Clinical Impact of Polymerase Chain Reaction-Based Aspergillus and Azole Resistance Detection in Invasive Aspergillosis: A Prospective Multicenter Study.

BACKGROUND: Invasive aspergillosis (IA) by a triazole-resistant Aspergillus fumigatus is associated with high mortality. Real-time resistance detection will result in earlier initiation of appropriate therapy. METHODS: In a prospective study, we evaluated the clinical value of the AsperGenius polymerase chain reaction (PCR) assay in hematology patients from 12 centers. This PCR assay detects the most frequent cyp51A mutations in A. fumigatus conferring azole resistance. Patients were included when a computed tomography scan showed a pulmonary infiltrate and bronchoalveolar fluid (BALf) sampling was performed. The primary end point was antifungal treatment failure in patients with azole-resistant IA. RESULTS: Of 323 patients enrolled, complete mycological and radiological information was available for 276 (94%), and probable IA was diagnosed in 99/276 (36%). Sufficient BALf for PCR testing was available for 293/323 (91%). Aspergillus DNA was detected in 116/293 (40%) and A. fumigatus DNA in 89/293 (30%). The resistance PCR was conclusive in 58/89 (65%) and resistance detected in 8/58 (14%). Two had a mixed azole-susceptible/azole-resistant infection. In the 6 remaining patients, treatment failure was observed in 1. Galactomannan positivity was associated with mortality (P = .004) while an isolated positive Aspergillus PCR was not (P = .83). CONCLUSIONS: Real-time PCR-based resistance testing may help to limit the clinical impact of triazole resistance. In contrast, the clinical impact of an isolated positive Aspergillus PCR on BALf seems limited. The interpretation of the EORTC/MSGERC PCR criterion for BALf may need further specification (eg, minimum cycle threshold value and/or PCR positive on >1 BALf sample).

Contact tracing for vancomycin-resistant Enterococcus faecium (VRE): evaluation of the Dutch policy of quintuple screening cultures.

Detection of vancomycin-resistant Enterococcus faecium (VRE) is hampered by low sensitivity of rectal swab cultures. This study aimed to define the number of screening cultures needed to increase sensitivity to detect VRE transmission, and to determine time from presumed exposure to detectable colonization. In a tertiary care setting, we retrospectively analyzed data from 9 VRE outbreaks. As a proxy or estimation for time to detectable colonization, the time between first positive culture of the presumed index patient and that of their contacts was determined. Only 64% of secondary cases were positive in the first out of five cultures. By using the first three out of five rectal swabs, 89% (95%CI: 78-95%) of all secondary cases would have been identified. The median number of days between the positive culture of the index patient and the first positive culture of secondary cases was 9 days. Eleven percent of secondary cases would have been missed if only three rectal samples would have been obtained. Furthermore, our results show that one or more rectal swabs taken around day 9 after presumed exposure should at least be included in the screening approach. In our setting, obtaining a fourth and a fifth rectal swab showed a relevant additional value compared to only one to three swabs. Our findings are useful for determining the most effective VRE contact tracing approach to prevent transmission.

Madurella mycetomatis grains within a eumycetoma lesion are clonal.

Eumycetoma is a neglected tropical infection of the subcutaneous tissue, characterized by tumor-like lesions and most commonly caused by the fungus Madurella mycetomatis. In the tissue, M. mycetomatis organizes itself in grains, and within a single lesion, thousands of grains can be present. The current hypothesis is that all these grains originate from a single causative agent, however, this hypothesis was never proven. Here, we used our recently developed MmySTR assay, a highly discriminative typing method, to determine the genotypes of multiple grains within a single lesion. Multiple grains from surgical lesions obtained from 11 patients were isolated and genotyped using the MmySTR panel. Within a single lesion, all tested grains shared the same genotype. Only in one single grain from one patient, a difference of one repeat unit in one MmySTR marker was noted relative to the other grains from that patient. We conclude that within these lesions the grains originate from a single clone and that the inherent unstable nature of the microsatellite markers may lead to small genotypic differences. LAY ABSTRACT: In lesions of the implantation mycosis mycetoma many Madurella mycetomatis grains are noted. It was unknown if grains arose after implantation of a single isolate or a mixture of genetically diverse isolates. By typing the mycetoma grains we showed that all grains within a single lesion were clonal and originated from a single isolate.

Rapid and Accurate Detection of Aminoglycoside-Modifying Enzymes and 16S rRNA Methyltransferases by Targeted Liquid Chromatography-Tandem Mass Spectrometry.

New and rapid diagnostic methods are needed for the detection of antimicrobial resistance to aid in curbing drug-resistant infections. Targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) is a method that could serve this purpose, as it can detect specific peptides of antimicrobial resistance mechanisms with high accuracy. In the current study, we developed an accurate and rapid targeted LC-MS/MS assay based on parallel reaction monitoring for detection of the most prevalent aminoglycoside-modifying enzymes and 16S rRNA methyltransferases in Escherichia coli and Klebsiella pneumoniae that confer resistance to aminoglycosides. Specific tryptic peptides needed for detection were selected and validated for AAC(3)-Ia, AAC(3)-II, AAC(3)-IV, AAC(3)-VI, AAC(6')-Ib, AAC(6')-Ib-cr, ANT(2″)-I, APH(3')-VI, ArmA, RmtB, RmtC, and RmtF. In total, 205 isolates containing different aminoglycoside resistance mechanisms that consisted mostly of E. coli and K. pneumoniae were selected for assay development and evaluation. Mass spectrometry results were automatically analyzed and were compared to whole-genome sequencing results. Of the 2,460 isolate and resistance mechanism combinations tested, 2,416 combinations matched. Discrepancies were further analyzed by repeating LC-MS/MS analysis and performing additional PCRs. Mass spectrometry results were also used to predict resistance and susceptibility to gentamicin, tobramycin, and amikacin in only the E. coli and K. pneumoniae isolates (n = 191). The category interpretations were correctly predicted for gentamicin in 97.4% of the isolates, for tobramycin in 97.4% of the isolates, and for amikacin in 82.7% of the isolates. Targeted LC-MS/MS can be applied for accurate and rapid detection of aminoglycoside resistance mechanisms.

A Short-Tandem-Repeat Assay (MmySTR) for Studying Genetic Variation in Madurella mycetomatis.

Madurella mycetomatis is the major causative agent of eumycetoma, a neglected tropical infection characterized by painless subcutaneous lesions, inflammation, and grains draining from multiple sinuses. To study the epidemiology of mycetoma, a robust discriminatory typing technique is needed. We describe the use of a short-tandem-repeat assay (MmySTR) for genotyping of M. mycetomatis isolates predominantly from Sudan. Eleven microsatellite markers (3 dinucleotides, 4 trinucleotide repeats, and 4 tetranucleotide repeats) were selected from the M. mycetomatis MM55 genome using the Tandem Repeats Finder software. PCR amplification primers were designed for each microsatellite marker using primer3 software and amplified in a multicolor multiplex PCR approach. To establish the extent of genetic variation within the population, a collection of 120 clinical isolates from different regions was genotyped with this assay. The 11 selected MmySTR markers showed a large genotypic heterogeneity. From a collection of 120 isolates, 108 different genotypes were obtained. Simpson's diversity index (D) value for individual markers ranged from 0.081 to 0.881, and the combined panel displayed an overall D value of 0.997. The MmySTR assay demonstrated high stability, reproducibility, and specificity. The MmySTR assay is a promising new typing technique that can be used to genotype isolates of M. mycetomatis Apart from the possible contribution of host factors, the genetic diversity observed among this group of isolates might contribute to the different clinical manifestations of mycetoma. We recommend that the MmySTR assay be used to establish a global reference database for future study of M. mycetomatis isolates.

Characterization of Staphylococcus roterodami sp. nov., a new species within the Staphylococcus aureus complex isolated from a human foot infection.

This article introduces a new Staphylococcus species cultivated from a human foot wound infection in a Dutch traveller returning from the island of Bali, Indonesia: Staphylococcus roterodami sp. nov. Based on the genomic sequence, there is strong molecular evidence for assigning the strain to a novel species within the S. aureus complex. Differences in cellular fatty acid spectrum and biochemical tests underline these findings. Its ecological niche and pathogenicity require further study. The type strain is DSM111914T (JCM34415T).

Temporal Variation in Staphylococcus aureus Protein A Genotypes from Nose and Skin in Atopic Dermatitis Patients.

BACKGROUND: Staphylococcus aureus colonization is associated with disease severity in patients with atopic dermatitis (AD). OBJECTIVE: To investigate temporal variation in S. aureus protein A gene (spa)-types isolated from the nose and lesional skin and the correlation of spa-types with disease severity. RESULTS: This study included 96 adult AD patients who were assessed at baseline (T0) and after a strict 2-week follow-up period (T1) in which treatment was standardized with a topical corticosteroid. Fifty-five different spa-types were detected in the nose and skin cultures. Seventy-three patients were colonized with S. aureus in the nasal cavity at both time points (persistent carriership), 59 of whom (81%) had identical spa-types over time. For skin samples, 42 (75%) of the 56 persistent skin carriers had identical spa-types over time. The same spa-type was carried in the nose and skin in 79 and 77% of the patients at T0 and T1, respectively. More severe disease was not associated with specific spa-types or with temporal variation in spa-type. CONCLUSION: S. aureus strains in AD are highly heterogeneous between patients. The majority of patients carry the same spa-type in the nose and skin without temporal variation, suggesting clonal colonization within individual patients. No predominant spa-type or temporal variation is associated with increased disease severity.

Impact of bacterial species and baseline resistance on fosfomycin efficacy in urinary tract infections.

OBJECTIVES: To assess the antibacterial effects of a single 3 g oral fosfomycin dose on Escherichia coli and Klebsiella pneumoniae clinical isolates within a dynamic bladder infection model. METHODS: An in vitro model simulating dynamic urinary fosfomycin concentrations was used. Target fosfomycin exposure (Cmax = 1984 mg/L and Tmax = 7.5 h) was validated by LC-MS/MS. Pharmacodynamic responses of 24 E. coli and 20 K. pneumoniae clinical isolates were examined (fosfomycin MIC ≤0.25-128 mg/L). Mutant prevention concentration (MPC), fosfomycin heteroresistance, fosfomycin resistance genes and fosA expression were examined. Pathogen kill and emergence of high-level resistance (HLR; MIC >1024 mg/L) were quantified. RESULTS: Following fosfomycin exposure, 20 of 24 E. coli exhibited reductions in bacterial counts below the lower limit of quantification without regrowth, despite baseline fosfomycin MICs up to 128 mg/L. Four E. coli regrew (MIC = 4-32 mg/L) with HLR population replacement. At baseline, these isolates had detectable HLR subpopulations and MPC >1024 mg/L. All E. coli isolates were fosA negative. In contrast, 17 of 20 K. pneumoniae regrew post exposure, 6 with emergence of HLR (proportion = 0.01%-100%). The three isolates without regrowth did not have a detectable HLR subpopulation after dynamic drug-free incubation. All K. pneumoniae had MPC >1024 mg/L and were fosA positive. WGS analysis and fosA expression failed to predict fosfomycin efficacy. CONCLUSIONS: E. coli and K. pneumoniae isolates demonstrate discrepant responses to a single fosfomycin dose in a dynamic bladder infection in vitro model. Treatment failure against E. coli was related to an HLR subpopulation, not identified by standard MIC testing. Activity against K. pneumoniae appeared limited, regardless of MIC testing, due to universal baseline heteroresistance.

Diagnostic Value of Microbial Cell-free DNA Sequencing for Suspected Invasive Fungal Infections: A Retrospective Multicenter Cohort Study.

Background: An early diagnosis and treatment of invasive fungal disease (IFD) is associated with improved outcome, but the moderate sensitivity of noninvasive diagnostic tests makes this challenging. Invasive diagnostic procedures such as bronchoalveolar lavage (BAL) have a higher yield but are not without risk. The detection and sequencing of microbial cell-free DNA (mcfDNA) may facilitate a noninvasive diagnosis. Materials: In a prospective observational study, we collected plasma in the 120 hours preceding or following a BAL in patients with hematological malignancies suspected for a pulmonary IFD. The EORTC/MSGERC2020 criteria were used for IFD classification. Sequencing was performed by Karius (Redwood City, CA) using their Karius Test (KT) on plasma and a "research use only test" on BAL fluid if available. Cases with a probable/proven IFD were identified based on standard diagnostic tests on serum and BAL (microscopy, polymerase chain reaction, galactomannan, culture) and used to calculate the sensitivity, specificity, and additional diagnostic value of the KT. Results: Of 106 patients enrolled, 39 (37%) had a proven/probable invasive aspergillosis, 7 (7%) a non-Aspergillus IFD, and 4 (4%) a mixed IFD. The KT detected fungal mcfDNA in 29 (28%) patients. Compared with usual diagnostic tests, the sensitivity and specificity were 44.0% (95% confidence interval [CI], 31.2-57.7) and 96.6% (95% CI, 88.5%-99.1%). Sensitivity of the KT was higher in non-Aspergillus IFD (Mucorales:2/3, Pneumocystis jirovecii: 3/5). On BAL, the sensitivity was 72.2% (95% CI, 62.1-96.3), and specificity 83.3% (95% CI, 49.1-87.5). Conclusions: Sequencing of mcfDNA may facilitate a noninvasive diagnosis of IFD in particular non-Aspergillus IFD. However, on plasma and similar to currently available diagnostics, it cannot be used as a "rule-out" test.

Identification and typing of isolates of Cyphellophora and relatives by use of amplified fragment length polymorphism and rolling circle amplification.

The species diversity and identification of black fungi belonging to Cyphellophora and Phialophora, which colonize and infect human skin and nails, were studied using amplified fragment length polymorphism (AFLP). A total of 76 Cyphellophora and Phialophora isolates were evaluated, and their delimitation was compared to earlier studies using multilocus sequencing. The results of the AFLP analysis and sequencing were in complete agreement with each other. Seven species-specific padlock probes for the most prevalent species were designed on the basis of the ribosomal DNA internal transcribed spacer region, and identification of the respective species could easily be achieved with the aid of rolling circle amplification.

Veronaea botryosa: molecular identification with amplified fragment length polymorphism (AFLP) and in vitro antifungal susceptibility.

Inter- and intraspecific genomic variability of 18 isolates of Veronaea botryosa originating from clinical and environmental sources was studied using amplified fragment length polymorphism (AFLP). The species was originally described from the environment, but several severe cases of disseminated infection in apparently healthy individuals have been reported worldwide. All tested strains of V. botryosa, identified on the basis of sequencing and phenotypic and physiological criteria prior to our study, were confirmed by AFLP analysis, yielding a clear separation of V. botryosa as a rather homogeneous group from related species. In vitro antifungal susceptibility testing resulted in MIC90s across all strains in increasing order posaconazole (0.25 µg/ml), itraconazole (1 µg/ml), voriconazole (4 µg/ml), terbinafine (4 µg/ml), caspofungin (8 µg/ml), anidulafungin (8 µg/ml), isavuconazole (16 µg/ml), amphotericin B (16 µg/ml), and fluconazole (32 µg/ml). Overall, the isolates showed a uniform pattern of low MICs of itraconazole and posaconazole, but high MICs for remaining agents. The echinocandins (caspofungin and anidulafungin) had no activity against V. botryosa. There was no statistically significant difference between susceptibilities of environmental (n = 11) and clinical (n = 7) isolates of V. botryosa (P > 0.05).

Environmental contamination with highly resistant microorganisms after relocating to a new hospital building with 100% single-occupancy rooms: A prospective observational before-and-after study with a three-year follow-up.

INTRODUCTION: Inanimate surfaces within hospitals can be a source of transmission for highly resistant microorganisms (HRMO). While many hospitals are transitioning to single-occupancy rooms, the effect of single-occupancy rooms on environmental contamination is still unknown. We aimed to determine differences in environmental contamination with HRMO between an old hospital building with mainly multiple-occupancy rooms and a new hospital building with 100% single-occupancy rooms, and the environmental contamination in the new hospital building during three years after relocating. METHODS: Environmental samples were taken twice in the old hospital, and fifteen times over a three-year period in the new hospital. Replicate Organism Direct Agar Contact-plates (RODACs) were used to determine colony forming units (CFU). Cotton swabs premoistened with PBS were used to determine presence of methicillin-resistant Staphylococcus aureus, carbapenemase-producing Pseudomonas aeruginosa, highly resistant Enterobacterales, carbapenem-resistant Acinetobacter baumannii, and vancomycin-resistant Enterococcus faecium. All identified isolates were subjected to whole genome sequencing (WGS) using Illumina technology. RESULTS: In total, 4993 hospital sites were sampled, 724 in the old and 4269 in the new hospital. CFU counts fluctuated during the follow-up period in the new hospital building, with lower CFU counts observed two- and three years after relocating, which was during the COVID-19 pandemic. The CFU counts in the new building were equal to or surpassed the CFU counts in the old hospital building. In the old hospital building, 24 (3.3%) sample sites were positive for 49 HRMO isolates, compared to five (0.1%) sample sites for seven HRMO isolates in the new building (P < 0.001). In the old hospital, 89.8% of HRMO were identified from the sink plug. In the new hospital, 71.4% of HRMO were identified from the shower drain, and no HRMO were found in sinks. DISCUSSION: Our results indicate that relocating to a new hospital building with 100% single-occupancy rooms significantly decreases HRMO in the environment. Given that environmental contamination is an important source for healthcare associated infections, this finding should be taken into account when considering hospital designs for renovations or the construction of hospitals.

Limiting spread of VIM-positive Pseudomonas aeruginosa from colonized sink drains in a tertiary care hospital: A before-and-after study.

BACKGROUND: In healthcare environments, sinks are being increasingly recognized as reservoirs for multidrug-resistant Gram-negative bacteria. In our hospital, carbapenemase-producing, Verona Integron-encoded Metallo-beta-lactamase (VIM)-positive Pseudomonas aeruginosa (VIM-PA) was detected at low endemicity in patients, and environmental culturing revealed that sink drains were primary reservoirs. Therefore, an intervention was initiated in several wards to install sink drain plugs as physical barriers against splashing to prevent transmission of VIM-PA from drain reservoirs to the surrounding sink environment. AIM: To assess the efficacy of the intervention on limiting spread of VIM-PA. METHODS: Swabs were taken from inner sink environments (i.e. drains), and outer sink environments (i.e. wash basins, faucet aerators, and countertops) twice before and three times after the intervention. Siphon water and drain wells were also sampled before and at the moment of the intervention, respectively. All samples were screened for VIM-PA, and isolates were typed with multiple-locus variable-number tandem repeat analysis (MLVA). RESULTS: There was a significant reduction in VIM-PA positivity in both inner (P-value <0.001) and outer (P-value 0.001) sink environments after the intervention. However, VIM-PA recolonization was observed in the inner sink environments of patient rooms, and also in rooms exclusive to healthcare personnel, over time. Surfaces in the outer sink environment were rarely positive for VIM-PA after the intervention. MLVA revealed three genetic clusters, with one found in all wards and room types during the study period. CONCLUSIONS: Drain plugs are a simple and effective infection prevention and control measure to contain spread of VIM-PA from drain reservoirs.

Universal screening or a universal risk assessment combined with risk-based screening for multidrug-resistant microorganisms upon admission: Comparing strategies.

OBJECTIVE: Timely identification of patients who carry multidrug-resistant microorganisms (MDRO) is needed to prevent nosocomial spread to other patients and to the hospital environment. We aimed to compare the yield of a universal screening strategy upon admission to the currently installed universal risk assessment combined with risk-based screening upon admission. METHODS: This observational study was conducted within a prospective cohort study. From January 1, 2018, until September 1, 2019, patients admitted to our hospital were asked to participate. Nasal and perianal samples were taken upon admission and checked for the presence of MDRO. The results of the universal risk assessment and risk-based screening were collected retrospectively from electronic health records. RESULTS: In total, 1017 patients with 1069 separate hospital admissions participated in the study. Universal screening identified 38 (3.6%) unknown MDRO carriers upon admission (37 individual patients), all carrying extended-spectrum beta-lactamase-producing Enterobacterales. For 946 of 1069 (88.5%) patients, both the universal risk assessment and universal screening were performed. For 19 (2.0%) admissions, ≥1 risk factor was identified. The universal risk assessment identified one (0.1%) unknown carrier, compared to 37 out of 946 carriers for the universal screening (P<0.001). Of the 37 carriers identified through the universal screening, 35 (94.6%) reported no risk factors. CONCLUSIONS: Our results show that in our low endemic setting, a universal screening strategy identified significantly more MDRO carriers than the currently implemented universal risk-assessment. When implementing a universal risk-assessment, risk factors should be carefully selected to be able to identify ESBL-E carriers. While the universal screening identified more MDRO carriers, further research is needed to determine the cost-effectiveness of this strategy.

Dynamics of Staphylococcus aureus in patients and the hospital environment in a tertiary care hospital in the Netherlands.

BACKGROUND: The dynamics of Staphylococcus aureus in patients and the hospital environment are relatively unknown. We studied these dynamics in a tertiary care hospital in the Netherlands. METHODS: Nasal samples were taken from adult patients at admission and discharge. Isolates cultured from clinical samples taken before and during hospitalization from these patients were included. Environmental samples of patient rooms were taken over a three-year period. Finally, isolates from clinical samples from patients with an epidemiological link to S. aureus positive rooms were included. Staphylococcal protein A (spa) typing was performed. RESULTS: Nasal samples were taken from 673 patients. One hundred eighteen (17.5%) were positive at admission and discharge, 15 (2.2%) patients acquired S. aureus during hospitalization. Nineteen patients had a positive clinical sample during hospitalization, 15.9% of the S. aureus were considered as from an exogenous source. One hundred and forty (2.8%) environmental samples were S. aureus positive. No persistent contamination of surfaces was observed. Isolates were highly diverse: spa typing was performed for 893 isolates, identifying 278 different spa types, 161 of these spa types were observed only once. CONCLUSION: Limited transmission could be identified between patients and the hospital environment, and from patient-to-patient. Exogenous acquisition was assumed to occur in 15% of clinical samples. Environmental contamination was infrequent, temporarily, and coincided with the strain from the patient admitted to the room at that time. MRSA was rare and not found in the environment.

Autochthonous and dormant Cryptococcus gattii infections in Europe.

Until recently, Cryptococcus gattii infections occurred mainly in tropical and subtropical climate zones. However, during the past decade, C. gattii infections in humans and animals in Europe have increased. To determine whether the infections in Europe were acquired from an autochthonous source or associated with travel, we used multilocus sequence typing to compare 100 isolates from Europe (57 from 40 human patients, 22 from the environment, and 21 from animals) with 191 isolates from around the world. Of the 57 human patient isolates, 47 (83%) were obtained since 1995. Among the 40 patients, 24 (60%) probably acquired the C. gattii infection outside Europe; the remaining 16 (40%) probably acquired the infection within Europe. Human patient isolates from Mediterranean Europe clustered into a distinct genotype with animal and environmental isolates. These results indicate that reactivation of dormant C. gattii infections can occur many years after the infectious agent was acquired elsewhere.

Microsatellite genotyping clarified conspicuous accumulation of Candida parapsilosis at a cardiothoracic surgery intensive care unit.

Candida parapsilosis has become a significant cause of invasive fungal infections in seriously ill patients. Nosocomial outbreaks through direct and indirect contact have been described. The aim of this study was the molecular characterization of what appeared to be an ongoing C. parapsilosis outbreak at the cardiothoracic intensive care unit of the University Hospital of Vienna between January 2007 and December 2008. Using two different molecular typing methods-automated repetitive sequence-based PCR (DiversiLab; bioMérieux) and microsatellite genotyping-we investigated the genetic relationship of 99 C. parapsilosis isolates. Eighty-three isolates originated from the cardiothoracic intensive care unit, while 16 isolates were random control isolates from other intensive care units and a different Austrian hospital. The 99 C. parapsilosis isolates analyzed by repetitive-element PCR all showed identical genotypes, suggesting an ongoing outbreak. In contrast, microsatellite genotyping showed a total of 56 different genotypes. Two major genotypes were observed in 10 and 15 isolates, respectively, whereas another 13 genotypes were observed in 2 to 4 isolates each. Forty-one genotypes were observed only once. Closely related genotypes that differed in only a single microsatellite marker were grouped into clonal complexes. When it comes to C. parapsilosis, microsatellite genotyping is a more discriminative method than repetitive-element PCR genotyping to investigate outbreaks.

Genotyping reveals the presence of a predominant genotype of Coxiella burnetii in consumer milk products.

Real-time PCR shows the widespread presence of Coxiella burnetii DNA in a broad range of commercially available milk and milk products. MLVA genotyping shows that this is the result of the presence of a predominant C. burnetii genotype in the dairy cattle population.

Molecular epidemiology of Aspergillus fumigatus isolates harboring the TR34/L98H azole resistance mechanism.

A rapid emergence of azole resistance has been observed in Aspergillus fumigatus in The Netherlands over the past decade. The dominant resistance mechanism appears to be of environmental origin and involves the TR(34)/L98H mutations in cyp51A. This resistance mechanism is now also increasingly being found in other countries. Therefore, genetic markers were used to gain more insights into the origin and spread of this genotype. Studies of 142 European isolates revealed that those with the TR(34)/L98H resistance mechanism showed less genetic variation than azole-susceptible isolates or those with a different genetic basis of resistance and were assigned to only four CSP (putative cell surface protein) types. Sexual crossing experiments demonstrated that TR(34)/L98H isolates could outcross with azole-susceptible isolates of different genetic backgrounds, suggesting that TR(34)/L98H isolates can undergo the sexual cycle in nature. Overall, our findings suggest a common ancestor of the TR(34)/L98H mechanism and subsequent migration of isolates harboring TR(34)/L98H across Europe.

Extensive genetic diversity within the Dutch clinical Cryptococcus neoformans population.

A set of 300 Dutch Cryptococcus neoformans isolates, obtained from 237 patients during 1977 to 2007, was investigated by determining the mating type, serotype, and AFLP and microsatellite genotype and susceptibility to seven antifungal compounds. Almost half of the studied cases were from HIV-infected patients, followed by a patient group of individuals with other underlying diseases and immunocompetent individuals. The majority of the isolates were mating type α and serotype A, followed by αD isolates and other minor categories. The most frequently observed genotype was AFLP1, distantly followed by AFLP2 and AFLP3. Microsatellite typing revealed a high genetic diversity among serotype A isolates but a lower diversity within the serotype D set of isolates. One patient was infected by multiple AFLP genotypes. Fluconazole and flucytosine had the highest geometric mean MICs of 2.9 and 3.5 µg/ml, respectively, while amphotericin B (0.24 µg/ml), itraconazole (0.08 µg/ml), voriconazole (0.07 µg/ml), posaconazole (0.06 µg/ml), and isavuconazole (0.03 µg/ml) had much lower geometric mean MICs. One isolate had a high flucytosine MIC (>64 µg/ml), while decreased susceptibility (≥16 µg/ml) for flucytosine and fluconazole was found in 9 and 10 C. neoformans isolates, respectively.