Browsing by Author "Toscano, Cristina"
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- Aspergillus section Fumigati – Epidemiological trends - A perspective from a National Reference LaboratoryPublication . Sabino, Raquel; Simões, Helena; Francisco, Mariana; Viegas, Carla; Toscano, Cristina; Batista, JuditeTeresa; Ferreira, Teresa; Veríssimo, CristinaObjectives: Aspergillus fumigatus is the most frequent agent of aspergilosis and reports on infections caused by this species or its siblings are becoming more frequent, together with the increasing number of at risk patients. Nowadays, due to the rising concerns on emerging antifungal resistance, the epidemiological surveillance for clinical and environmental isolates is mandatory. The overall objective of the project is to understand the epidemiology of the Aspergillus isolates (species and antifungal resistance) collected in the Portuguese National Reference Laboratory through our surveillance system on Aspergillus. Methods: During the period 2013-2017, 117 Aspergillus section Fumigati isolates were collected at the National Health Reference Dr. Ricardo Jorge, through the surveillance system on Aspergillus. Isolates were obtained from different patient samples from 15 healthcare institutions of all country, and from different environmental sources (air or surfaces sampling). All isolates were plated for growth as single colonies on malt extract agar with chloramphenicol. These isolates were identified on the basis of macro and microscopic morphology and through the use of molecular tools. Genomic DNA was prepared from each isolate and the sequencing of the Internal Transcribed Spacers (ITS) regions as well as of the gene codifying to calmodulin. Surveillance of azole resistance was performed firstly using Sabouraud dextrose agar supplemented with itraconazole (ICZ), voriconazole (VCZ), and posaconazole (PCZ). When growth was observed, the minimal inhibitory concentration (MIC) was determined by broth microdilution method. In case of doubt, a specific PCR for detection of mutations in the Cyp51A gene of A. fumigatus was performed using the AsperGenius® multiplex real-time PCR assay. Results: From the isolates collected during the study period, 94 were from clinical (human) sources, 2 from animals diagnosed with aspergillosis and 21 from environmental sources Clinical isolates were obtained from 90 patients (53 males, 34 females, 3 not known), with ages ranging from 37 days to 88 years old. Most of these isolates (98%) were from respiratory specimens. The underlying diseases reported are, among others, cystic fibrosis, COPD, HIV, asthma, and neoplasms. In total, 111 A. fumigatus sensu stricto isolates were identified, followed by 3 A. lentulus, 2 A. felis and 1 A. hiratsukae (from hospital environment). In 7 cases, the morphological identification did not matched with the correct species-section. Interestingly, the 5 clinical cryptic species were from the same hospital. Regarding susceptibility, relevant and residual growths were obtained in azole resistance screening media (Table 1). The positive results were then screened by microdilutions and by detection of Cyp51A mutations and resistances were not confirmed. Conclusions: The understanding of local resistance patterns is valuable to assess shifts in the epidemiology of Aspergillus (and therefore, to manage therapeutic approaches). In our collection of Fumigati isolates, 5% of them were cryptic species. Although we did not confirm azole resistance by microdilution or detection of Cyp51A mutations, the MIC values obtained suggest that the median values are higher than what is described in other studies (1.4 to ICZ, 0.4 to PCZ),which may explain the growth in screening media and may suggest a local epidemiology.
- Comparison of Multi-locus Genotypes Detected in Aspergillus fumigatus Isolated from COVID Associated Pulmonary Aspergillosis (CAPA) and from Other Clinical and Environmental SourcesPublication . Morais, Susana; Toscano, Cristina; Simões, Helena; Carpinteiro, Dina; Viegas, Carla; Veríssimo, Cristina; Sabino, RaquelBackground: 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.
- COVID-19-Associated Invasive Pulmonary Aspergillosis in the Intensive Care Unit: A Case Series in a Portuguese HospitalPublication . Ranhel, David; Ribeiro, Ana; Batista, Judite; Pessanha, Maria; Cristovam, Elisabete; Duarte, Ana; Dias, Ana; Coelho, Luís; Monteiro, Filipa; Freire, Pedro; Veríssimo, Cristina; Sabino, Raquel; Toscano, CristinaInvasive pulmonary aspergillosis (IPA) has become a recognizable complication in coronavirus disease 2019 (COVID-19) patients admitted to intensive care units (ICUs). Alveolar damage in the context of acute respiratory distress syndrome (ARDS) appears to be the culprit in facilitating fungal invasion in COVID-19 patients, leading to a COVID-19-associated pulmonary aspergillosis (CAPA) phenomenon. From November 2020 to 15 February 2021, 248 COVID-19 patients were admitted to our ICUs, of whom ten patients (4% incidence) were classified as either probable (six) or possible (four) CAPA cases. Seven patients had positive cultural results: Aspergillus fumigatus sensu stricto (five), A. terreus sensu stricto (one), and A. welwitschiae (one). Five patients had positive bronchoalveolar lavage (BAL) and galactomannan (GM), and two patients had both positive cultural and GM criteria. All but two patients received voriconazole. Mortality rate was 30%. Strict interpretation of classic IPA definition would have resulted in eight overlooked CAPA cases. Broader diagnostic criteria are essential in this context, even though differentiation between Aspergillus colonization and invasive disease might be more challenging. Herein, we aim to raise awareness of CAPA in view of its potential detrimental outcome, emphasizing the relevance of a low threshold for screening and early antifungal treatment in ARDS patients.
- Distribution of Chlamydia trachomatis ompA-genotypes over three decades in PortugalPublication . Lodhia, Zohra; Cordeiro, Dora; Correia, Cristina; João, Inês; Carreira, Teresa; Vieira, Luís; Nunes, Alexandra; Ferreira, Rita; Schäfer, Sandra; Aliyeva, Elzara; Portugal, Clara; Monge, Isabel; Pessanha, Maria Ana; Toscano, Cristina; Côrte-Real, Rita; Antunes, Marília; Gomes, Joao Paulo; Borges, Vítor; José Borrego, MariaObjectives: Chlamydia trachomatis is classified into 15 major genotypes, A to L3, based on the diversity of ompA gene. Here, we evaluated and characterised the distribution and diversity of ompA-genotypes over 32 years (1990-2021) in Portugal. Methods: The collection of the Portuguese National Reference Laboratory for Sexually Transmitted Infections includes 5824 C. trachomatis-positive samples that were successfully ompA-genotyped between 1990 and 2021. An in-depth analysis of ompA-genotypes distribution across the years, as well as by biological sex, age and anatomical site of infection was performed. Results: ompA-genotype E was consistently the most frequently detected across the years, with a median frequency of 34.6%, followed by D/Da (17.6%), F (14.3%) and G (10.7%). The prevalence of lymphogranuloma venereum (LGV) genotypes (mostly L2, 62.0%, followed by L2b, 32.1%) increased since 2016, reaching the highest value in 2019 (20.9%). LGV, G and Da genotypes were associated with biological sex, specifically with being male, and were the most frequent among anorectal specimens (37.7%, 19.4% and 17.7%, respectively). Notably, LGV ompA-genotypes represented 38.9% of the male anorectal specimens since 2016, and were also detected among oropharynx and urogenital samples. ompA-genotype E was the most frequently detected at the oropharynx (28.6%) and urogenital (33.9%) sites during the study period, followed by D/Da (17.4%) and F (16.0%) in the urogenital specimens, and by G (26.1%) and D/Da (25.7%) in oropharynx specimens. Our data also highlight the emergence of the recombinant L2b/D-Da strain since 2017 (representing between 2.0% and 15.5% of LGV cases per year) and the non-negligible detection of ompA-genotype B in urogenital and anorectal specimens. Conclusions: This study provides a comprehensive landscape of C. trachomatis molecular surveillance in Portugal, highlighting the continued relevance of ompA-genotyping as a complement to rapid LGV-specific detection tests. It also contributes to a deeper understanding of C. trachomatis epidemiology, diversity and pathogenicity.
- Distribution of Chlamydia trachomatis ompA-genotypes over three decades in PortugalPublication . Lodhia, Zohra; Cordeiro, Dora; Correia, Cristina; João, Inês; Carreira, Teresa; Vieira, Luís; Nunes, Alexandra; Ferreira, Rita; Schäfer, Sandra; Aliyeva, Elzara; Portugal, Clara; Monge, Isabel; Pessanha, Maria Ana; Toscano, Cristina; Côrte-Real, Rita; Antunes, Marília; Gomes, Joao Paulo; Borges, Vítor; Borrego, Maria JoséObjectives: Chlamydia trachomatis is classified into 15 major genotypes, A to L3, based on the diversity of ompA gene. Here, we evaluated and characterised the distribution and diversity of ompA-genotypes over 32 years (1990–2021) in Portugal. Methods: The collection of the Portuguese National Reference Laboratory for Sexually Transmitted Infections includes 5824 C. trachomatis-positive samples that were successfully ompA-genotyped between 1990 and 2021. An in-depth analysis of ompA-genotypes distribution across the years, as well as by biological sex, age and anatomical site of infection was performed. Results: ompA-genotype E was consistently the most frequently detected across the years, with a median frequency of 34.6%, followed by D/Da (17.6%), F (14.3%) and G (10.7%). The prevalence of lymphogranuloma venereum (LGV) genotypes (mostly L2, 62.0%, followed by L2b, 32.1%) increased since 2016, reaching the highest value in 2019 (20.9%). LGV, G and Da genotypes were associated with biological sex, specifically with being male, and were the most frequent among anorectal specimens (37.7%, 19.4% and 17.7%, respectively). Notably, LGV ompA-genotypes represented 38.9% of the male anorectal specimens since 2016, and were also detected among oropharynx and urogenital samples. ompA-genotype E was the most frequently detected at the oropharynx (28.6%) and urogenital (33.9%) sites during the study period, followed by D/Da (17.4%) and F (16.0%) in the urogenital specimens, and by G (26.1%) and D/Da (25.7%) in oropharynx specimens. Our data also highlight the emergence of the recombinant L2b/D-Da strain since 2017 (representing between 2.0% and 15.5% of LGV cases per year) and the non-negligible detection of ompA-genotype B in urogenital and anorectal specimens. Conclusions: This study provides a comprehensive landscape of C. trachomatis molecular surveillance in Portugal, highlighting the continued relevance of ompA-genotyping as a complement to rapid LGV-specific detection tests. It also contributes to a deeper understanding of C. trachomatis epidemiology, diversity and pathogenicity.
- Epidemiologia das Infeções Fúngicas Superficiais em Portugal: revisão de 3 anos (2014-2016)Publication . Rato, Margarida; Costin, Adelina; Furtado, Constança; Sousa, Cristina; Toscano, Cristina; Veríssimo, Cristina; Trindade, Felicidade; Tavares Almeida, Filipa; da Cunha Velho, Glória; Catorze, Goreti; Raposo, Inês; Selada, Joana; Ferreira, João A.; Batista, Judite; Santos, Luís; Sereijo, Manuel; Silva, Manuela; Apetato, Margarida; Sanches, Maria; Costa-Silva, Miguel; Filipe, Paulo L.; Santos, Paulo; Fonseca, Pedro D.; Mascarenhas, Rosa; Bajanca, Rui; Lopes, Virgínia; Lewis, Viviana; Duarte, Maria da Luz; Galhardas, Célia; Anes, MargaridaIntrodução: As infeções fúngicas superficiais são as dermatoses infeciosas mais frequentes e a sua incidência continua a aumentar. Os dermatófitos são os principais agentes causais apresentando, contudo, uma distribuição geográfica variável. Material e Métodos: O presente estudo teve como objetivo a caracterização epidemiológica das infeções fúngicas superficiais diagnosticadas nos Serviços/Unidades de Dermatologia pertencentes ao Serviço Nacional de Saúde Português entre janeiro de 2014 e dezembro 2016 através da análise retrospetiva dos resultados das culturas realizadas durante esse período. Resultados: Foram estudados 2375 isolamentos, pertencentes a 2319 doentes. O dermatófito mais frequentemente isolado foi o Trichophyton rubrum (53,6%), tendo sido o principal agente causal da tinha da pele glabra (52,4%) e das onicomicoses (51,1%). Relativamente às tinhas do couro cabeludo, globalmente o Microsporum audouinii foi o agente mais prevalente (42,6%), seguido do Trichophyton soudanense (22,1%). Enquanto na área metropolitana de Lisboa estes dermatófitos foram os principais agentes de tinha do couro cabeludo, nas regiões Norte e Centro o agente mais frequente foi o Microsporum canis (58,5%). Os fungos leveduriformes foram os principais responsáveis pelas onicomicoses das mãos (76,7%). Conclusão: Os resultados deste estudo estão globalmente concordantes com a literatura científica. O Trichophyton rubrum apresenta-se como o dermatófito mais frequentemente isolado em cultura. Na tinha do couro cabeludo, na área metropolitana de Lisboa, as espécies antropofílicas de importação assumem particular destaque.
- Epidemiology and genetic variability of respiratory syncytial virus in Portugal, 2014-2018Publication . Sáez-López, Emma; Cristóvão, Paula; Costa, Inês; Pechirra, Pedro; Conde, Patrícia; Guiomar, Raquel; Peres, Maria João; Viseu, Regina; Lopes, Paulo; Soares, Vânia; Vale, Fátima; Fonseca, Patricia; Freitas, Ludivina; Alves, Jose; Pessanha, Maria Ana; Toscano, Cristina; Mota-Vieira, Luísa; Veloso, Rita Cabral; Côrte-Real, Rita; Branquinho, Paula; Pereira‑Vaz, João; Rodrigues, Fernando; Cunha, Mário; Martins, Luís; Mota, Paula; Couto, Ana Rita; Bruges-Armas, Jácome; Almeida, Sofia; Rodrigues, Débora; Portuguese Laboratory Network for the Diagnosis of Influenza InfectionIntroduction: Respiratory syncytial virus (RSV) is associated with substantial morbidity and mortality since it is a predominant viral agent causing respiratory tract infections in infants, young children and the elderly. Considering the availability of the RSV vaccines in the coming years, molecular understanding in RSV is necessary. Objective: The objective of the present study was to describe RSV epidemiology and genotype variability in Portugal during the 2014/15-2017/18 period. Material and methods: Epidemiological data and RSV-positive samples from patients with a respiratory infection were collected through the non-sentinel and sentinel influenza surveillance system (ISS). RSV detection, subtyping in A and B, and sequencing of the second hypervariable region (HVR2) of G gene were performed by molecular methods. Phylogenetic trees were generated using the Neighbor-Joining method and p-distance model on MEGA 7.0. Results: RSV prevalence varied between the sentinel (2.5%, 97/3891) and the non-sentinel ISS (20.7%, 3138/16779), being higher (P < 0.0001) among children aged <5 years. Bronchiolitis (62.9%, 183/291) and influenza-like illness (24.6%, 14/57) were associated (P < 0.0001) with RSV laboratory confirmation among children aged <6 months and adults ≥65 years, respectively. The HVR2 was sequenced for 562 samples. RSV-A (46.4%, 261/562) and RSV-B (53.6%, 301/562) strains clustered mainly to ON1 (89.2%, 233/261) and BA9 (92%, 277/301) genotypes, respectively, although NA1 and BA10 were also present until 2015/2016. Conclusion: The sequence and phylogenetic analysis reflected the relatively high diversity of Portuguese RSV strains. BA9 and ON1 genotypes, which have been circulating in Portugal since 2010/2011 and 2011/2012 respectively, predominated during the whole study period.
- Etiologia das infeções fúngicas invasivas e subcutâneas: análise dos dados da Rede Nacional de Vigilância Laboratorial das Infeções Fúngicas Invasivas e Subcutâneas (IFIs), 2013-2018Publication . Veríssimo, Cristina; Toscano, Cristina; Ferreira, Teresa; Abreu, Gabriela; Simões, Helena; Diogo, José; Queirós, Ana Maria; Santiago, Felicidade; Lima, Ana; Sabino, RaquelA epidemiologia das infeções fúngicas invasivas tem vindo a alterar-se com o surgimento de novos agentes etiológicos. A Rede Nacional de Vigilância Laboratorial de Infeções Fúngicas Invasivas e Subcutâneas (IFI) teve início em 2013 com o objetivo de melhor compreender a epidemiologia destas infeções em Portugal. O objetivo deste estudo é analisar os dados obtidos através da Rede entre junho de 2013 e setembro de 2018. Os laboratórios participantes asseguram a comunicação dos casos de IFIs ao Laboratório Nacional de Referência do INSA, enviando amostras biológicas ou isolados, acompanhados de um inquérito contendo informações demográficas, laboratoriais e avaliações clínicas dos doentes com suspeita de infeção fúngica. O estudo incluiu: i) casos de infeção fúngica invasiva (IFI) por fungos filamentosos/dimórficos, ii) casos de IFI provável, de acordo com os critérios estabelecidos pelo EORTC/MSG 2008, e iii) casos de infeções fúngicas subcutâneas. As infeções por leveduras não foram incluídas. Entre junho de 2013 e setembro de 2018, foram incluídos 52 casos, distribuídos da seguinte forma: IFI comprovada (por fungos filamentosos) (n=9); Infeção fúngica subcutânea (n=17); Infeção por fungos dimórficos endémicos (n=9), totalizando 67% de casos de IFI comprovados (n=35) e 33% de casos IFI provável (n=17). Os dados obtidos chamam a atenção para a grande diversidade de espécies envolvidas em infeções fúngicas profundas, com implicações para o diagnóstico clínico/laboratorial bem como para o tratamento destas infeções
- Genomic Study of a Clostridium difficile Multidrug Resistant Outbreak-Related Clone Reveals Novel Determinants of ResistancePublication . Isidro, Joana; Menezes, Juliana; Serrano, Mónica; Borges, Vítor; Paixão, Pedro; Mimoso, Margarida; Martins, Filomena; Toscano, Cristina; Santos, Andrea; Henriques, Adriano O.; Oleastro, MónicaBackground: Clostridium difficile infection (CDI) is prevalent in healthcare settings. The emergence of hypervirulent and antibiotic resistant strains has led to an increase in CDI incidence and frequent outbreaks. While the main virulence factors are the TcdA and TcdB toxins, antibiotic resistance is thought to play a key role in the infection by and dissemination of C. difficile. Methods: A CDI outbreak involving 12 patients was detected in a tertiary care hospital, in Lisbon, which extended from January to July, with a peak in February, in 2016. The C. difficile isolates, obtained from anaerobic culture of stool samples, were subjected to antimicrobial susceptibility testing with Etest®strips against 11 antibiotics, determination of toxin genes profile, PCR-ribotyping, multilocus variable-number tandem-repeat analysis (MLVA) and whole genome sequencing (WGS). Results: Of the 12 CDI cases detected, 11 isolates from 11 patients were characterized. All isolates were tcdA -/tcdB + and belonged to ribotype 017, and showed high level resistance to clindamycin, erythromycin, gentamicin, imipenem, moxifloxacin, rifampicin and tetracycline. The isolates belonged to four genetically related MLVA types, with six isolates forming a clonal cluster. Three outbreak isolates, each from a different MLVA type, were selected for WGS. Bioinformatics analysis showed the presence of several antibiotic resistance determinants, including the Thr82Ile substitution in gyrA, conferring moxifloxacin resistance, the substitutions His502Asn and Arg505Lys in rpoB for rifampicin resistance, the tetM gene, associated with tetracycline resistance, and two genes encoding putative aminoglycoside-modifying enzymes, aadE and aac(6')-aph(2″). Furthermore, a not previously described 61.3 kb putative mobile element was identified, presenting a mosaic structure and containing the genes ermG, mefA/msrD and vat, associated with macrolide, lincosamide and streptogramins resistance. A substitution found in a class B penicillin-binding protein, Cys721Ser, is thought to contribute to imipenem resistance. Conclusion: We describe an epidemic, tcdA -/tcdB +, multidrug resistant clone of C. difficile from ribotype 017 associated with a hospital outbreak, providing further evidence that the lack of TcdA does not impair the infectious potential of these strains. We identified several determinants of antimicrobial resistance, including new ones located in mobile elements, highlighting the importance of horizontal gene transfer in the pathogenicity and epidemiological success of C. difficile.
- Influenza virus type/subtype and different infection profiles by age group during 2017/2018 seasonPublication . Guiomar, Raquel; Pechirra, Pedro; Cristóvão, Paula; Costa, Inês; Conde, Patrícia; Côrte-Real, Rita; Branquinho, Paula; Garcia, David; Conde, Sílvia; Rodrigues, Fernando; Pereira-Vaz, João; Alves, José; Freitas, Ludivina; Mota Vieira, Luísa; Cabral Veloso, Rita; Bruges Armas, Jácome; Couto, Ana Rita; Ribeiro, Carlos; Barreto, Rosário; Cunha, Mário; Martins, Luís; Almeida, Sofia; Peres, Maria João; Viseu, Regina; Mota, Paula; Lopes, Paulo; Soares, Vânia; Vale, Fátima; Fonseca, Patrícia; Toscano, Cristina; Dias, AnaBackground: Influenza has a major impact in hospitalization during each influenza season. We analysed the influenza type/subtype distribution by age group and medical care wards (ambulatory, hospital, intensive care unit). Material and Methods: During 2017/2018 season, 14 hospitals from Portugal mainland and Atlantic Island (Azores and Madeira) reported to the National Influenza Centre 13747 cases of respiratory infection, all tested for influenza type and/or subtype. Epidemiological data: age, sample collection, hospital dwelling service and patient outcome were reported. Results: From the 13747 reported cases, 3717(27%) were influenza positive of which 2033 (55%) were influenza B, 722 (19%) A unsubtyped, 505 (14%) AH3, 442 (12%) AH1pdm09 and 15(0,1%) mixed infections. Influenza A was detected in 71% (204/208) of toddlers(<5 years) although in the remaining age groups influenza B was detected in more than 50% of the confirmed flu cases. Influenza B was the predominant virus in hospitalized and ICU influenza cases between 5-14 years (69% and 75%, respectively) and played a major role in elderly (65+ years) hospitalized and ICU cases(57% and 67%, respectively). AH1pdm09 virus was detected in 30% of the influenza confirmed ICU patients, 2.1 times more than in hospitalized cases in other wards and 3.3 times more than influenza AH1pdm09 cases in ambulatory care. Influenza mixed infection were detected sporadically,mainly in hospitalized and ICU patients. From 2080 known outcomes, 40(1.9%) patients deceased, influenza was confirmed in 11(28%) of these cases. Conclusions: Cocirculation of different influenza virus type/subtype may indicate different infection profiles by age groups and should guide influenza preventive/treatment measures.