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.

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.

Trends on Aspergillus Epidemiology-Perspectives from a National Reference Laboratory Surveillance Program.

Identification of Aspergillus to species level is important since sibling species may display variable susceptibilities to multiple antifungal drugs and also because correct identification contributes to improve the knowledge of epidemiological studies. Two retrospective laboratory studies were conducted on Aspergillus surveillance at the Portuguese National Mycology Reference Laboratory. The first, covering the period 2017-2018, aimed to study the molecular epidemiology of 256 Aspergillus isolates obtained from patients with respiratory, subcutaneous, or systemic infections and from environmental samples. The second, using our entire collection of clinical and environmental A. fumigatus isolates (N = 337), collected between 2012 and 2019, aimed to determine the frequency of azole-resistant A. fumigatus isolates. Aspergillus fumigatus sensu stricto was the most frequent species in both clinical and environmental samples. Overall, and considering all Aspergillus sections identified, a high frequency of cryptic species was detected, based on beta-tubulin or calmodulin sequencing (37% in clinical and 51% in environmental isolates). Regarding all Fumigati isolates recovered from 2012-2019, the frequency of cryptic species was 5.3% (18/337), with the identification of A. felis (complex), A. lentulus, A. udagawae, A. hiratsukae, and A. oerlinghauensis. To determine the frequency of azole resistance of A. fumigatus, isolates were screened for azole resistance using azole-agars, and 53 possible resistant isolates were tested by the CLSI microdilution reference method. Nine A. fumigatus sensu stricto and six Fumigati cryptic isolates showed high minimal inhibitory concentrations to itraconazole, voriconazole, and/or posaconazole. Real-time PCR to detect cyp51A mutations and sequencing of cyp51A gene and its promoter were performed. The overall frequency of resistance to azoles in A. fumigatus sensu stricto was 3.0%. With this retrospective analysis, we were able to detect one azole-resistant G54R mutant A. fumigatus environmental isolate, collected in 2015. The TR34/L98H mutation, linked to environmental transmission route of azole resistance, was the most frequently detected mutation (N = 4; 1.4%). Our findings underline the demand for correct identification and susceptibility testing of Aspergillus isolates.

Delayed-Interval Delivery in Dichorionic Twin Pregnancies: A Case Report of 154 Latency Days.

Premature delivery often complicates multifetal pregnancies, placing neonates at risk of serious morbidity and mortality. In select cases, preterm birth of one sibling may not require delivery of the remaining fetus(es), which may remain in utero for a delayed-interval delivery, consequently improving neonatal morbidity and mortality. Currently, there is no consensus on the best protocol for the optimal management of these cases. We report one case of delayed-interval delivery of a dichorionic pregnancy assisted in our center. In this case, prophylactic cerclage, tocolytic therapy and administration of broad-spectrum prophylactic antibiotics enabled delivery at 37 weeks, corresponding to 154 days of latency, which is, to our knowledge, the longest interval described in the literature. The attempt to defer the delivery of the second fetus in peri-viability is an option that should be offered to parents after counseling, providing that the clinical criteria of eligibility are fulfilled. The correct selection of candidates, combined with the correct performance of procedures, as well as fetal and maternal monitoring and early identification of complications increase the probability of success of this type of delivery.

O parto pré-termo espontâneo complica frequentemente as gestações multifetais, condicionando elevada morbimortalidade perinatal. Em determinados casos, o nascimento prematuro do primeiro feto pode não requerer o nascimento do(s) feto(s) restante(s), que podem permanecer in utero, com o objetivo de diminuir a morbidade e mortalidade neonatal. Atualmente, não existe consenso quanto à melhor atitude clínica nas situações de parto diferido. Descrevemos um caso de parto diferido de gravidez bicoriônica vigiado no nosso centro. Neste caso, a realização de cerclagem, a terapêutica tocolítica e a administração de antibioticoterapia de largo espectro permitiu o parto às 37 semanas do segundo gêmeo, o que corresponde a 154 dias de latência, que, segundo o nosso conhecimento, é o intervalo de diferimento mais longo descrito na literatura. A tentativa de diferir o parto do segundo feto na periviabilidade é uma opção que deve ser oferecida aos progenitores, após aconselhamento e desde que se cumpram os critérios clínicos de elegibilidade. A seleção correta das candidatas, em conjunto com a realização de corretos procedimentos, monitorização fetal e materna e identificação precoce de complicações aumentam a possibilidade de sucesso deste tipo de parto.

Molecular Detection of Aspergillus: Application of a Real-Time PCR Multiplex Assay in Tissue Samples.

Diagnosis of invasive fungal infections is complex, and the lack of standardization of molecular methods is still a challenge. Several methods are available for the diagnosis of invasive aspergillosis, but their effectiveness will depend on the studied population, the patients' comorbidities, and the use of mold active prophylaxis, among others. The ability to determine the identity of the infecting Aspergillus species, and to detect mutations conferring specific resistance patterns directly from DNA extracted from the biological product, is an advantage of nucleic acid testing compared with antigen-based assays. In this study, we to present laboratory cases where the diagnosis of aspergillosis was performed using a real-time multiplex PCR for the detection of Aspergillus DNA in tissue samples, showing its usefulness as one more tool in the diagnosis of aspergillosis in tissue samples. Aspergillus real-time multiplex PCR was also used to detect azole-resistance in some cases. In the majority of the PCR positive cases, cultures remained negative after 60 days. The PCR assay directed to Aspergillus gave positive signals for Aspergillus fumigatus sensu stricto. Results were confirmed by panfungal PCR, followed by sequencing, revealing 100% homology with Aspergillus fumigatus sensu stricto. Mutations conferring azole resistance were not detected.

Delayed-Iinterval delivery in dichorionic twin pregnancies: A case report of 154 latency days / Parto diferido emg ravidez gemelar bicoriônica: Um caso com 154 dias de latência

Abstract Premature delivery often complicates multifetal pregnancies, placing neonates at risk of seriousmorbidity andmortality. In select cases, pretermbirth of one sibling may not require delivery of the remaining fetus(es), which may remain in utero for a delayedinterval delivery, consequently improving neonatalmorbidity andmortality. Currently, there is no consensus on the best protocol for the optimalmanagement of these cases. We report one case of delayed-interval delivery of a dichorionic pregnancy assisted in our center. In this case, prophylactic cerclage, tocolytic therapy and administration of broad-spectrum prophylactic antibiotics enabled delivery at 37 weeks, corresponding to 154 days of latency, which is, to our knowledge, the longest interval described in the literature. The attempt to defer the delivery of the second fetus in peri-viability is an option that should be offered to parents after counseling, providing that the clinical criteria of eligibility are fulfilled. The correct selection of candidates, combined with the correct performance of procedures, as well as fetal and maternal monitoring and early identification of complications increase the probability of success of this type of delivery.

Resumo O parto pré-termo espontâneo complica frequentemente as gestações multifetais, condicionando elevada morbimortalidade perinatal. Em determinados casos, o nascimento prematuro do primeiro feto pode não requerer o nascimento do(s) feto(s) restante(s), que podem permanecer in utero, com o objetivo de diminuir a morbidade e mortalidade neonatal. Atualmente, não existe consenso quanto à melhor atitude clínica nas situações de parto diferido. Descrevemos um caso de parto diferido de gravidez bicoriônica vigiado no nosso centro. Neste caso, a realização de cerclagem, a terapêutica tocolítica e a administração de antibioticoterapia de largo espectro permitiu o parto às 37 semanas do segundo gêmeo, o que corresponde a 154 dias de latência, que, segundo o nosso conhecimento, é o intervalo de diferimento mais longo descrito na literatura. A tentativa de diferir o parto do segundo feto na periviabilidade é uma opção que deve ser oferecida aos progenitores, após aconselhamento e desde que se cumpram os critérios clínicos de elegibilidade. A seleção correta das candidatas, em conjunto com a realização de corretos procedimentos, monitorização fetal e materna e identificação precoce de complicações aumentam a possibilidade de sucesso deste tipo de parto.

Detection of deep fungal infections: a polyphasic approach.

PURPOSE: Tissue samples from patients with suspicion of deep or subcutaneous fungal infections were analysed at the Portuguese Reference Mycology Laboratory according to a proposed diagnostic approach, which aims to constitute a rapid and accurate diagnosis for these fungal infections. METHODOLOGY: Forty-six tissue biopsy samples were analysed over a period of 26 months, using a diagnostic approach that includes culture, panfungal PCR and Aspergillus-directed PCR.Results/Key findings. Overall, 23 samples were reported as negative while the remaining 23 were reported as positive for fungi (PCR, culture and/or histology). PCR showed an estimated detection limit of 12 pg DNA µl-1. From the 46 samples, 30 were negative for fungal DNA while 16 gave positive results. From these, 12 cases were detected by panfungal PCR and six cases by PCR directed toward Aspergillus. In 61 % of the cases, there was concordance between molecular and cultural methods. Aetiological agents identified were Candida albicans, C. glabrata, C. tropicalis, Trichosporon montevideense, Alternaria spp., Exophiala sp., Trichoderma sp., Histoplasma spp., Aspergillus fumigatus, Trichophyton rubrum and Paracoccidioides brasiliensis. CONCLUSION: Our results showed that the proposed polyphasic approach appears to be a useful strategy in the detection of fungi from tissue samples, allowing a better prognosis. In further studies, the inclusion of a higher number of samples and the implementation of more genus-specific PCRs will certainly contribute to an increase in the specificity and sensitivity of this method.

Resiliência: novos olhares face aos desafios do nosso tempo / Resilience: new looks at the challenges of our time

Este livro - organizado e coordenado por Helena Ralha-Simões - é uma coletânea de estudos, investigações e reflexões em torno de uma realidade que começou a emergir a partir dos anos 70 e foi recebendo sentidos e interpretações distintas no decorrer dos últimos 40 anos. Os temas aqui desenvolvidos vão da triangulação epistemológica dos conceitos de resiliência, identidade e pessoalidade (Carlos Marques Simões), a um conceito interdisciplinar, ainda vago e à procura do futuro (Helena Ralha-Simões), passando pela resiliência em português: do soletrar ao senso comum (Ana Maria Albuquerque), como salto para a adaptação (Celeste Simões) ou em situações de violência (Simone Gonçalves de Assis, Suely Ferreira Deslandes e Fernanda Serpeloni), focalizando finalmente o envelhecimento com resiliência e sabedoria (Maria Helena Martins), os conceitos e contextos de resiliência (Nuno Álvaro C. Murcho) e a espiritualidade e a resiliência (Carla Fonseca Tomás). (AU).

Cutaneous infection by different Alternaria species in a liver transplant recipient.

Fungal invasive infections are rare in general population but are an emergent cause of infection in the immunocompromized population, especially in the solid organ transplant recipients. Herein the authors report a clinical case of a liver transplanted patient suffering a cutaneous co-existent infection with A. alternata as well as A. infectoria. To our knowledge this is the first case of cutaneous concomitant infection due to those two species reported not only in Portugal but also worldwide. The patient was treated with surgical excision of the lesions and oral itraconazol without relapse.

Ethics issues experienced in HBM within Portuguese health surveillance and research projects.

BACKGROUND: In keeping with the fundamental practice of transparency in the discussion and resolution of ethics conflicts raised by research, a summary of ethics issues raised during Portuguese biomonitoring in health surveillance and research is presented and, where applicable, their resolution is described. METHODS: Projects underway aim to promote the surveillance of public health related to the presence of solid waste incinerators or to study associations between human exposure to environmental factors and adverse health effects. The methodological approach involves biomonitoring of heavy metals, dioxins and/or other persistent organic pollutants in tissues including blood, human milk and both scalp and pubic hair in groups such as the general population, children, pregnant women or women attempting pregnancy. As such, the projects entail the recruitment of individuals representing different demographic and health conditions, the collection of body tissues and personal data, and the processing of the data and results. RESULTS: The issue of autonomy is raised during the recruitment of participants and during the collection of samples and data. This right is protected by the requirement for prior written, informed consent from the participant or, in the case of children, from their guardian. Recruitment has been successful, among eligible participants, in spite of incentives rarely being offered. The exception has been in obtaining guardians' consent for children's participation, particularly for blood sampling. In an attempt to mitigate the harm-benefit ratio, current research efforts include alternative less invasive biomarkers.Surveys are currently being conducted under contract as independent biomonitoring actions and as such, must be explicitly disclosed as a potential conflict of interests. Communication of results to participants is in general only practised when a health issue is present and corrective action possible. Concerning human milk a careful approach is taken, considering breast-feeding's proven benefits. CONCLUSION: No national legislation currently accounts for the surveillance component of biomonitoring as distinct from research. Ethics issues arising within the domain of research are resolved according to available regulations. For issues encountered during surveillance, the same principles are used as guidance, completed by the authors' best judgement and relevant ethics committees' findings.