A multicentre external quality assessment: A first step to standardise PCR protocols for the diagnosis of histoplasmosis and coccidioidomycosis.

BACKGROUND: In-house real-time PCR (qPCR) is increasingly used to diagnose the so-called endemic mycoses as commercial assays are not widely available. OBJECTIVES: To compare the performance of different molecular diagnostic assays for detecting Histoplasma capsulatum and Coccidioides spp. in five European reference laboratories. METHODS: Two blinded external quality assessment (EQA) panels were sent to each laboratory that performed the analysis with their in-house assays. Both panels included a range of concentrations of H. capsulatum (n = 7) and Coccidioides spp. (n = 6), negative control and DNA from other fungi. Four laboratories used specific qPCRs, and one laboratory a broad-range fungal conventional PCR (cPCR) and a specific cPCR for H. capsulatum with subsequent sequencing. RESULTS: qPCR assays were the most sensitive for the detection of H. capsulatum DNA. The lowest amount of H. capsulatum DNA detected was 1-4 fg, 0.1 pg and 10 pg for qPCRs, specific cPCR and broad-range cPCR, respectively. False positive results occurred with high concentrations of Blastomyces dermatitidis DNA in two laboratories and with Emergomyces spp. in one laboratory. For the Coccidioides panel, the lowest amount of DNA detected was 1-16 fg by qPCRs and 10 pg with the broad-range cPCR. One laboratory reported a false positive result by qPCR with high load of Uncinocarpus DNA. CONCLUSION: All five laboratories were able to correctly detect H. capsulatum and Coccidioides spp. DNA and qPCRs had a better performance than specific cPCR and broad-range cPCR. EQAs may help standardise in-house molecular tests for the so-called endemic mycoses improving patient management.

Anti-Aspergillus fumigatus IgG in patients with bronchiectasis and its relationship with clinical outcome.

Aspergillosis is a mycosis, most commonly affecting the airways. This mycosis can worsen the clinical condition of patients with concurrent lung diseases. We assayed for the presence of serum anti-A. fumigatus IgG in bronchiectasis patients from a tertiary hospital in south Brazil and evaluated the relationship with clinical outcome. Thirty-one patients with bronchiectasis, without cystic fibrosis, were included. Clinical and epidemiological data were collected from all participants. Positive serological tests were detected in 13% (4/31) of the patients. The mortality rate for the year following the assay was, in the seropositive group, 75% (3/4), whereas in the seronegative group, 15% (4/27). An illustrative case is also shown and discussed. Our study highlights the diagnostic challenge and the possible impact of Aspergillus infection on these patients, indicating the necessity of more and larger investigations in the field.

Algorithm to assess the presence of Aspergillus fumigatus resistant strains: The case of Norwegian sawmills.

Association between selection pressure caused by the use of azole fungicides in sawmills and the development of fungal resistance has been described. The aim of this study was to implement an algorithm to assess the presence of Aspergillus section Fumigati resistant strains in sawmills.Eighty-six full-shift inhalable dust samples were collected from eleven industrial sawmills in Norway. Different culture media were used and molecular identification to species level in Aspergillus section Fumigati was done by calmodulin sequencing and TR34/L98H and TR46/Y121F/T289A mutations were screened by real-time PCR assay and confirmed by cyp51A sequencing. Six Fumigati isolates were identified as A. fumigatus sensu stricto and two of these grew on azole-supplemented media and were further analyzed by real-time PCR. One was confirmed to be a TR34/L98H mutant.The obtained results reinforce the need to assess the presence of A. fumigatus sensu stricto resistant isolates at other workplaces with fungicide pressure.

Molecular epidemiology of aspergillosis in Magellanic penguins and susceptibility patterns of clinical isolates.

Aspergillus section Fumigati is reported in up to 99% of aspergillosis cases in penguins. So far, no data regarding molecular epidemiology and azole resistance are available for A. fumigatus isolates collected from Magellanic penguins. The aim of this work was to perform molecular identification of Aspergillus section Fumigati at species level, to genotype those isolates using microsatellite markers, to evaluate the in vitro susceptibility patterns of A. fumigatus sensu stricto, and to characterize the cyp51A gene in clinical A. fumigatus strains isolated from Magellanic penguins with proven aspergillosis. All 34 isolates included in the study were identified as A. fumigatus sensu stricto. Analyzing the genetic diversity of the isolates of A. fumigatus sensu stricto, we identified two possible outbreaks in the rehabilitation center and we also observed the maintenance of clonal strains through the years. One A. fumigatus sensu stricto isolate was resistant to posaconazole, but the mutations found in the cyp51A gene of this isolate have not been described as conferring phenotypic resistance, suggesting that other mechanisms of resistance could be involved in the resistance of this isolate. With this study, we were able to understand the molecular diversity of Aspergillus fumigatus isolates collected from Magellanic penguins, to characterize them and to associate them with the described global population of Aspergillus fumigatus.

A. fumigatus sensu stricto is of great importance in penguins' aspergillosis. We could identify two outbreaks in the rehabilitation center and the maintenance of clonal strains through the years. Regarding antifungal prophylaxis, it may proceed, but preferably with surveillance for azole resistance.

Aspergillus collected in specific indoor settings: their molecular identification and susceptibility pattern.

Exposure to Aspergillus conidia is an increased risk factor for the development of respiratory symptoms. The emergence of azole resistance in Aspergillus fumigatus is a major concern for the scientific community. The aim of this study was to perform the molecular identification of Aspergillus species collected from different occupational and non-occupational indoor settings and to study the azole susceptibility profile of the collected Fumigati isolates. The selected Aspergillus isolates were identified as belonging to the sections Fumigati, Nigri Versicolores, Terrei, Clavati and Nidulantes. All the Aspergillus fumigatus were screened for azole resistance using an agar media supplemented with itraconazole, voriconazole and posaconazole. None of the tested isolates showed resistance to those azoles. Knowledge of Aspergillus epidemiology in specific indoor environments allows a better risk characterization regarding Aspergillus burden. This study allowed the analysis of the molecular epidemiology and the determination of the susceptibility pattern of Aspergillus section Fumigati found in the studied indoor settings.

Invasive pulmonary aspergillosis treatment duration in haematology patients in Europe: An EFISG, IDWP-EBMT, EORTC-IDG and SEIFEM survey.

Invasive pulmonary aspergillosis (IPA) optimal duration of antifungal treatment is not known. In a joint effort, four international scientific societies/groups performed a survey to capture current practices in European haematology centres regarding management of IPA. We conducted a cross-sectional internet-based questionnaire survey in 2017 to assess practices in sixteen European countries concerning IPA management in haematology patients including tools to evaluate treatment response, duration and discontinuation. The following four groups/societies were involved in the project: European Society of Clinical Microbiology and Infectious Diseases (ESCMID) Fungal Infection Study Group (EFISG), Infectious Diseases Working Party-European Society for Blood and Bone Marrow Transplantation (IDWP-EBMT), European Organisation for Research and Treatment-Infectious Disease group (EORTC-IDG) and Sorveglianza Epidemiologica Infezioni nelle Emopatie (SEIFEM). A total of 112 physicians from 14/16 countries answered the survey. Galactomannan antigen was available in serum and bronchoalveolar lavage in most centres (106/112 [95%] and 97/112 [87%], respectively), quantitative Aspergillus PCR in 27/112 (24%) centres, ß-D-glucan in 24/112 (21%) and positron emission tomography in 50/112 (45%). Treatment duration differed between haematological malignancies, with a median duration of 6 weeks [IQR 3-12] for patients with AML, 11 [4-12] for patients with allogenic stem cell transplantation and GvHD and 6 [3-12] for patients with lymphoproliferative disease. Treatment duration significantly differed according to country. Essential IPA biomarkers are not available in all European countries, and treatment duration is highly variable according to country. It will be important to provide guidelines to help with IPA treatment cessation with algorithms according to biomarker availability.

Molecular identification of clinical and environmental avian Aspergillus isolates.

Aspergillosis causes high morbidity and mortality in avian species. The main goal of this study was to use molecular techniques to identify Aspergillus species collected from different avian species with aspergillosis. A subsample of those isolates was also screened for resistance to itraconazole. Over a 2-year period, clinical samples were recovered from 44 birds with clinical signs of the disease, clinical pathology results suspicious of aspergillosis, or from birds that died from Aspergillus spp. infection. Environmental sampling was also performed in seabird rehabilitation centers and natural seabird environments. Seventy-seven isolates (43 clinical and 34 environmental) were identified as Aspergillus fumigatus sensu stricto. No cryptic species from the Fumigati section were detected. Two environmental isolates were identified as Aspergillus nidulans var. dentatus and Aspergillus spinulosporus. None of the Aspergillus isolates tested were resistant to itraconazole. Our study emphasizes the dominant association of Aspergillus fumigatus sensu stricto in avian mycoses and shows the lack of itraconazole resistance in the studied isolates.

The role of occupational Aspergillus exposure in the development of diseases.

Aspergillus spp. have a high nutritional versatility and good growth on a large variety of construction materials. They also colonize soil or food, but decaying vegetation is their primary ecological niche. Therefore, exposure to fungi may occur at home, during hospitalization, during specific leisure activities, or at the workplace. The development of Aspergillus infections depends on the interplay between host susceptibility and the organism. Environments with high counts of fungal elements (conidia, hyphal fragments and others), high levels of bioarerosols, and elevated concentrations of mycotoxins or other volatile organic compounds should be considered as potential hazards, since they may present a risk to the exposed person. Rural tasks as well as work related to wood and food industries, poultries, swineries, waste handling plants, and other occupational environments involving contaminated organic material are among the ones posing higher respiratory risks to the workers. This paper presents a review of several studies related to occupational and indoor exposure to Aspergillus, potential health effects related to that exposure, and associated exposure assessment procedures.

Fungal burden exposure assessment in podiatry clinics from Ireland.

Fungi are amongst the bioaerosols of most importance, as indicated by the growing interest in this field of research. The aim was to characterize the exposure to fungal burden in podiatry clinics using culture-based and molecular methods. METHODS: Airborne fungi were collected using an impaction air sampler and surface samples were also performed. Fourteen air samples were collected for direct detection of fungal DNA from filamentous fungi and dermatophytes. Overall, 63.6 % of the evening samples and 46 % of the morning samples surpassed the threshold values (150 CFU/m3). Molecular detection, by real time PCR, of the target fungal species/strains (Aspergillus and Stachybotrys species) was negative for all samples collected. Trichophyton rubrum was detected by PCR analysis in one DNA sample collected on day six. Results suggest the use of both culture-based and molecular methodologies are desirable for a complete evaluation of fungal burden in this particular health care setting.

Susceptibility of Candida albicans from Cystic Fibrosis Patients.

Candida albicans is a common microbe, colonizer and potential pathogen found in respiratory cultures of cystic fibrosis (CF) patients. Because of possible development of resistance in patient isolates resulting from residence in the abnormal milieu of CF patient airways, or from exposure to antifungals, and considering the possibility of patient-to-patient spread of microbes and reports of elevated resistance to other fungal pathogens, it was important to assay the susceptibility of isolates of Candida and compare that profile to isolates from the community. In our center, and unlike another fungal pathogen, no increase in resistance of Candida isolates of the CF cohort was found.

Antifungal susceptibility of 175 Aspergillus isolates from various clinical and environmental sources.

Some environmental Aspergillus spp. isolates have been described as resistant to antifungals, potentially causing an emerging medical problem. In the present work, the antifungal susceptibility profile of 41 clinical and 134 environmental isolates of Aspergillus was determined using the CLSI microdilution method. The aim of this study was to compare environmental and clinical isolates with respect to their susceptibility, and assess the potential implications for therapy of isolates encountered in different environments. To our knowledge, this is the first report comparing antifungal susceptibility profiles of Aspergillus collected from different environmental sources (poultries, swineries, beach sand, and hospital environment). Significant differences were found in the distribution of the different species sections for the different sources. Significant differences were also found in the susceptibility profile of the different Aspergillus sections recovered from the various sources. Clear differences were found between the susceptibility of clinical and environmental isolates for caspofungin, amphotericin B and posaconazole, with clinical isolates showing overall greater susceptibility, except for caspofungin. In comparison to clinical isolates, hospital environmental isolates showed significantly less susceptibility to amphotericin B and posaconazole. These data indicate that species section identity and the site from which the isolate was recovered influence the antifungal susceptibility profile, which may affect initial antifungal choices.

Molecular screening of 246 Portuguese Aspergillus isolates among different clinical and environmental sources.

Clinical and environmental samples from Portugal were screened for the presence of Aspergillus and the distributions of the species complexes were determined in order to understand how their distributions differ based on their source. Fifty-seven Aspergillus isolates from clinical samples were collected from 10 health institutions. Six species complexes were detected by internal transcribed spacer sequencing; Fumigati, Flavi, and Nigri were found most frequently (50.9%, 21.0%, and 15.8%, respectively). ß-tubulin and calmodulin sequencing resulted in seven cryptic species (A. awamorii, A. brasiliensis, A. fructus, A. lentulus, A. sydowii, A. tubingensis, Emericella echinulata) being identified among the 57 isolates. Thirty-nine isolates of Aspergillus were recovered from beach sand and poultry farms, 31 from swine farms, and 80 from hospital environments, for a total 189 isolates. Eleven species complexes were found in these 189 isolates, and those belonging to the Versicolores species complex were found most frequently (23.8%). There was a significant association between the different environmental sources and distribution of the species complexes; the hospital environment had greater variability of species complexes than other environmental locations. A high prevalence of cryptic species within the Circumdati complex was detected in several environments; from the isolates analyzed, at least four cryptic species were identified, most of them growing at 37ºC. Because Aspergillus species complexes have different susceptibilities to antifungals, knowing the species-complex epidemiology for each setting, as well as the identification of cryptic species among the collected clinical isolates, is important. This may allow preventive and corrective measures to be taken, which may result in decreased exposure to those organisms and a better prognosis.

Occupational exposure to aflatoxin B1 in swine production and possible contamination sources.

Although the adverse health consequences of ingestion of food contaminated with aflatoxin B1 (AFB1) are known, relatively few studies are available on the adverse effects of exposure in occupational settings. Taking this into consideration, our study was developed aiming to elucidate the possible effects of occupational exposure to AFB1 in Portuguese swine production facilities using a specific biomarker to assess exposure to AFB1. In total, 28 workers participated in this study, providing blood samples, and a control group (n = 30) was composed of subjects without any type of agricultural activity. Fungal contamination was also studied by conventional methods through air, surfaces, and new and used floor coverage. Twenty-one workers (75%) showed detectable levels of AFB1 with values ranging from <1 ng/ml to 8.94 ng/ml and with a mean value of 1.91 ± 1.68 ng/ml. In the control group, the AFB1 values were all below 1 ng/ml. Twelve different Aspergillus species were identified. Aspergillus versicolor presented the highest airborne spore counts (3210 CFU/m3) and was also detected in higher values in surfaces (>300 CFU/cm2). Data indicate that exposure to AFB1 occurs in swine barns, and this site serves as a contamination source in an occupational setting.

Comparison of Multi-locus Genotypes Detected in Aspergillus fumigatus Isolated from COVID Associated Pulmonary Aspergillosis (CAPA) and from Other Clinical and Environmental Sources.

BACKGROUND: Aspergillus fumigatus is a saprophytic fungus, ubiquitous in the environment and responsible for causing infections, some of them severe invasive infections. The high morbidity and mortality, together with the increasing burden of triazole-resistant isolates and the emergence of new risk groups, namely COVID-19 patients, have raised a crescent awareness of the need to better comprehend the dynamics of this fungus. The understanding of the epidemiology of this fungus, especially of CAPA isolates, allows a better understanding of the interactions of the fungus in the environment and the human body. METHODS: In the present study, the M3 markers of the STRAf assay were used as a robust typing technique to understand the connection between CAPA isolates and isolates from different sources (environmental and clinical-human and animal). RESULTS: Of 100 viable isolates that were analyzed, 85 genotypes were found, 77 of which were unique. Some isolates from different sources presented the same genotype. Microsatellite genotypes obtained from A. fumigatus isolates from COVID+ patients were all unique, not being found in any other isolates of the present study or even in other isolates deposited in a worldwide database; these same isolates were heterogeneously distributed among the other isolates. CONCLUSIONS: Isolates from CAPA patients revealed high heterogeneity of multi-locus genotypes. A genotype more commonly associated with COVID-19 infections does not appear to exist.

Effectiveness of school-based sleep promotion programs for adolescents: a systematic review protocol.

OBJECTIVE: This systematic review will assess the effectiveness of school-based sleep promotion programs on sleep knowledge, hygiene, quality, behavior, and efficiency in adolescents. INTRODUCTION: Adolescents need to sleep between 8 and 10 hours per night; however, most sleep fewer hours due to peer and school pressure, stress, and excessive use of electronic devices at night. Sleep deprivation can lead to cardiovascular problems, issues with stress management, and suicidal ideation. Schools are an ideal space to implement intervention programs, providing the opportunity to reach a large number of adolescents. Considering the negative impacts of poor sleep, it is essential to assess the effectiveness of school-based sleep promotion programs on sleep knowledge, hygiene, quality, behavior, and efficiency. INCLUSION CRITERIA: Studies with adolescents (10-19 years old) exposed to school-based sleep promotion programs will be included. Studies in English, Spanish, and Portuguese will be considered, with no time limits. METHODS: Nine databases, CINAHL, Cochrane Central Register of Controlled Trials, ERIC (all via EBSCOhost), MEDLINE (PubMed), LILACS, SciELO, Psychology and Behavioral Sciences Collection, Web of Science Core Collection, and Scopus, will be searched from inception to identify relevant studies. Two independent reviewers will perform the study selection, critical appraisal using JBI tools, and data extraction. Studies will, where possible, be pooled in meta-analysis. Heterogeneity will be assessed using the standard χ 2 and I2 tests. Statistical analyses will be performed using the random effects model. If fewer than 5 studies are included, the fixed effects model will be used. The Grading of Recommendations, Assessment, Development and Evaluation (GRADE) approach will be used to grade the certainty of evidence and a Summary of Findings will be created. REVIEW REGISTRATION: PROSPERO CRD42023374838.

Strategies for Monitoring Microbial Life in Beach Sand for Protection of Public Health.

The 2021 revised guidelines of the World Health Organization recommend monitoring the quality of sand in addition to water at recreational beaches. This review provides background information about the types of beaches, the characteristics of sand, and the microbiological parameters that should be measured. Analytical approaches are described for quantifying fungi and fecal indicator bacteria from beach sand. The review addresses strategies to assess beach sand quality, monitoring approaches, sand remediation, and the proposed way forward for beach sand monitoring programs. In the proposed way forward, recommendations are provided for acceptable levels of fungi given their distribution in the environment. Additional recommendations include evaluating FIB distributions at beaches globally to assess acceptable ranges of FIB levels, similar to those proposed for fungi.

Candida auris in Intensive Care Setting: The First Case Reported in Portugal.

Candida auris is an opportunistic human pathogen that has rapidly spread to multiple countries and continents and has been associated with a high number of nosocomial outbreaks. Herein, we report the first case of C. auris in Portugal, which was associated with a patient transferred from Angola to an ICU in Portugal for liver transplantation after a SARS-CoV-2 infection. C. auris was isolated during the course of bronchoalveolar lavage, and it was subjected to antifungal susceptibility testing and whole-genome sequence analysis. This isolate presents low susceptibility to azoles and belongs to the genetic clade III with a phylogenetic placement close to African isolates. Although clade III has already been reported in Europe, taking into account the patient's clinical history, we cannot discard the possibility that the patient's colonization/infection occurred in Angola, prior to admission in the Portuguese hospital. Considering that C. auris is a fungal pathogen referenced by WHO as a critical priority, this case reinforces the need for continuous surveillance in a hospital setting.

Novel Clinical and Laboratorial Challenges in Aspergillosis.

In recent years, research in the areas of Aspergillus and aspergillosis has continued to advance rapidly, including advancements in genomics, immunological studies, clinical areas, and diagnostic areas. Recently, new risk groups for the development of aspergillosis have emerged-patients with influenza- or COVID-19-ssociated pulmonary aspergillosis. The rise and spread of antifungal resistances have also become a clinical concern in some geographic areas and have drawn the attention of clinicians due to difficulties in treating these infections. In this paper, a snapshot of these issues is presented, emphasizing these novel clinical and laboratorial challenges in the aspergillosis field and focusing on their actual relevance.

Invasive and Subcutaneous Infections Caused by Filamentous Fungi: Report from a Portuguese Multicentric Surveillance Program.

Invasive fungal infections (IFI) have significantly increased over the past years due to advances in medical care for the at-risk immunocompromised population. IFI are often difficult to diagnose and manage, and can be associated with substantial morbidity and mortality. This study aims to contribute to understanding the etiology of invasive and subcutaneous fungal infections, their associated risk factors, and to perceive the outcome of patients who developed invasive disease, raising awareness of these infections at a local level but also in a global context. A laboratory surveillance approach was conducted over a seven-year period and included: (i) cases of invasive and subcutaneous fungal infections caused by filamentous/dimorphic fungi, confirmed by either microscopy or positive culture from sterile samples, (ii) cases diagnosed as probable IFI according to the criteria established by EORTC/MSG when duly substantiated. Fourteen Portuguese laboratories were enrolled. Cases included in this study were classified according to the new consensus definitions of invasive fungal diseases (IFD) published in 2020 as follows: proven IFI (N = 31), subcutaneous fungal infection (N = 23). Those proven deep fungal infections (N = 54) totalized 71.1% of the total cases, whereas 28.9% were classified as probable IFI (N = 22). It was possible to identify the etiological fungal agent in 73 cases (96%). Aspergillus was the most frequent genera detected, but endemic dimorphic fungi represented 14.47% (N = 11) of the total cases. Despite the small number of cases, a high diversity of species were involved in deep fungal infections. This fact has implications for clinical and laboratory diagnosis, and on the therapeutic management of these infections, since different species, even within the same genus, can present diverse patterns of susceptibility to antifungals.