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Infections caused by filamentous fungi are rising in incidence and became a serious health concern. Their rapid and reliable identification in the clinical laboratory is essential for an early and accurate diagnosis to guide timely therapy. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been reported as a rapid and reliable method for identification of bacteria and yeasts isolated from clinical samples. However, it has less used for molds identification. The aim of this study was to evaluate Vitek® MS (a MALDI-TOF MS system) ability to identify molds and differentiate species within a complex. A collection of 90 filamentous fungi, 70 clinical and 20 environmental isolates, was studied by morphological and molecular methods and by Vitek® MS. Seventy-four isolates (82.2%) were identified using Vitek® MS v3.0 at Genus/Complex/Species group level; within these, 47/74 (63.5%) were correctly identified at species level and only one was misidentified. In contrast, 16/90 isolates (17.8%) were not identified, of which 13 were not present in the database. Results here expressed favor Vitek® MS v3.0 as a very useful system for identification of most common clinical isolates of filamentous fungi. Accordingly, it may be an important tool for clinical microbiology laboratories in their task to answer to clinicians, adequately and rapidly, helping in proper patient's management.

Aims Aspergillus sp. are ubiquitous saprophytic fungi and their conidia easily inhaled. This is particularly important in immunocompromised patients, more susceptible to developing invasive aspergillosis. In addition to A. fumigatus sensu stricto, cryptic species, many resistant to antifungal drugs, have been associated with invasive infections, making it important to assess their presence and diversity in different environments. Therefore, the aim of this study was to assess the presence, diversity and susceptibility to antifungal drugs of airborne fungi. Moreover, in azole-resistant A. fumigatus sensu stricto isolates the presence of underlying molecular mechanisms of resistance was investigated. Methods and results Eighty-four Aspergillus isolates were collected from the environment air in hospitals and the Water Treatment Plant. The use of molecular tools allowed to detect 12 different cryptic species, showing a prevalence of 21·4%. The majority of isolates (69·0%) belonged to A. fumigatus complex and 65·4% were A. fumigatus sensu stricto. Among these, 21·8% were resistant to itraconazole (ITZ), 38·2% to posaconazole and 87·3% to isavuconazole; none of them were resistant to voriconazole or amphotericin B. Sequencing of the cyp51A gene on the 12 A. fumigatus sensu stricto ITZ-resistant isolates revealed the presence of mutations. Conclusion Our study reports a large number of environmental-resistant Aspergillus species, including A. fumigatus sensu stricto that display an important role in invasive fungal infections. None of the environmental isolates showed mutations on cyp51A gene related to azole resistance. Significant and impact of the study This study is the first assessment of molecular resistance mechanisms in A. fumigatus sensu stricto environmental isolates, in Portugal. Since TR34/L98H and TR46/Y121F/T289A cyp51A mutations were already reported in the clinical setting in Portugal (Monteiro et al. J Glob Antimicron Resist 13: 190-191, 2018; Pinto et al. Front Microbiol 9: 1656, 2018), and have been linked to environmental route, it is utmost importance to perform surveillance network for azole-resistant A. fumigatus.

BACKGROUND: Extracorporeal membrane oxygenation (ECMO), could be extremely helpful in the management of COVID-19 patients with refractory hypoxemic respiratory failure; however, to date, evidence on the true effecttiveness of ECMO in the COVID19 setting still hangs in the balance. METHODS: This was a prospective cohort study of 39 COVID-19 patients admitted to the intensive care unit (ICU) in an experienced ECMO center at a tertiary hospital during March/April 2020. Among the recruited participants, 10 (25.6%) required ECMO (ICU-ECMO group) and 29 (74.4%) did not have ECMO support (ICU group). Immunological parameters were assessed both at ICU admission and on a daily basis for 7 consecutive days. RESULTS: The absolute lymphocyte count increased significantly in the ICU-ECMO group compared to the ICU group in which it remained relatively stable: s for the time variable was 127.1 [95% CI 68.9 - 185.3], p < 0.001 and for the interaction term -141.36 [-208.95 - -73.77], p < 0.001. On the other hand, globally, no significant differences were observed over time for the lymphocyte percentage, although it was higher in the ICU patients. Neutrophil counts were overall higher in the ICU-ECMO group (s -4,275.38 [-6,845.21 - 1,705.55], p = 0.001). In regard to neutrophil percentage, a significant decrease over time was reported (s -1.76 [-3.16 - -0.36], p = 0.014), namely in the ICU-ECMO group (s for the interaction 2.09 [0.45 - 3.73], p = 0.013). CONCLUSIONS: Herein, we found ECMO support seems to provide a less aggressive immune response in COVID-19 patients with severe and refractory respiratory dysfunction.

Objectives: To compare the concordance of ceftaroline MIC values by reference broth microdilution (BMD) and Etest (bioMérieux, France) for MSSA and MRSA isolates obtained from PREMIUM (D372SL00001), a European multicentre study. Methods: Ceftaroline MICs were determined by reference BMD and by Etest for 1242 MSSA and MRSA isolates collected between February and May 2012 from adult patients with community-acquired pneumonia or complicated skin and soft tissue infections; tests were performed across six European laboratories. Selected isolates with ceftaroline resistance in broth (MIC >1 mg/L) were retested in three central laboratories to confirm their behaviour. Results: Overall concordance between BMD and Etest was good, with >97% essential agreement and >95% categorical agreement. Nevertheless, 12 of the 26 MRSA isolates found resistant by BMD scored as susceptible by Etest, with MICs ≤1 mg/L, thus counting as very major errors, whereas only 5 of 380 MRSA isolates found ceftaroline susceptible in BMD were miscategorized as resistant by Etest. Twenty-one of the 26 isolates with MICs of 2 mg/L by BMD were then retested twice by each of three central laboratories: BMD MICs of 2 mg/L were consistently found for 19 of the 21 isolates. Among 147 Etest results for these 21 isolates (original plus six repeats per isolate) 112 were >1 mg/L. Conclusions: BMD and Etest have good overall agreement for ceftaroline against Staphylococcus aureus; nevertheless, reliable Etest-based discrimination of the minority of ceftaroline-resistant (MIC 2 mg/L) MRSA is extremely challenging, requiring careful reading of strips, ideally with duplicate testing., 0, SCOPUS: ar.j, info:eu-repo/semantics/published

Introduction: Non-tuberculous mycobacteria are ubiquitous micro-organisms that rarely cause disease in humans, unless they are immunocompromised or have undergone trauma. Case presentation: We describe a case of spontaneous breast abscess due to Mycobacterium fortuitum in an immunocompetent patient, identified using DNA-STRIP technology. This is the third report that, apart from being caused by the same agent, has the localization on the left breast in common, raising the question of a laterality effect. Conclusion: Careful inspection of cultures and accurate agent identification are procedures to be emphasized in clinical microbiology laboratories. In fact, they may reveal unusual growth due to uncommon micro-organisms.

Beauveria bassiana is a ubiquitous fungus available as an insecticide. In humans, it has limited virulence; to our knowledge, only 3 cases of invasive disease and 10 cases of keratitis have been documented.We report the first case of B bassiana keratitis in a patient with aphakic bullous keratopathy. The fungal keratitis proved to be highly resistant to topical clotrimazole. Molecular identification was based on DNA sequence analysis. The minimal inhibitory concentrations (MIC) obtained were 2 µg/mL for voriconazole, 0.250 µg/mL for posaconazole, and128 µg/mL for fluconazole; amphotericin B MIC was16 µg/mL. In the absence of clinical improvement, a penetrating keratoplasty (PK) was performed. The patient was discharged on topical and systemic voriconazole and prednisolone 40 mg PO/day. The eye remained calm with a transparent cornea and clear anterior chamber.B bassiana keratitis is extremely rare, with only a few cases reported. Its risk factors are unknown. We report the first case in a patient with aphakic bullous keratopathy, which proved highly resistant to antifungal therapy (antifungal susceptibility results are presented). A PK was necessary for clinical improvement. A review of the literature is performed in an effort to define therapeutic strategies.

Probably the most important decision in the management of Community-Acquired Pneumonia (CAP) is patient site of care. Patients with Streptococcus pneumoniae-caused CAP admitted to our hospital between 1st January and 31st December 2006 were retrospectively analysed. Samples of blood, sputum, bronchial and bronchoalveolar lavage and urine were collected for microbiological testing using standard culture techniques and urine antigen detection. Pneumonia Severity Index (PSI) and British Thoracic Society (BTS) CURB-65 scoring tools were evaluated. The statistical treatment was performed using the SPSS 14.0 program. We included 104 patients, 67.3% male, median age 63 years old, mortality 13.4%. There was a significant association between the PSI and CURB-65 score and mortality. Despite advances, CAP is still an important health problem with a high atten - dant morbi-mortality. This study confirms the value of PSI and CURB-65 in the prediction of severe pneumonia. Resumo: A avaliação da gravidade perante qualquer caso de pneumonia adquirida na comunidade (PAC) é de suma importância, pois dela decorrem decisões como a necessidade de internamento e o tratamento empírico inicial. Os autores apresentam um estudo retrospectivo, que incluiu doentes internados devido a pneumonia por Streptococcus pneumoniae durante o ano de 2006, no Hospital de São João. A confirmação etiológica de infecção foi feita por isolamentos no sangue, líquido pleural, secreções traqueobrônquicas, lavado brônquico, lavado broncoalveolar e pesquisa de antigenúria. Foram analisados os factores de risco e avaliados, com base nas normas PSI (Pneumonia Severity Index) e da British Thoracic Society (BTS) - CURB-65. A análise estatística foi efectuada utilizando teste T para amostras independentes e ANOVA, usando o programa de análise estatística SPSS 14.0.Foram incluídos 104 doentes com idade mediana de 63 anos, sendo 67,3% do sexo masculino. O estudo revelou existir uma associação com significado estatístico entre os resultados de PSI e CURB-65 e a evolução para a mortalidade.Apesar da melhoria dos meios diagnósticos e profilácticos, e da terapêutica antibiótica, a pneumonia pneumocócica permanece uma entidade de grande morbilidade e mortalidade. O valor preditivo das normas PSI e CURB-65 foi confirmado nesta população de doentes, documentando uma correlação entre o número de factores de risco e a evolução da doença. Key-words: Community-acquired pneumonia, Streptococcus pneumoniae, Palavras-chave: Pneumonia adquirida na comunidade, Streptococcus pneumoniae

Introduction: Nocardiosis emerges as a serious infection in the immunocompromised patient, causing an aggressive clinical picture, which requires prolonged treatment. Case presentation: We present five cases of disseminated nocardiosis in patients under corticosteroid therapy with four different forms of clinical presentation. Conclusion: Immunocompromised patients are at increased risk of disseminated nocardiosis, which may require extended culture time for diagnosis and molecular methods. The cases emphasize the relevance of a careful evaluation of the central nervous system, a common target in nocardiosis dissemination.

Background Cryptococcal meningoencephalitis is an opportunistic infection that predominantly affects immunocompromised patients. Hyper immunoglobulin M syndrome is a primary immunodeficiency syndrome that increases susceptibility to several opportunistic infections. Here, we report a case of cryptococcal meningoencephalitis in the context of hyper immunoglobulin M syndrome, a situation that has been reported very few times and whose management is not clearly defined. We describe our management of this case and the outcome of the patient to help in future similar situations. Case presentation The patient is a 19-year-old Caucasian male student diagnosed with X-linked hyper immunoglobulin M syndrome and treated chronically with weekly intravenous immunoglobulin and daily sulfamethoxazole-trimethoprim. He was admitted to the infectious diseases ward because of headache, diplopia and a cerebral-spinal fluid analysis revealing cryptococcal meningoencephalitis. The patient was treated with liposomal amphotericin and flucytosine with a favorable outcome. Maintenance therapy with fluconazole has continued and will be sustained for 6 months following his upcoming bone marrow transplantation. Conclusion Monitoring for cryptococcal meningoencephalitis should be considered in patients with primary immunodeficiencies, as clinical manifestations may go unnoticed. In these patients, it is expected that chronic treatment with fluconazole will be the only treatment that will prevent reinfection or reactivation, and therefore should be kept at least until bone marrow transplant, the only curative treatment, is performed. It may, however, lead to intolerable side effects and hepatic toxicity.

Stress can affect all people, regardless of age, gender or ethnicity. The human body uses stress as an adaptive response against several situations, but it can cause physical, mental or emotional distress. In stress situation the nervous system evokes the same physiological responses, whatever the type of stress and these responses cause changes in the physical, cognitive, emotional and social performance. This paper proposes to develop a methodology for analyzing the stress level using electroencephalographic signals (EEG) based on asymmetry patterns of alpha waves. The subjects of this research are professionals from Vitoria's Fire Department. Activities of the Central Nervous System are capture using Emotiv Epoc Headset, placed on the volunteer's scalp. The signals are analyzed from the frontal cortex at positions Fp1 (AF3), Fp2 (AF4), F3 and F4, according to of the international 10-20 system. The results are analyzed together with some peripheral physiological signals, such as: heart beat rate (ECG), peripheral blood flow, skin conductance, breath rate, body temperature and change in level of salivary cortisone. In addition, a psychological assessment of voluntary's stress is performed through a questionnaire of LIPP-ISS (Inventory of Symptom Stress). A validation study is conducted through a comparison with data available in literature, as well as with evaluation conducted by a psychologist. The results are analyzed in order to get a correlation between EEG signals and physical, mental or emotional stress.

The identification of stress-related emotional states allows the use of a passive single-switch BCI (passive-ssBCI) together with a main multi-class BCI. Thus, the passive-ssBCI could work in the background of the main BCI, allowing it to recognize stress-related emotions and to switch to a “stress mode”, which allows a better adaptation to the user current mental state, leading the system to better success rates. Frontal cortex asymmetry gave evidence that greater right frontal activity seems to be more highly related to negative emotional states. In this work, index of asymmetry of alpha band was computed by comparing the power of contra-lateral frontal electrodes. Discrete Wavelet transformation was used to decompose the EEG signals in frequency bands. The power spectral density was then calculated using modified Hamming periodogram. Signals from a public database for the analysis of human affective states, which was labeled with volunteers self-assessment scores, were employed. In order to label negative stress-related emotional state, rules based on valence, arousal and dominance dimensional emotions were defined. The results were promissory, because computed index of asymmetry in the alpha band indicated activity into the right frontal hemisphere was higher than the left one for seven out of nine volunteers during negative emotional states. This index could be employed as switch to a “stress mode” in a passive-ssBCI.

Listeria monocytogenes infection (listeriosis) is an uncommon but severe foodborne illness that affects mainly individuals with recognized underlying conditions: the elderly, immunocompromised individuals, and pregnant women and their fetuses. The aim of this study was to obtain epidemiological data on cases of listeriosis occurring in Portugal from 2008 through 2012, collected in hospitals on a voluntary basis. L. monocytogenes isolates were characterized by genoserotyping by multiplex polymerase chain reaction, DNA macrorestriction pulsed-field gel electrophoresis (PFGE), and determination of minimal inhibitory concentration (MIC, μg/mL) for 12 antibiotics. During this period, 203 cases of listeriosis were detected. The annual incidence rate observed ranged from 0.2 to 0.7 cases per 100,000 inhabitants. Nineteen cases (9.5%) corresponded to maternal/neonatal (MN) infections. The mean age of the nonmaternal/neonatal (non-MN) cases with documented age was 59 years, and 46.4% occurred in patients aged over 65 years. The majority of listeriosis cases were caused by genoserogroup IVb isolates, and PFGE analysis revealed a high molecular diversity, suggesting that most were sporadic. Nevertheless, several clusters of isolates presenting different geographic and time distributions were detected. The incidence of antibiotic-resistant isolates of L. monocytogenes was low but significantly higher than in previous years (2003-2007). The implementation of a national surveillance system monitoring the incidence of listeriosis and antimicrobial resistance of strains would be most valuable, allowing identification of sporadic and outbreak cases, to detect general trends in antibiotic susceptibilities, and potentially identify food sources of clinical strains.

We describe a novel heterothallic species in Aspergillus section Fumigati, namely A. felis (neosartorya-morph) isolated from three host species with invasive aspergillosis including a human patient with chronic invasive pulmonary aspergillosis, domestic cats with invasive fungal rhinosinusitis and a dog with disseminated invasive aspergillosis. Disease in all host species was often refractory to aggressive antifungal therapeutic regimens. Four other human isolates previously reported as A. viridinutans were identified as A. felis on comparative sequence analysis of the partial β-tubulin and/or calmodulin genes. A. felis is a heterothallic mold with a fully functioning reproductive cycle, as confirmed by mating-type analysis, induction of teleomorphs within 7 to 10 days in vitro and ascospore germination. Phenotypic analyses show that A. felis can be distinguished from the related species A. viridinutans by its ability to grow at 45°C and from A. fumigatus by its inability to grow at 50°C. Itraconazole and voriconazole cross-resistance was common in vitro. © 2013 Barrs et al.

In contrast with the common hematogenous dissemination of invasive aspergillosis (IA), we present case with a protracted course through anatomical planes in an immunocompromised adult male. The unusual clinical features and laboratory findings led to fungal genotyping and identification of the mold as Aspergillus viridinutans. It appears to be the first described case of IA caused by this agent in an adult patient.

Quinolone resistance is an emerging problem in Streptococcus pyogenes , and recombination with Streptococcus dysgalactiae DNA has been implicated as a frequent mechanism leading to resistance. We have characterized a collection of S. dysgalactiae subsp. equisimilis isolates responsible for infections in humans ( n = 314) and found a high proportion of levofloxacin-resistant isolates (12%). Resistance was associated with multiple emm types and genetic lineages, as determined by pulsed-field gel electrophoretic profiling. Since we could not find evidence for a role of efflux pumps in resistance, we sequenced the quinolone resistance-determining regions of the gyrA and parC genes of representative resistant and susceptible isolates. We found much greater diversity among the parC genes (19 alleles) than among the gyrA genes (5 alleles). While single mutations in either GyrA or ParC were sufficient to raise the MIC so that the strains were classified as intermediately resistant, higher-level resistance was associated with mutations in both GyrA and ParC. Evidence for recombination with S. pyogenes DNA was found in some parC alleles, but this was not exclusively associated with resistance. Our data support the existence of a common reservoir of genes conferring quinolone resistance shared between S. dysgalactiae subsp. equisimilis and S. pyogenes , while no recombination with the animal pathogen S. dysgalactiae subsp. dysgalactiae could be found.

Bacteremia due to Vibrio cholerae non-O1 and non-O139 is a rare condition and potentially fatal. We report a case of bacteremia due to V. cholerae non-O1 and non-O139 in a Portuguese male with Hepatitis C cirrhosis, admitted due to acute diarrhea, after consuming shrimp. He had no recent travels. To our knowledge, this is the first reported case of bacteremia due to V. cholerae non-O1 and non-O139 in Portugal.

The population of group B streptococci (GBS) associated with invasive infections in nonpregnant adults from 2001 to 2008 was analyzed in isolates submitted from 24 hospital laboratories in Portugal ( n = 225). The isolates were characterized by antimicrobial susceptibility, pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), and surface protein gene profiling. GBS invasive cases were found more frequently among men in all age groups. In addition, serotype Ia was the most frequent in our collection, whereas serotype V is dominant elsewhere. Serotype Ia was represented mainly by a single PFGE cluster defined by sequence type 23 (ST23) and surface protein gene eps and by ST24 and bca , similarly to neonatal invasive infections in Portugal, indicating that the same genetic lineages can be responsible for both vaginal colonization and invasive disease in all age groups. In contrast, the hypervirulent serotype III/ST17 neonatal lineage was responsible for a minority of infections. Serotype V isolates were distributed into two genetic lineages, one defined by ST1 and surface protein gene alp3 and macrolide resistant, and another presenting with ST2 and eps and fully susceptible to all antimicrobials tested. The erm (TR) gene was the most frequently found among erythromycin-resistant isolates, while the bovine-associated tet (O) gene was found in a minority of tetracycline-resistant isolates. Our data emphasize the importance of local identification of the genetic lineages responsible for GBS invasive infections in nonpregnant adults. The dominance of serotype Ia in invasive disease in Portugal highlights the importance of this serotype in GBS pathogenesis.

Resumo: A avaliação da gravidade perante qualquer caso de pneumonia adquirida na comunidade (PAC) é de suma importância, pois dela decorrem decisões como a necessidade de internamento e o tratamento empírico inicial. Os autores apresentam um estudo retrospectivo, que incluiu doentes internados devido a pneumonia por Streptococcus pneumoniae durante o ano de 2006, no Hospital de São João. A confirmação etiológica de infecção foi feita por isolamentos no sangue, líquido pleural, secreções traqueobrônquicas, lavado brônquico, lavado broncoalveolar e pesquisa de antigenúria. Foram analisados os factores de risco e avaliados, com base nas normas PSI (Pneumonia Severity Index) e da British Thoracic Society (BTS) -CURB-65. A análise estatística foi efectuada utilizando teste T para amostras independentes e ANOVA, usando o programa de análise estatística SPSS 14.0.Foram incluídos 104 doentes com idade mediana de 63 anos, sendo 67,3% do sexo masculino. O estudo revelou existir uma associação com significado estatístico entre os resultados de PSI e CURB-65 e a evolução para a mortalidade.Apesar da melhoria dos meios diagnósticos e profilácticos, e da terapêutica antibiótica, a pneumonia pneumocócica permanece uma entidade de grande morbilidade e mortalidade. O valor preditivo das normas PSI e CURB-65 foi confirmado nesta população de doentes, documentando uma correlação entre o número de factores de risco e a evolução da doença.Rev Port Pneumol 2008; XIV (5): 601-615 Abstract: Probably the most important decision in the management of Community-Acquired Pneumonia (CAP) is patient site of care. Patients with Streptococcus pneumoniae-caused CAP admitted to our hospital between 1st January and 31st December 2006 were retrospectively analysed. Samples of blood, sputum, bronchial and bronchoalveolar lavage and urine were collected for microbiological testing using standard culture techniques and urine antigen detection. Pneumonia Severity Index (PSI) and British Thoracic Society (BTS) CURB-65 scoring tools were evaluated. The statistical treatment was performed using the SPSS 14.0 program. We included 104 patients, 67.3% male, median age 63 years old, mortality 13.4%. There was a significant association between the PSI and CURB-65 score and mortality. Despite advances, CAP is still an important health problem with a high attendant morbi-mortality. This study confirms the value of PSI and CURB-65 in the prediction of severe pneumonia.Rev Port Pneumol 2008; XIV (5): 601-615 Palavras-chave: Pneumonia adquirida na comunidade, Streptococcus pneumoniae, Key-words: Community-acquired pneumonia, Streptococcus pneumoniae

Stress can affect all people, regardless of age, gender or ethnicity. The human body utilizes stress as a response in three different situations, classified according to the way it generates physical, mental or emotional stress. It is important to notice that the nervous system evokes the same physiological responses, no matter what type of stress, and those responses cause a change of level in physical and cognitive performance. This paper presents an analysis methodology of the stress level using brain's signals captured by Electroencephalogram (EEG). Professionals from Vitoria´s Fire Department, members of Projeto VIDA participate as volunteers. The EEG signals are captured through a cap placed over the volunteer's head in order to capture the brain´s signals, using electrodes specifically placed in the frontal cortex at positions Fp1, Fp2, F3 and F4. The results are analyzed together with the peripheral physiological signals, such as: heart beat rate (ECG), peripheral blood flow (at fingers and toes), skin conductance, breath rate and body temperature. A validation study is conducted through a comparison of data available in literature, as well as with evaluation conducted by a psychologist. The results are analyzed in order to get a correlation between EEG signals and physical mental or emotional stress.