Long-term treatment with nebulized colistin in oncological patients with recurrent respiratory infections

Objectives: To assess the efficacy of long-term treatment with nebulized colistin in reducing the number of respiratory infections, emergency consultations and hospitalizations in oncological patients.MethodsA retrospective, observational, single-centre study including patients with solid or haematologic malignancies, or pulmonary GVHD after HSTC who received treatment with nebulized colistin for at least six-months to prevent recurrent respiratory infections (July 2010 to June 2017).ResultsTwelve patients were included (median age: 54.4, range: 23–85), 7 with solid malignancies and 5 with haematologic malignancies (2 with pulmonary GVHD). Pseudomonas aeruginosa was the most frequent microorganism in sputum cultures (11/12 patients), all strains were susceptible to colistin. There was a statistically significant reduction (p=0.01) in respiratory infections in the six-month period after starting colistin (median: 1, range: 0–4) compared to the six-month period before (median: 4, range: 1–8). There was also a reduction in emergency consultations (precolistin: median: 1.50, range: 0–3; postcolistin: median: 0, range: 0–3) and hospitalizations (precolistin: median: 1.50, range: 0–3; postcolistin: median: 0, range: 0–3) due to respiratory infections. No colistin-resistant strains were identified.ConclusionsLong-term treatment with nebulized colistin may be useful to reduce the number of exacerbations in oncological patients with recurrent respiratory infections. (AU)

Objetivos: Evaluar la eficacia de un tratamiento prolongado con colistina nebulizada para reducir el número de infecciones respiratorias, consultas en Urgencias y hospitalizaciones en pacientes oncológicos.MétodosEstudio retrospectivo, observacional y unicéntrico en pacientes con neoplasias sólidas o hematológicas o EICR pulmonar tras TPH tratados con colistina nebulizada al menos 6 meses para prevenir infecciones respiratorias recurrentes (julio del 2010-junio del 2017).ResultadosSe incluyó a 12 pacientes (edad mediana 54,4, rango: 23-85), 7 con cáncer sólido y 5 con neoplasias hematológicas (2 con EICR pulmonar). El microorganismo aislado más frecuentemente en esputos fue Pseudomonasaeruginosa (11/12 pacientes); todas las cepas fueron colistina-sensibles. Se evidenciaron una reducción estadísticamente significativa (p = 0,01) de las infecciones respiratorias en los 6 meses tras iniciar colistina (mediana: 1, rango: 0-4) comparado con los 6 meses previos (mediana: 4, rango: 1-8), y una reducción del número de visitas a Urgencias (precolistina: mediana: 1,50, rango: 0-3; postcolistina: mediana: 0, rango: 0-3) y hospitalizaciones (precolistina: mediana: 1,50, rango: 0-3; postcolistina: mediana: 0, rango: 0-3) por infección respiratoria. No se detectaron cepas resistentes a colistina.ConclusionesUn tratamiento prolongado con colistina nebulizada puede ser útil para reducir el número de exacerbaciones en pacientes oncológicos con infecciones respiratorias recurrentes. (AU)

An Immunochemical Approach to Detect the Quorum Sensing-Regulated Virulence Factor 2-Heptyl-4-Quinoline N-Oxide (HQNO) Produced by Pseudomonas aeruginosa Clinical Isolates.

Understanding quorum sensing (QS) and its role in the development of pathogenesis may provide new avenues for diagnosing, surveillance, and treatment of infectious diseases. For this purpose, the availability of reliable and efficient analytical diagnostic tools suitable to specifically detect and quantify these essential QS small molecules and QS regulated virulence factors is crucial. Here, we reported the development and evaluation of antibodies and an enzyme-linked immunosorbent assay (ELISA) for HQNO (2-heptyl-4-quinoline N-oxide), a QS product of the PqsR system, which has been found to act as a major virulence factor that interferes with the growth of other microorganisms. Despite the nonimmunogenic character of HQNO, the antibodies produced showed high avidity and the microplate-based ELISA developed could detect HQNO in the low nM range. Hence, a limit of detection (LOD) of 0.60 ± 0.13 nM had been reached in Müeller Hinton (MH) broth, which was below previously reported levels using sophisticated equipment based on liquid chromatography coupled to mass spectrometry. The HQNO profile of release of different Pseudomonas aeruginosa clinical isolates analyzed using this ELISA showed significant differences depending on whether the clinical isolates belonged to patients with acute or chronic infections. These data point to the possibility of using HQNO as a specific biomarker to diagnose P. aeruginosa infections and for patient surveillance. Considering the role of HQNO in inhibiting the growth of coinfecting bacteria, the present ELISA will allow the investigation of these complex bacterial interactions underlying infections. IMPORTANCE Bacteria use quorum sensing (QS) as a communication mechanism that releases small signaling molecules which allow synchronizing a series of activities involved in the pathogenesis, such as the biosynthesis of virulence factors or the regulation of growth of other bacterial species. HQNO is a metabolite of the Pseudomonas aeruginosa-specific QS signaling molecule PQS (Pseudomonas quinolone signal). In this work, the development of highly specific antibodies and an immunochemical diagnostic technology (ELISA) for the detection and quantification of HQNO was reported. The ELISA allowed profiling of the release of HQNO by clinical bacterial isolates, showing its potential value for diagnosing and surveillance of P. aeruginosa infections. Moreover, the antibodies and the ELISA reported here may contribute to the knowledge of other underlying conditions related to the pathology, such as the role of the interactions with other bacteria of a particular microbiota environment.

Long-term treatment with nebulized colistin in oncological patients with recurrent respiratory infections.

OBJECTIVES: To assess the efficacy of long-term treatment with nebulized colistin in reducing the number of respiratory infections, emergency consultations and hospitalizations in oncological patients. METHODS: A retrospective, observational, single-centre study including patients with solid or haematologic malignancies, or pulmonary GVHD after HSTC who received treatment with nebulized colistin for at least six-months to prevent recurrent respiratory infections (July 2010 to June 2017). RESULTS: Twelve patients were included (median age: 54.4, range: 23-85), 7 with solid malignancies and 5 with haematologic malignancies (2 with pulmonary GVHD). Pseudomonas aeruginosa was the most frequent microorganism in sputum cultures (11/12 patients), all strains were susceptible to colistin. There was a statistically significant reduction (p=0.01) in respiratory infections in the six-month period after starting colistin (median: 1, range: 0-4) compared to the six-month period before (median: 4, range: 1-8). There was also a reduction in emergency consultations (precolistin: median: 1.50, range: 0-3; postcolistin: median: 0, range: 0-3) and hospitalizations (precolistin: median: 1.50, range: 0-3; postcolistin: median: 0, range: 0-3) due to respiratory infections. No colistin-resistant strains were identified. CONCLUSIONS: Long-term treatment with nebulized colistin may be useful to reduce the number of exacerbations in oncological patients with recurrent respiratory infections.

Pertactin-Deficient Bordetella pertussis with Unusual Mechanism of Pertactin Disruption, Spain, 1986-2018.

Bordetella pertussis not expressing pertactin has increased in countries using acellular pertussis vaccines (ACV). The deficiency is mostly caused by pertactin gene disruption by IS481. To assess the effect of the transition from whole-cell vaccine to ACV on the emergence of B. pertussis not expressing pertactin in Spain, we studied 342 isolates collected during 1986-2018. We identified 93 pertactin-deficient isolates. All were detected after introduction of ACV and represented 38% of isolates collected during the ACV period; 58.1% belonged to a genetic cluster of isolates carrying the unusual prn::del(-292, 1340) mutation. Pertactin inactivation by IS481 insertion was identified in 23.7% of pertactin-deficient isolates, arising independently multiple times and in different phylogenetic branches. Our findings support the emergence and dissemination of a cluster of B. pertussis with an infrequent mechanism of pertactin disruption in Spain, probably resulting from introduction of ACV.

An Immunochemical Approach to Quantify and Assess the Potential Value of the Pseudomonas Quinolone Signal as a Biomarker of Infection.

Quorum sensing (QS) is a bacterial cell density-based communication system using low molecular weight signals called autoinducers (AIs). Identification and quantification of these molecules could provide valuable information related to the stage of colonization or infection as well as the stage of the disease. With this scenario, we report here for the first time the development of antibodies against the PQS (pseudomonas quinolone signal), the main signaling molecule from the pqs QS system of Pseudomonas aeruginosa, and the development of a microplate-based enzyme-linked immunosorbent assay (ELISA) able of quantifying this molecule in complex biological media in the low nanometer range (LOD, 0.36 ± 0.14 nM in culture broth media). Moreover, the PQS ELISA here reported has been found to be robust and reliable, providing accurate results in culture media. The technique allowed us to follow up the PQS profile of the release of bacterial clinical isolates obtained from patients of different disease status. A clear correlation was found between the PQS immunoreactivity equivalents and the chronic or acute infection conditions, which supports the reported differences on virulence and behavior of these bacterial strains due to their adaptation capability to the host environment. The results obtained point to the potential of the PQS as a biomarker of infection and to the value of the antibodies and the technology developed for improving diagnosis and management of P. aeruginosa infections based on the precise identification of the pathogen, appropriate stratification of the patients according to their disease status, and knowledge of the disease progression.

A new pleosporalean fungus isolated from superficial to deep human clinical specimens.

Human infections by pleosporalean fungi (class Dothideomycetes, phylum Ascomycota) are rarely reported. Because their identification is challenging using morphological characterization, several phylogenetic markers must be sequenced for an accurate identification and taxonomical placement of the isolates. Three isolates of clinical origin were phenotypically characterized, but due to the absence of relevant morphological traits, D1-D2 domains of the 28S nrRNA gene (LSU), the internal transcribed spacer region (ITS) of the nrRNA, and fragments of the RNA polymerase II subunit 2 (rpb2) and translation elongation factor 1-alpha (tef1) genes were sequenced to allow a phylogenetic analysis that would solve their phylogenetic placement. That analysis revealed that these isolates did not match any previously known pleosporalean genera, and they are proposed here as the new fungal genus, Gambiomyces. Unfortunately, the isolates remained sterile, which, consequently, made the morphological description of the reproductive structures impossible. Future studies should try to understand the behaviour of this fungus in nature as well as its characteristics as an opportunistic fungal pathogen. Molecular identification is becoming an essential tool for proper identification of Dothideomycetes of clinical origin. LAY ABSTRACT: We describe a new pleosporalen pathogenic fungus, Gambiomyces profunda, found in superficial to deep samples from a human patient. Because all strains remained sterile, the fungus was finally identified following a phylogenetic analysis by using four different molecular markers.

High-Throughput Immunochemical Method to Assess the 2-Heptyl-4-quinolone Quorum Sensing Molecule as a Potential Biomarker of Pseudomonas aeruginosa Infections.

Bacterial quorum sensing (QS) is being contemplated as a promising target for developing innovative diagnostic and therapeutic strategies. Here we report for the first time the development of antibodies against 2-heptyl-4-quinolone (HHQ), a signaling molecule from the pqs QS system of Pseudomonas aeruginosa, involved in the production of important virulent factors and biofilm formation. The antibodies produced were used to develop an immunochemical diagnostic approach to assess the potential of this molecule as a biomarker of P. aeruginosa infection. The ELISA developed is able to reach a detectability in the low nM range (IC50 = 4.59 ± 0.29 nM and LOD = 0.34 ± 0.13 nM), even in complex biological samples such as Müeller Hinton (MH) culture media. The ELISA developed is robust and reproducible and has been found to be specific to HHQ, with little interference from other related alkylquinolones from the pqs QS system. The ELISA has been used to analyze the HHQ production kinetics of P. aeruginosa clinical isolates grown in MH media, pointing to its potential as a biomarker of infection and at the possibility to use the technology developed to obtain additional information about the disease stage.

Consensus Multilocus Sequence Typing Scheme for Pneumocystis jirovecii.

Pneumocystis jirovecii is an opportunistic human pathogenic fungus causing severe pneumonia mainly in immunocompromised hosts. Multilocus sequence typing (MLST) remains the gold standard for genotyping of this unculturable fungus. However, the lack of a consensus scheme impedes a global comparison, large scale population studies and the development of a global MLST database. To overcome this problem this study compared all genetic regions (19 loci) currently used in 31 different published Pneumocystis MLST schemes. The most diverse/commonly used eight loci, ß-TUB, CYB, DHPS, ITS1, ITS1/2, mt26S and SOD, were further assess for their ability to be successfully amplified and sequenced, and for their discriminatory power. The most successful loci were tested to identify genetically related and unrelated cases. A new consensus MLST scheme consisting of four genetically independent loci: ß-TUB, CYB, mt26S and SOD, is herein proposed for standardised P. jirovecii typing, successfully amplifying low and high fungal burden specimens, showing adequate discriminatory power, and correctly identifying suspected related and unrelated isolates. The new consensus MLST scheme, if accepted, will for the first time provide a powerful tool to investigate outbreak settings and undertake global epidemiological studies shedding light on the spread of this important human fungal pathogen.

Detection of Streptococcus pneumoniae antigen in pleural fluid: usefulness of an immunofluorescence-based lateral flow assay for the diagnosis of pneumococcal pneumonia.

The performance of an immunofluorescence-based Streptococcus pneumoniae antigen detection test in pleural fluid (IF-PF) was evaluated. For proven and possible pneumococcal pneumonias global sensitivity and specificity were 92.6 (95 CI 76.6-97.9) and 80 (95 CI 62.7-90.5), respectively, with no significant differences between children and adults. Global diagnostic accuracy of IF-PF was 86% (74.2-93.7), and a substantial k index of concordance with culture/RT-PCR of 0.716 (0.535-0.896). IF-PF might be useful as a rapid complementary test for the etiologic diagnosis of pneumococcal pneumonia.

Timely Diagnosis of Histoplasmosis in Non-endemic Countries: A Laboratory Challenge.

Human histoplasmosis is a fungal infection caused by the inhalation of microconidia of the thermally dimorphic fungi Histoplasma capsulatum. Autochthonous cases of histoplasmosis have been diagnosed in almost every country, but it is considered an endemic infection in specific areas of the world. Many of them are popular travel destinations or the source of migratory movements. Thus, the vast majority of the registered cases in non-endemic countries are imported. They correspond to people having been exposed to the fungus in endemic locations as immigrants, expatriates, transient workers or tourists, with reported cases also associated to organ donation. Misdiagnosis and delays in initiation of treatment are not uncommon in cases of imported histoplasmosis. They are associated to high fatality-rates specially in patients with compromised cellular immunity in which progressive disseminated forms develop. The diagnosis of this infection in non-endemic countries is hampered by the lack of clinical suspicion and a dearth of available diagnostic tools adequate to offer rapid and accurate results. Non-culture-based assays such as nucleic-acid amplification tests present as a suitable alternative in this situation, offering improved sensitivity and specificity, shortened turnaround time, and increased biosafety by avoiding culture manipulation. In non-endemic regions, molecular techniques are being used mainly in laboratories from countries that have registered an increase in the incidence of imported cases. However, the number of published techniques is limited and lack consensus. Efforts are currently under way to standardize nucleic acid amplification-based techniques for its implementation in areas registering a rising number of imported cases.

Taking a look on fungi in cystic fibrosis: more questions than answers / Una mirada a los hongos en la fibrosis quística: más preguntas que respuestas

Cystic fibrosis (CF) is one of the most frequent recessive inherited diseases in western countries. Advances in medical care have led to a substantial increase in the life expectancy of CF patients. Survival beyond adolescence has permitted to see fungi not only as late colonizers, but also as potential pathogens responsible of allergic reactions and chronic infections related to lung function deterioration. The role of fungi, nevertheless, has been overlooked until recently. As a result, a number of questions on their epidemiology, clinical significance, or diagnosis, among others, remain unanswered. Besides more in depth studies about the extent of the deleterious effect of fungi on the CF host, new technologies may provide the key to understand its pathogenic role, its interaction with other microbial components of the respiratory microbiota, and should pave the way to define subsets of patients at risk who would benefit from specific therapy. This review is intended to provide a quick overview on what we know about the presence of fungi in the CF airway and its repercussion in the host, and to point out some of the many knowledge gaps needed to understand and advance in the management of fungi in the airway of CF subjects

La fibrosis quística (FQ) es una de las enfermedades genéticas recesivas más frecuentes en los países occidentales. Los avances médicos han supuesto un notable incremento de la esperanza de vida de estos pacientes. La supervivencia más allá de la adolescencia ha revelado la presencia de los hongos no solo como colonizadores tardíos sino como patógenos potenciales, responsables de reacciones alérgicas e infecciones crónicas relacionadas con el deterioro de la función pulmonar. El papel de los hongos en los pacientes con FQ, no obstante, ha sido pasado por alto hasta fechas recientes. Como consecuencia, quedan por responder numerosas cuestiones sobre su epidemiología, significado clínico o diagnóstico, entre otras. Son precisos estudios en profundidad sobre el verdadero alcance del efecto deletéreo de los hongos en el paciente con FQ. Las nuevas tecnologías pueden resultar claves para entender su papel patogénico, su interacción con otros componentes de la microbiota respiratoria, y para la identificación de los subgrupos de pacientes de más riesgo que se beneficiarían particularmente de las terapias dirigidas. Esta revisión pretende proporcionar una rápida mirada a lo que sabemos actualmente sobre la presencia de los hongos en las vías respiratorias de los pacientes FQ y su repercusión en el huésped, así como señalar las múltiples lagunas de conocimiento que es necesario rellenar para entender y avanzar en el manejo de la enfermedad fúngica respiratoria de los pacientes con FQ

Taking a look on fungi in cystic fibrosis: More questions than answers.

Cystic fibrosis (CF) is one of the most frequent recessive inherited diseases in western countries. Advances in medical care have led to a substantial increase in the life expectancy of CF patients. Survival beyond adolescence has permitted to see fungi not only as late colonizers, but also as potential pathogens responsible of allergic reactions and chronic infections related to lung function deterioration. The role of fungi, nevertheless, has been overlooked until recently. As a result, a number of questions on their epidemiology, clinical significance, or diagnosis, among others, remain unanswered. Besides more in depth studies about the extent of the deleterious effect of fungi on the CF host, new technologies may provide the key to understand its pathogenic role, its interaction with other microbial components of the respiratory microbiota, and should pave the way to define subsets of patients at risk who would benefit from specific therapy. This review is intended to provide a quick overview on what we know about the presence of fungi in the CF airway and its repercussion in the host, and to point out some of the many knowledge gaps needed to understand and advance in the management of fungi in the airway of CF subjects.

Population dynamics and antigenic drift of Bordetella pertussis following whole cell vaccine replacement, Barcelona, Spain, 1986-2015.

Among the factors associated with the resurgence of whooping cough, special emphasis has been given to pathogen adaptation after the introduction of the acellular vaccine (ACV). To assess the impact of the vaccine transition strategy from whole-cell vaccine (WCV) to ACV on population dynamics of Bordetella pertussis in Barcelona (Spain), we studied 339 isolates collected from 1986 to 2015 by PFGE and multi-locus variable-number tandem repeat analysis (MLVA). Additionally, allelic variants for the pertussis toxin and its promoter, pertactin, type 3 fimbriae and fimbrial serotyping were assessed to determine its antigenic drift. A shift was observed in the B. pertussis population as well as in its antigenic profile concurrently with the introduction of ACV in Barcelona. Four out of the five most prevalent PFGE profiles were replaced by new profiles following the ACV introduction. MLVA type 27 was the dominant genotype, and its frequency increased from 25% to 79.3% after WCV replacement. Antigen typing demonstrated the emergence of prn2, ptxP3, fim3-2 and a shift from the fimbriae 3 to the fimbriae 2 serotypes after the ACV introduction. Our findings support the presence of population and antigenic dynamic changes in B. pertussis likely driven by the introduction of ACV.

Molecular Diagnosis of Pneumocystis jirovecii Pneumonia by Use of Oral Wash Samples in Immunocompromised Patients: Usefulness and Importance of the DNA Target.

Pneumocystis jirovecii pneumonia (PJP) is an important cause of pneumonia in the HIV-negative immunocompromised population, for whom the fungal load is low, the differential diagnosis is difficult, and a bronchoalveolar lavage (BAL) sample is often not readily available. Molecular techniques have improved the microbiological diagnosis in this scenario. The usefulness of two real-time PCR techniques targeting nuclear single-copy and mitochondrial multicopy genes, respectively, applied to oral wash specimens (OW) for PJP diagnosis was assessed, and its accuracy was compared to a BAL fluid-based diagnosis. Immunocompromised patients having PJP in the differential diagnosis of an acute respiratory episode, and from whom OW and BAL or lung biopsy specimens were obtained ≤48 h apart, were retrospectively included. PCRs targeting the dihydropteroate synthase gene (DHPS) and the mitochondrial small-subunit (mtSSU) rRNA gene were performed in paired OW-BAL specimens. Thirty-six patients were included (88.6% HIV negative). Fifteen patients (41.7%) were classified as PJP, and a further 8 were considered P. jirovecii colonized. Quantification of DHPS and mtSSU in BAL fluid showed an accuracy of 96.9% and 93.0%, respectively, for PJP diagnosis, whereas a qualitative approach performed better when applied to OW (accuracy, 91.7%) irrespective of the PCR target studied (kappa = 1). Qualitative molecular diagnosis applied to OW showed an excellent performance for PJP diagnosis regardless of the target studied, being easier to interpret than the quantitative approach needed for BAL fluid.

Phaeohyphomycosis caused by Medicopsis romeroi in solid organ transplant recipients: Report of two cases and comprehensive review of the literature.

Medicopsis romeroi is a melanized coelomycetous fungus, mainly found in tropical and subtropical regions and an uncommon cause of infection in solid organ transplant (SOT) recipients. We describe two cases of SOT recipients diagnosed with phaeohyphomycosis due to M romeroi and provide a comprehensive literature review. These infections should be considered in patients native to tropical countries with a localized skin and soft tissue infection. Sequencing is needed for accurate identification of uncommon melanized fungi. Surgical treatment is recommended to cure the infection and co-adjunctive oral antifungals should be considered.

Cutaneous infections by dematiaceous opportunistic fungi: Diagnosis and management in 11 solid organ transplant recipients.

BACKGROUND: The incidence of cutaneous infections by dematiaceous fungi is rising in our environment due to the high number of solid organ transplant recipients (SOTR). OBJECTIVE: To review our experience in the management of cutaneous phaeohyphomycoses in a Spanish reference centre for dermatological care of SOTR. METHODS: Retrospective clinical, histopathological and microbiological review of all SOTR diagnosed of a phaeohyphomycosis in a 7-year period. RESULTS: Eleven SOTR were identified (8 lung and 3 kidney). The lesions were solitary in six patients and multiple in five, affecting mostly the lower extremities. Early lesions showed epidermal hyperplasia and a diffuse dermal suppurative granulomatous infiltrate that was progressively substituted by fibrosis when the lesions were treated. Septated fungal structures with refractile walls were identified. DNA sequencing confirmed the presence of Alternaria spp (8 cases), Cladosporium cladosporioides, Microsphaeropsis arundinis and Exophiala oligosperma. Three patients with single lesions were treated with surgery, while the other 8 required long-term antifungal therapy, including itraconazole, voriconazole and/or terbinafine, combined with surgery and reduction in tacrolimus doses. CONCLUSION: A clinical, histopathological and microbiological correlation is essential to corroborate this diagnosis. Solitary lesions are easily treated with surgery, but larger or multiple lesions may require long medical treatments combined with surgery and modification of immunosuppressive medication. The list of dematiaceous fungi implicated in cutaneous infections is expanding, in line with the availability of more sophisticated identification methods and the increasing number of immunosuppressed patients.

Immune Parameters for Diagnosis and Treatment Monitoring in Invasive Mold Infection.

Infections caused by invasive molds, including Aspergillus spp., can be difficult to diagnose and remain associated with high morbidity and mortality. Thus, early diagnosis and targeted systemic antifungal treatment remains the most important predictive factor for a successful outcome in immunocompromised individuals with invasive mold infections. Diagnosis remains difficult due to low sensitivities of diagnostic tests including culture and other mycological tests for mold pathogens, particularly in patients on mold-active antifungal prophylaxis. As a result, antifungal treatment is rarely targeted and reliable markers for treatment monitoring and outcome prediction are missing. Thus, there is a need for improved markers to diagnose invasive mold infections, monitor response to treatment, and assist in determining when antifungal therapy should be escalated, switched, or can be stopped. This review focuses on the role of immunologic markers and specifically cytokines in diagnosis and treatment monitoring of invasive mold infections.

Molecular Identification and Susceptibility Testing of Molds Isolated in a Prospective Surveillance of Triazole Resistance in Spain (FILPOP2 Study).

Antifungal resistance is increasing by the emergence of intrinsically resistant species and by the development of secondary resistance in susceptible species. A previous study performed in Spain revealed levels of azole resistance in molds of between 10 and 12.7%, but secondary resistance in Aspergillus fumigatus was not detected. We used itraconazole (ITZ)-supplemented medium to select resistant strains. A total of 500 plates supplemented with 2 mg/liter of ITZ were sent to 10 Spanish tertiary hospitals, and molecular identification and antifungal susceptibility testing were performed. In addition, the cyp51A gene in those A. fumigatus strains showing azole resistance was sequenced. A total of 493 isolates were included in the study. Sixteen strains were isolated from patients with an infection classified as proven, 104 were isolated from patients with an infection classified as probable, and 373 were isolated from patients with an infection classified as colonization. Aspergillus was the most frequent genus isolated, at 80.3%, followed by Scedosporium-Lomentospora (7.9%), Penicillium-Talaromyces (4.5%), Fusarium (2.6%), and the order Mucorales (1%). Antifungal resistance was detected in Scedosporium-Lomentospora species, Fusarium, Talaromyces, and Mucorales Three strains of A. fumigatus sensu stricto were resistant to azoles; two of them harbored the TR34+L98H mechanism of resistance, and the other one had no mutations in cyp51A The level of azole resistance in A. fumigatus remains low, but cryptic species represent over 10% of the isolates and have a broader but overall higher range of antifungal resistance.

Azole-Resistance in Aspergillus terreus and Related Species: An Emerging Problem or a Rare Phenomenon?

Objectives: Invasive mold infections associated with Aspergillus species are a significant cause of mortality in immunocompromised patients. The most frequently occurring aetiological pathogens are members of the Aspergillus section Fumigati followed by members of the section Terrei. The frequency of Aspergillus terreus and related (cryptic) species in clinical specimens, as well as the percentage of azole-resistant strains remains to be studied. Methods: A global set (n = 498) of A. terreus and phenotypically related isolates was molecularly identified (beta-tubulin), tested for antifungal susceptibility against posaconazole, voriconazole, and itraconazole, and resistant phenotypes were correlated with point mutations in the cyp51A gene. Results: The majority of isolates was identified as A. terreus (86.8%), followed by A. citrinoterreus (8.4%), A. hortai (2.6%), A. alabamensis (1.6%), A. neoafricanus (0.2%), and A. floccosus (0.2%). One isolate failed to match a known Aspergillus sp., but was found most closely related to A. alabamensis. According to EUCAST clinical breakpoints azole resistance was detected in 5.4% of all tested isolates, 6.2% of A. terreus sensu stricto (s.s.) were posaconazole-resistant. Posaconazole resistance differed geographically and ranged from 0% in the Czech Republic, Greece, and Turkey to 13.7% in Germany. In contrast, azole resistance among cryptic species was rare 2 out of 66 isolates and was observed only in one A. citrinoterreus and one A. alabamensis isolate. The most affected amino acid position of the Cyp51A gene correlating with the posaconazole resistant phenotype was M217, which was found in the variation M217T and M217V. Conclusions:Aspergillus terreus was most prevalent, followed by A. citrinoterreus. Posaconazole was the most potent drug against A. terreus, but 5.4% of A. terreus sensu stricto showed resistance against this azole. In Austria, Germany, and the United Kingdom posaconazole-resistance in all A. terreus isolates was higher than 10%, resistance against voriconazole was rare and absent for itraconazole.