and protected areas. The dataset is a baseline for future assessments of biodiversity in the face of environmental degradation, climate change, and accelerated extinction processes.

Corrigendum: Identifying anaerobic bacteria using MALDI-TOF mass spectrometry: a four-year experience.

[This corrects the article DOI: 10.3389/fcimb.2021.521014.].

One-year longitudinal changes of peripheral CD4+ T-lymphocyte counts, gut microbiome, and plaque vulnerability after an acute coronary syndrome.

Background: Longitudinal changes in gut microbiome and inflammation may be involved in the evolution of atherosclerosis after an acute coronary syndrome (ACS). We aimed to characterize repeated profiles of gut microbiota and peripheral CD4+ T lymphocytes during the first year after an ACS, and to address their relationship with atherosclerotic plaque changes. Methods: Over one year we measured the microbiome, peripheral counts of CD4+ T populations and cytokines in 67 patients shortly after a first ACS. We compared baseline measurements to those of a matched population of 40 chronic patients. A subgroup of 20 ACS patients underwent repeated assessment of fibrous cap thickness (FCT) of a non-culprit lesion. Results: At admission, ACS patients showed gut dysbiosis compared with the chronic group, which was rapidly reduced and remained low at 1-year. Also, their Th1 and Th2 CD4+ T counts were increased but decreased over time. The CD4+ T counts were related to ongoing changes in gut microbiome. Unsupervised clustering of repeated CD4+ Th0, Th1, Th2, Th17 and Treg counts in ACS patients identified two different cell trajectory patterns, related to cytokines. The group of patients following a high-CD4+ T cell trajectory showed a one-year reduction in their FCT [net effect = -24.2 µm; p = 0.016]. Conclusions: Patients suffering an ACS show altered profiles of microbiome and systemic inflammation that tend to mimic values of chronic patients after 1-year. However, in one-third of patients, this inflammatory state remains particularly dysregulated. This persistent inflammation is likely related to plaque vulnerability as evident by fibrous cap thinning (Clinical Trial NCT03434483).

Cutibacterium species valvular and cardiac device-related infective endocarditis: contemporary data from the GAMES prospective cohort (2008-2023).

BACKGROUND: Information on infective endocarditis (IE) caused by Cutibacterium spp. is limited and new Duke-ISCVID criteria have not yet been properly assessed. We examined clinical characteristics, outcomes and performance of diagnostic tests for Cutibacterium valvular and cardiac implantable electronic device-related IE (CIED-IE). METHODS: Data corresponding to all episodes of Cutibacterium IE recorded from 2008 to 2023 in a prospective national cohort including 46 Spanish hospitals were examined. Possible IE cases were reassessed using the new criteria. The sensitivity of blood cultures, valvular and CIED cultures, and PCR of the 16SrRNA gene and sequencing (16SPCR) was evaluated. RESULTS: There were 67/6,692 (1%) episodes of IE caused by Cutibacterium spp., 85% affecting men. Of these, 50 were valve-related (45 prosthetic, 5 native) and 17 CIED-related. The new criteria identified 8 additional cases and reclassified 15 as definite IE. Intracardiac complications (abscess, pseudoaneurysm, perforation or intracardiac fistula) occurred in 23/50 (46%) valvular IE episodes, leading to 18% mortality, and up to 40% mortality if surgery was indicated but could not be performed. All CIED-IE cases underwent device removal and no deaths were recorded. Positive diagnosis rates for blood cultures, valve/device cultures and 16SPCR were 52%, 70% and 82%, respectively. CONCLUSION: Cutibacterium IE is a rare yet potentially life-threatening condition that warrants a high index of suspicion in men with endovascular prosthetic material. The new Duke-ISCVID criteria and molecular techniques are useful for its diagnosis. Considering a significant complication rate, cardiac surgery and removal of CIEDs play a key role in reducing mortality.

Lateral kangaroo position for thermal stability of extremely preterm: Non-inferiority randomized controlled trial.

BACKGROUND: Kangaroo care (KC) is an evidence-based best practice that can prevent major health complications in preterm infants. However, there is a lack of evidence on the feasibility and safety of placing extremely preterm infants under 28 weeks gestational age in KC position. AIM: To compare thermal stability 60 min after the first KC session in the lateral versus prone position in extremely preterm infants under 28 weeks gestational age. STUDY DESIGN: This is a single-centre, randomized, non-inferiority, parallel clinical trial. The patients were extremely preterm infants during their first 5 days of life. Infants in the intervention group received KC in the lateral position while those in the control group received KC in the prone position. All infants receiving KC were inside their polyethylene bags but maintained skin-to-skin contact. The primary outcome was the axillary temperature of the infants, and the secondary outcome was the development of intraventricular haemorrhage. RESULTS: Seventy infants were randomized (35 per group). The mean gestational age was 26 +1(1+1) in both groups. In the first KC session, the infant temperature at 60 minutes was 36.79°C (0.43) in lateral KC position, and 36.78°C (0.38) in prone KC position (p = .022). In lateral KC position, 7.69% (2) of the children who, according to the cranial ultrasound performed before the first session, had no haemorrhage presented with intraventricular haemorrhage after the first session. In prone KC position, new haemorrhages appeared after the first session in 29.17% (7) (p = .08). CONCLUSIONS: The lateral KC position is an alternative to the conventional prone KC position and maintains normothermia in infants under 28 weeks gestational age. RELEVANCE TO CLINICAL PRACTICE: Extremely preterm infants are candidates for KC. Lateral KC position is an evidence-based best practice that can be applied to preterm infants under 28 weeks GA. This evidence is particularly useful in performing umbilical catheterization on these patients.

Clinical Outcome of Patients with Escherichia coli Isolated from Catheter Lumens and/or Peripheral Blood Cultures: A Retrospective Analysis.

BACKGROUND: Escherichia coli commonly causes catheter-related bloodstream infection (C-RBSI) in specific populations. The differential time to positivity (DTTP) technique is the recommended conservative procedure for diagnosing C-RBSIs. METHODS: We conducted a retrospective study of episodes in which E. coli was isolated from catheter lumens obtained using the DTTP technique. Microbiological and clinical data were obtained based on the DTTP technique as either catheter colonization, C-RBSI, or non-C-RBSI. RESULTS: A total of 89 catheter blood cultures were included, classified as follows: catheter colonization, 33.7%; C-RBSI, 9.0%; and non-C-RBSI, 57.3%. Only 15.7% of the catheters were withdrawn, with no positive catheter-tip cultures. We found no statistically significant differences in catheter type, antibiotic treatment, or clinical outcome among the groups, except for the frequency of catheter lock therapy or in the frequency of successful treatment. Mortality was associated with C-RBSI in only one patient. CONCLUSION: E. coli bacteremia diagnosed by the DTTP technique was classified as non-catheter-related in most patients. As the majority of the catheters were retained, E. coli bacteremia could not be microbiologically confirmed as catheter-related by the catheter-tip culture. Future studies are needed to assess the profitability of the DTTP technique for diagnosing E. coli C-RBSIs.

A solution to achieve sequencing from SARS-CoV-2 specimens with low viral loads: concatenation of reads from independent reactions.

BACKGROUND: During the pandemic, whole genome sequencing was critical to characterize SARS-CoV-2 for surveillance, clinical and therapeutical purposes. However, low viral loads in specimens often led to suboptimal sequencing, making lineage assignment and phylogenetic analysis difficult. We propose an alternative approach to sequencing these specimens that involves sequencing in triplicate and concatenation of the reads obtained using bioinformatics. This proposal is based on the hypothesis that the uncovered regions in each replicate differ and that concatenation would compensate for these gaps and recover a larger percentage of the sequenced genome. RESULTS: Whole genome sequencing was performed in triplicate on 30 samples with Ct > 32 and the benefit of replicate read concatenation was assessed. After concatenation: i) 28% of samples reached the standard quality coverage threshold (> 90% genome covered > 30x); ii) 39% of samples did not reach the coverage quality thresholds but coverage improved by more than 40%; and iii) SARS-CoV-2 lineage assignment was possible in 68.7% of samples where it had been impaired. CONCLUSIONS: Concatenation of reads from replicate sequencing reactions provides a simple way to access hidden information in the large proportion of SARS-CoV-2-positive specimens eliminated from analysis in standard sequencing schemes. This approach will enhance our potential to rule out involvement in outbreaks, to characterize reinfections and to identify lineages of concern for surveillance or therapeutical purposes.

Azole resistance screening in Aspergillus fumigatus sensu stricto using the azole-containing agar method (EUCAST E.Def 10.2): conidial suspension filtration and inoculum adjustment before inoculum preparation may not be needed.

Azole resistance screening in Aspergillus fumigatus sensu stricto can be routinely carried out by using azole-containing agar plates (E.Def 10.2 procedure); however, conidial suspension filtering and inoculum adjustment before inoculum preparation are time-consuming. We evaluated whether skipping the filtration and inoculum adjustment steps negatively influenced the performance of the E.Def 10.2 procedure. A. fumigatus sensu stricto isolates (n = 98), previously classified as azole susceptible or azole resistant (E.Def 9.4 method), were studied. Azole-resistant isolates had either the wild-type cyp51A gene sequence (n = 1) or the following cyp51A gene substitutions: TR34-L98H (n = 41), G54R (n = 5), TR46-Y121F-T289A (n = 1), or G448S (n = 1). In-house azole-containing agar plates were prepared according to the EUCAST E.Def 10.2 procedure. Conidial suspensions obtained by adding distilled water (Tween 20 0.1%) were either filtered and the inocula adjusted to 0.5 McFarland or left unfiltered and unadjusted. Agreements between the agar screening methods using inocula prepared by each procedure were high for itraconazole (99%), voriconazole (100%), and posaconazole (94.9%). Sensitivity and specificity (considering the susceptibility category as per the microdilution E.Def 9.4 method as the gold standard) of E.Def 10.2 were 100% to rule in or rule out resistance when unfiltered and unadjusted suspensions were used; the resistance phenotype of isolates harboring the TR34-L98H, G54R, or TR46-Y121F-T289A substitutions was correctly detected. Unfiltered and unadjusted conidial suspensions do not negatively influence the performance of the E.Def 10.2 method when screening for azole resistance in A. fumigatus sensu stricto. IMPORTANCE: Azole resistance screening in Aspergillus fumigatus sensu stricto can be routinely carried out by using azole-containing plates (E.Def 10.2 procedure); however, conidial suspension filtering and inoculum adjustment before inoculation of plates are time-consuming. We, here, showed that unfiltered and unadjusted conidial suspensions do not negatively influence the performance of the E.Def 10.2 method when screening for azole resistance in A. fumigatus sensu stricto.

Addressing catheter lock therapy: Does heparin reduce the bioactivity of dalbavancin when together in solution during freezing?

INTRODUCTION: The possible use of dalbavancin as a catheter lock solution was previously demonstrated by our study group. However, it was needed to assess whether heparin could affect dalbavancin bioactivity during freezing storage. METHODS: We tested the bioactivity of a dalbavancin+heparin (DH) vs. dalbavancin (D) against Staphylococcal biofilms comparing DH median value of cfu counts and metabolic activity with that obtained for D before and during storage under freezing up to 6 months. RESULTS: Despite there was a slight decrease in the median percentage reduction of metabolic activity at month 3 in Staphylococcus epidermidis between DH and D (97.6 vs. 100, p=0.037), considering the clinical criteria, no significant reduction in any of the variables tested was observed at the end of the experiment between D and DH solutions. CONCLUSION: The addition of heparin to a dalbavancin lock solution did not affect its bioactivity against staphylococcal biofilms irrespective of its preservation time under freezing.

Invasive aspergillosis: A comprehensive review.

Invasive aspergillosis (IA) is a severe fungal infection caused by Aspergillus species, particularly Aspergillus fumigatus, although new species, sometimes resistant to antifungals are becoming more common. IA predominantly affects immunocompromised patients, such as those with haematological malignancies, solid organ transplant recipients, and critically ill patients. However, new at-risk populations have emerged in recent years, such as IA associated with severe viral infections. Advanced diagnostic methods are crucial, especially considering the rising concern of antifungal resistance. Early detection is critical for successful treatment, typically involving antifungal medications like voriconazole or amphotericin B, but new antifungals are arriving to complete the therapeutic strategies. Despite advancements, mortality rates remain high, underscoring the importance of timely interventions and ongoing research. Healthcare providers should maintain a high index of suspicion, especially in immunocompromised patients and other new risk factors that are arising, to promptly diagnose and manage invasive aspergillosis.

Validation of the antibacterial effect of topically applied tranexamic acid using in vitro and in vivo models.

Background: Several studies have shown that tranexamic acid (TXA), an antifibrinolytic, reduces postoperative infection rates. Recent in vitro research showed that TXA alone and in combination with vancomycin and gentamicin had a synergistic effect against some staphylococcal strains. In the present study, this synergistic effect was validated in samples from patients with staphylococcal periprosthetic infection (PPI) and in an in vivo model. Methods: We tested 19 clinical strains (5 Staphylococcus aureus and 14 coagulase-negative staphylococci [CoNS]) against 10 mg/ml TXA alone and in combination with serial dilutions of vancomycin and gentamicin. The standardized microtiter plate method was used. The minimal inhibitory concentration (MIC) were calculated using standard visualization of well turbidity. We also used an S. aureus (ATCC29213) murine subcranial PPI model to compare the synergistic effect of TXA and gentamicin with that of TXA or gentamicin alone after 4 days of monitoring. The mice were euthanized, and disks were removed for analysis of cfu/ml counts and cell viability rate. Biofilm structure of both in vitro and in vivo samples was also analyzed using scanning electron microscopy (SEM). Results: When TXA was combined with vancomycin or gentamicin, the MIC decreased in 30% of the strains studied. According to species, the MIC50 for vancomycin and gentamicin alone and in combination with TXA against S. aureus strains was the same. This was also the case for CoNS with vancomycin and its corresponding combination, whereas with gentamicin and TXA, a reduction in MIC50 was observed (2 dilutions). In addition, in the in vivo model, the mean (SD) log cfu/ml and cell viability rate obtained from the implant was lower in the group of mice treated with TXA and gentamicin than in those treated only with TXA or gentamicin. SEM images also corroborated our findings in strains in which the MIC was reduced, as well as the in the mice implants, with the area occupied by biofilm being greater in samples treated only with gentamicin or TXA than in those treated with TXA+gentamicin. Conclusion: We confirm that combining TXA with vancomycin or gentamicin exerts a synergistic effect. However, this only occurs in selected strains.

Isavuconazole Versus Voriconazole as the First-line Therapy for Solid Organ Transplant Recipients With Invasive Aspergillosis: Comparative Analysis of 2 Multicenter Cohort Studies.

BACKGROUND: Isavuconazole (ISA) and voriconazole (VORI) are recommended as the first-line treatment for invasive aspergillosis (IA). Despite theoretical advantages of ISA, both triazole agents have not been compared in solid organ transplant recipients. METHODS: We performed a post hoc analysis of 2 retrospective multicenter cohorts of solid organ transplant recipients with invasive fungal disease (the SOTIS [Solid Organ Transplantation and ISavuconazole] and DiasperSOT [DIagnosis of ASPERgillosis in Solid Organ Transplantation] studies). We selected adult patients with proven/probable IA that were treated for ≥48 h with ISA (n = 57) or VORI (n = 77) as first-line therapy, either in monotherapy or combination regimen. The primary outcome was the rate of clinical response at 12 wk from the initiation of therapy. Secondary outcomes comprised 12-wk all-cause and IA-attributable mortality and the rates of treatment-emergent adverse events and premature treatment discontinuation. RESULTS: Both groups were comparable in their demographics and major clinical and treatment-related variables. There were no differences in the rate of 12-wk clinical response between the ISA and VORI groups (59.6% versus 59.7%, respectively; odds ratio [OR], 0.99; 95% confidence interval [CI], 0.49-2.00). This result was confirmed after propensity score adjustment (OR, 0.81; 95% CI, 0.32-2.05) and matching (OR, 0.79; 95% CI, 0.31-2.04). All-cause and IA-attributable mortality were also similar. Patients in the ISA group were less likely to experience treatment-emergent adverse events (17.5% versus 37.7%; P = 0.011) and premature treatment discontinuation (8.8% versus 23.4%; P = 0.027). CONCLUSIONS: Front-line treatment with ISA for posttransplant IA led to similar clinical outcomes than VORI, with better tolerability and higher treatment completion.

Prevalence and prognostic impact of stroke in a national cohort of infective endocarditis.

BACKGROUND: Stroke is a common complication of infective endocarditis (IE). Our aim was to describe the prevalence and prognostic impact of stroke in a national cohort of IE. METHODS: Consecutive inclusion at 46 Spanish hospitals between 2008 and 2021. RESULTS: Out of 5667 IE cases, 1125 had acute stroke (19.8%): 818 ischemic strokes (811 cardioembolic strokes (193 with hemorrhagic transformation), 4 transient ischemic attacks, 3 lacunar infarctions), 127 intracranial hemorrhages, and 27 other neurological complications (cerebral abscesses, encephalitis, and meningitis). Compared to patients without stroke, those with stroke had a similar mean age (69 years) but were more frequently female (68.2% vs 63.7%, p = 0.04) and had a higher incidence of intracardiac complications (35% vs 30%, p = 0.01), surgical indication (69.9% vs 65.9%, p = 0.001), in-hospital mortality (40.9% vs 22.0%, p < 0.001), and 1-year mortality (46.2% vs 27.9%, p < 0.001). The following variables were independently associated with stroke: mitral location (odds ratio (OR) = 1.5, 95% confidence interval (CI) = 1.34-1.8, p < 0.001), vascular phenomenon (OR = 2.9, 95% CI = 2.4-3.6, p = 0.0001), acute renal failure (OR = 1.2, 95% CI = 1.0-1.4, p = 0.021), septic shock (OR = 1.3, 95% CI = 1.1-1.6, p = 0.007), sepsis (OR = 1.3, 95% CI = 1.1-1.6, p = 0.005), surgery indicated but not performed (OR = 1.4, 95% CI = 1.2-1.7, p < 0.001), community-acquired IE (OR = 1.2, 95% CI = 1-1.4, p = 0.017), and peripheral embolization (OR = 1.6, 95% CI = 1.4-1.9, p < 0.001). Stroke was an independent predictor of in-hospital (OR = 2.1, 95% CI = 1.78-2.51, p < 0.001) and 1-year mortality (hazard ratio = 1.9, 95% CI = 1.6-2.5). CONCLUSION: One-fifth of patients with IE have concomitant stroke. Stroke is associated with mortality.

Surface Engineering of Escherichia coli to Display Its Phytase (AppA) and Functional Analysis of Enzyme Activities.

Escherichia coli phytase (AppA) is widely used as an exogenous enzyme in monogastric animal feed mainly because of its ability to degrade phytic acid or its salt (phytate), a natural source of phosphorus. Currently, successful recombinant production of soluble AppA has been achieved by gene overexpression using both bacterial and yeast systems. However, some methods for the biomembrane immobilization of phytases (including AppA), such as surface display on yeast cells and bacterial spores, have been investigated to avoid expensive enzyme purification processes. This study explored a homologous protein production approach for displaying AppA on the cell surface of E. coli by engineering its outer membrane (OM) for extracellular expression. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of total bacterial lysates and immunofluorescence microscopy of non-permeabilized cells revealed protein expression, whereas activity assays using whole cells or OM fractions indicated functional enzyme display, as evidenced by consistent hydrolytic rates on typical substrates (i.e., p-nitrophenyl phosphate and phytic acid). Furthermore, the in vitro results obtained using a simple method to simulate the gastrointestinal tract of poultry suggest that the whole-cell biocatalyst has potential as a feed additive. Overall, our findings support the notion that biomembrane-immobilized enzymes are reliable for the hydrolysis of poorly digestible substrates relevant to animal nutrition.

In Vivo Detection of Staphylococcus aureus Infections Using Radiolabeled Antibodies Specific for Bacterial Toxins.

Purpose: The Gram-positive Staphylococcus aureus bacterium is one of the leading causes of infection in humans. The lack of specific noninvasive techniques for diagnosis of staphylococcal infection together with the severity of its associated complications support the need for new specific and selective diagnostic tools. This work presents the successful synthesis of an immunotracer that targets the α-toxin released by S. aureus. Methods: [89Zr]Zr-DFO-ToxAb was synthesized based on radiolabeling an anti-α-toxin antibody with zirconium-89. The physicochemical characterization of the immunotracer was performed by high-performance liquid chromatography (HPLC), radio-thin layer chromatography (radio-TLC), and electrophoretic analysis. Its diagnostic ability was evaluated in vivo by positron emission tomography/computed tomography (PET/CT) imaging in an animal model of local infection-inflammation (active S. aureus vs. heat-killed S. aureus) and infective osteoarthritis. Results: Chemical characterization of the tracer established the high radiochemical yield and purity of the tracer while maintaining antibody integrity. In vivo PET/CT image confirmed the ability of the tracer to detect active foci of S. aureus. Those results were supported by ex vivo biodistribution studies, autoradiography, and histology, which confirmed the ability of [89Zr]Zr-DFO-ToxAb to detect staphylococcal infectious foci, avoiding false-positives derived from inflammatory processes. Conclusions: We have developed an immuno-PET tracer capable of detecting S. aureus infections based on a radiolabeled antibody specific for the staphylococcal alpha toxins. The in vivo assessment of [89Zr]Zr-DFO-ToxAb confirmed its ability to selectively detect staphylococcal infectious foci, allowing us to discern between infectious and inflammatory processes.

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.

Microevolution, reinfection and highly complex genomic diversity in patients with sequential isolates of Mycobacterium abscessus.

Mycobacterium abscessus is an opportunistic, extensively drug-resistant non-tuberculous mycobacterium. Few genomic studies consider its diversity in persistent infections. Our aim was to characterize microevolution/reinfection events in persistent infections. Fifty-three sequential isolates from 14 patients were sequenced to determine SNV-based distances, assign resistance mutations and characterize plasmids. Genomic analysis revealed 12 persistent cases (0-13 differential SNVs), one reinfection (15,956 SNVs) and one very complex case (23 sequential isolates over 192 months), in which a first period of persistence (58 months) involving the same genotype 1 was followed by identification of a genotype 2 (76 SNVs) in 6 additional alternating isolates; additionally, ten transient genotypes (88-243 SNVs) were found. A macrolide resistance mutation was identified from the second isolate. Despite high diversity, the genotypes shared a common phylogenetic ancestor and some coexisted in the same specimens. Genomic analysis is required to access the true intra-patient complexity behind persistent infections involving M. abscessus.