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Background Severe Acute Respiratory Infections (SARI) surveillance is recommended to assess severity of respiratory infections disease. In 2021, the National Institute of Health Doutor Ricardo Jorge, in collaboration with two central hospitals, implemented a SARI sentinel surveillance system based on electronic health registries. We describe its application in the 2021/2022 season and compare the evolution of SARI cases with the COVID-19 and influenza activity in two regions of Portugal. Methods We identified SARI cases based on ICD-10 codes for influenza-like illness, cardiovascular diagnosis, respiratory diagnosis and respiratory infection. Pearson correlation and cross-correlations between weekly SARI cases, weekly COVID-19 cases and the number of weekly positive samples for influenza were estimated. Results A high correlation between SARI cases or hospitalizations due to respiratory infection and COVID-19 incidence was obtained (ρ = 0.78 and ρ = 0.82, respectively). Weekly SARI hospitalizations detected the COVID-19 epidemic peak a week earlier. A weak correlation was observed between SARI cases and the number of positive samples for influenza (ρ = -0.20). However, if restricted to hospitalizations due to cardiovascular diagnosis, a moderate correlation was observed (ρ = 0.37). Moreover, hospitalizations due to cardiovascular diagnosis detected the increase of influenza epidemic activity a week earlier. Conclusion In the 2021/2022 season, the Portuguese SARI sentinel surveillance system pilot was able to early detect the 5th COVID-19 epidemic wave and the increase of influenza activity. Establishing complementary virological inpatient surveillance is vital to aid in understanding the relationship between respiratory virus epidemics and disease severity.

Previously, we identified a Chlamydia trachomatis Lymphogranuloma venereum (LGV) recombinant strain possessing a unique non-LGV ompA genotype. Here, culture-independent genome sequencing confirms its circulation in Europe, Middle East and North America, and unveils genetic evidence of emergence of antibiotic resistance. Multi-country and systematic molecular surveillance is needed. This work results in part from the GenomePT project (POCI-01–0145-FEDER-022184), supported by COMPETE 2020— Operational Programme for Competitiveness and Internationalisation (POCI), Lisboa Portugal Regional Operational Programme (Lisboa2020), Algarve Portugal Regional Operational Programme (CRESC Algarve2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF), and by Fundação para a Ciência e a Tecnologia (FCT). This work was also supported by Fundos FEDER through the Programa Operacional Factores de Competitividade—COMPETE and by Fundos Nacionais through the Fundação para a Ciência e a Tecnologia within the scope of the project UID/BIM/00009/2019 (Centre for Toxicogenomics and Human Health -ToxOmics). info:eu-repo/semantics/publishedVersion

ObjectiveTo describe and analyze transmitted drug resistance (TDR) between 2014 and 2019 in newly infected patients with HIV-1 in Portugal and to characterize its transmission networks.MethodsClinical, socioepidemiological, and risk behavior data were collected from 820 newly diagnosed patients in Portugal between September 2014 and December 2019. The sequences obtained from drug resistance testing were used for subtyping, TDR determination, and transmission cluster (TC) analyses.ResultsIn Portugal, the overall prevalence of TDR between 2014 and 2019 was 11.0%. TDR presented a decreasing trend from 16.7% in 2014 to 9.2% in 2016 (pfor–trend = 0.114). Multivariate analysis indicated that TDR was significantly associated with transmission route (MSM presented a lower probability of presenting TDR when compared to heterosexual contact) and with subtype (subtype C presented significantly more TDR when compared to subtype B). TC analysis corroborated that the heterosexual risk group presented a higher proportion of TDR in TCs when compared to MSMs. Among subtype A1, TDR reached 16.6% in heterosexuals, followed by 14.2% in patients infected with subtype B and 9.4% in patients infected with subtype G.ConclusionOur molecular epidemiology approach indicates that the HIV-1 epidemic in Portugal is changing among risk group populations, with heterosexuals showing increasing levels of HIV-1 transmission and TDR. Prevention measures for this subpopulation should be reinforced.

Recent studies have shown that persistent SARS-CoV-2 infections in immunocompromised patients can trigger the accumulation of an unusual high number of mutations with potential relevance at both biological and epidemiological levels. Here, we report a case of an immunocompromised patient (non-Hodgkin lymphoma patient under immunosuppressive therapy) with a persistent SARS-CoV-2 infection (marked by intermittent positivity) over at least 6 months. Viral genome sequencing was performed at days 1, 164, and 171 to evaluate SARS-CoV-2 evolution. Among the 15 single-nucleotide polymorphisms (SNPs) (11 leading to amino acid alterations) and 3 deletions accumulated during this long-term infection, four amino acid changes (V3G, S50L, N87S, and A222V) and two deletions (18-30del and 141-144del) occurred in the virus Spike protein. Although no convalescent plasma therapy was administered, some of the detected mutations have been independently reported in other chronically infected individuals, which supports a scenario of convergent adaptive evolution. This study shows that it is of the utmost relevance to monitor the SARS-CoV-2 evolution in immunocompromised individuals, not only to identify novel potentially adaptive mutations, but also to mitigate the risk of introducing "hyper-evolved" variants in the community. IMPORTANCE Tracking the within-patient evolution of SARS-CoV-2 is key to understanding how this pandemic virus shapes its genome toward immune evasion and survival. In the present study, by monitoring a long-term COVID-19 immunocompromised patient, we observed the concurrent emergence of mutations potentially associated with immune evasion and/or enhanced transmission, mostly targeting the SARS-CoV-2 key host-interacting protein and antigen. These findings show that the frequent oscillation in the immune status in immunocompromised individuals can trigger an accelerated virus evolution, thus consolidating this study model as an accelerated pathway to better understand SARS-CoV-2 adaptive traits and anticipate the emergence of variants of concern. This study was partially cofunded by Fundação para a Ciência e Tecnologia and Agência de Investigação Clínica e Inovação Biomédica (234_596874175) on behalf of the Research 4 COVID-19 call. Some infrastructural resources used in this study come from the GenomePT project (POCI-01-0145-FEDER-022184), supported by COMPETE 2020-Operational Programme for Competitiveness and Internationalisation (POCI), Lisboa Portugal Regional Operational Program (Lisboa2020), Algarve Portugal Regional Operational Programme (CRESC Algarve2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF), and by Fundação para a Ciência e a Tecnologia (FCT). info:eu-repo/semantics/publishedVersion

Commentary: G. Ciccarese et al. J Eur Acad Dermatol Venereol 2021; 35:1606-1607. https://doi.org/10.1111/jdv.17468. Background: Lymphogranuloma venereum (LGV) is a sexual transmitted infection (STI), currently endemic within the population of men who have sex with men (MSM) of Western Countries. L2B variant has been reported as the predominant strain in the current LGV epidemics, although a shift towards L2-434 has been observed in some European countries. Objectives: To evaluate and characterize the population with LGV infection diagnosed in Centro Hospitalar Universitário de Lisboa Central, Lisbon, Portugal. Methods: A retrospective analysis of all LGV diagnoses between 2016 and 2019 was performed. The diagnosis was established through ompA-genotyping of samples yielding a positive result to Chlamydia trachomatis (CT). All considered samples were retrieved from the clinician activity, through swabbing and urine analysis and CT infection diagnosis was obtained using real-time PCR. Results: During the period studied 16 279 CT diagnostics tests were employed, with a striking increase from 2016 (n = 467) to 2019 (n = 9362). A total of 1602 diagnoses of CT were established, from which 168 (10.5%) corresponded to LGV, with both infections showing a rising evolution, between 2016 and 2019, of 2.9 and 2.7 times, respectively. The majority of the LGV strains were genotyped as L2/434 (67.3%; n = 113). LGV predominantly affected MSM and men who have sex with men and women (97.0%; n = 163). Anorectal infection was the most prevalent one (90.5%; n = 152), being proctitis the main clinical presentation (76.2%; n = 128). Absence of symptoms was reported in almost 15% of the cases (n = 24). The presence of concomitant infection with human immunodeficiency virus was dominant (73.2%; n = 123) and the prevalence of one or more STI co-infections was about 60.1% (n = 99). Conclusions: An increasing evolution of CT and LGV testing and diagnosing was observable throughout the studied period. Characteristics of the population are similar with those described within LGV epidemics. In accordance with recent European studies, predominance towards L2 genotype was identified. info:eu-repo/semantics/publishedVersion

Aim: The diagnosis of coronavirus disease 2019 (COVID-19) depends on accurate and rapid testing. Choosing an appropriate sample may impact diagnosis. Naso-oropharyngeal swabs (NOS) are most frequently used, despite several limitations. Since studies suggest nasopharyngeal aspirate (NPA) as a superior alternative in children, we hypothesised collecting both nasopharyngeal swab and aspirate would improve our diagnostic accuracy. Methods: Observational, longitudinal, prospective study from 7 March to 7 May in a tertiary paediatric hospital in Lisbon. The objective was to compare the rate of detection of SARS-CoV-2 between NOS and NPA samples collected simultaneously. Results: A total of 438 samples collected from 85 patients with confirmed COVID-19. There were 47.7% overall positive specimens - 32% (70/219) positive NOS and 63.5% (139/219) positive NPA. The tests were 67.6% concordant (k = 0.45). 50.3% had positive NPA with negative NOS, while 1.3% had positive NOS with negative NPA. NPA proved to be more sensitive (98.6% with 95% confidence interval 91.2-99.9% vs. 49.6% with 95% confidence interval 41.1-58.2%, P < 0.001). Additionally, the difference between NPA and NOS positive samples was statistically significant across all population groups (age, health condition, clinical presentation, contact with COVID-19 patients or need for hospitalisation), meaning NPA is more sensitive overall. Conclusions: Nasopharyngeal aspirates had greater sensitivity than naso-oropharyngeal swabs in detecting SARS-CoV-2. Our results suggest paediatric patients would benefit from collecting nasopharyngeal aspirates in hospital settings, whenever feasible, to improve diagnosis of COVID-19. info:eu-repo/semantics/publishedVersion

Chlamydia trachomatis is the most prevalent sexually transmitted bacterium worldwide and the causative agent of trachoma. Its strains are classified according to their ompA genotypes, which are strongly linked to differential tissue tropism and disease outcomes [ocular disease, urogenital disease and lymphogranuloma venereum (LGV)]. While the genome-based species phylogenetic tree presents four main clades correlating with tropism/prevalence, namely ocular, LGV, urogenital T1 (more prevalent genotypes) and urogenital T2 (less prevalent genotypes), inter-clade exchange of ompA is considered a rare phenomenon probably mediating marked tropism alterations. An LGV epidemic, associated with the clonal expansion of the L2b genotype, has emerged in the last few decades, raising concerns particularly due to its atypical clinical presentation (ulcerative proctitis) and circulation among men who have sex with men (MSM). Here, we report an LGV outbreak, mostly affecting human immunodeficiency virus-positive MSM engaging in high-risk sexual practices, caused by an L2b strain with a rather unique non-LGV ompA signature that precluded the laboratory notification of this outbreak as LGV. C. trachomatis whole-genome capture and sequencing directly from clinical samples was applied to deeply characterize the genomic backbone of this novel LGV outbreak-causing clone. It revealed a chimeric genome structure due to the genetic transfer of ompA and four neighbouring genes from a serovar D/Da strain, likely possessing the genomic backbone associated with the more prevalent urogenital genotypes (T1 clade), to an LGV (L2b) strain. The hybrid L2b/D-Da strain presents the adhesin and immunodominant antigen MOMP (major outer membrane protein) (encoded by ompA) with an epitope repertoire typical of non-invasive genital strains, while keeping the genome-dispersed virulence fingerprint of a classical LGV strain. As previously reported for inter-clade ompA exchange among non-LGV clades, this novel C. trachomatis genomic mosaic involving a contemporary epidemiologically and clinically relevant LGV strain may have implications on its transmission, tissue tropism and pathogenic capabilities. The emergence of variants with epidemic and pathogenic potential highlights the need for more focused surveillance strategies to capture C. trachomatis evolution in action. info:eu-repo/semantics/publishedVersion

Chlamydia trachomatisis the most prevalent sexually transmitted bacteria worldwide and the causative agent of blinding trachoma. Strains are classified based onompAgenotypes, which are strongly linked with differential tissue tropism and disease outcomes. A lymphogranuloma venereum (LGV) epidemics, characterized by ulcerative proctitis, has emerged in the last two decades (mainly L2b genotype), raising high concern especially due to its circulation among men who have sex with men (MSM). Here, we report an ongoing outbreak (mostly affecting HIV-positive MSM engaging in high-risk practices) caused by an L2b strain with a rather unique genome makeup that precluded the laboratory notification of this outbreak as LGV due to its non-LGVompAsignature. Homologous recombination mediated the transfer of a ~4.5Kbp fragment enrollingCT681/ompAand neighboring genes (CT677/rrf, CT678/pyrH, CT679/tsf, CT680/rpsB) from a serovar D/Da strain likely possessing the typical T1 clade genome backbone associated with most prevalent genotypes (E and F). The hybrid L2b/D-Da strain presents the adhesin and immunodominant antigen MOMP (coded byompA) with an epitope repertoire typical of non-invasive genital strains, while keeping the genome-dispersed virulence fingerprint of a classical LGV (L2b) strain. As previously reported for inter-cladeompAexchange among non-LGV clades, this unprecedentedC. trachomatisgenomic mosaic involving a contemporary epidemiologically and clinically relevant LGV strain may have implications on its transmission, tissue tropism and pathogenic capabilities. The emergence of such variants with epidemic and pathogenic potential highlights the need of more oriented surveillance strategies focused on capturing the C.trachomatisevolution in action.

Em colaboração com a Rede Portuguesa de Laboratóros para o Diagnóstico da Gripe Introduction: Immune profile for influenza viruses is highly changeable over time. Serological studies can assess the prevalence of influenza, estimate the risk of infection, highlight asymptomatic infection rate and can also provide data on vaccine coverage. The aims of the study were to evaluate pre-existing cross-protection against influenza A(H3) drift viruses and to assess influenza immunity in the Portuguese population. Materials and methods: We developed a cross-sectional study based on a convenience sample of 626 sera collected during June 2014, covering all age groups, both gender and all administrative health regions of Portugal. Sera antibody titers for seasonal and new A(H3) drift influenza virus were evaluated by hemagglutination inhibition assay (HI). Seroprevalence to each seasonal influenza vaccine strain virus and to the new A(H3) drift circulating strain was estimated by age group, gender and region and compared with seasonal influenza-like illness (ILI) incidence rates before and after the study period. Results: Our findings suggest that seroprevalences of influenza A(H3) (39.9%; 95% CI: 36.2–43.8) and A(H1)pdm09 (29.7%; 95% CI: 26.3–33.4) antibodies were higher than for influenza B, in line with high ILI incidence rates for A(H3) followed by A(H1)pdm09, during 2013/2014 season. Low pre-existing crossprotection against new A(H3) drift viruses were observed in A(H3) seropositive individuals (46%). Both against influenza A(H1)pdm09 and A(H3) seroprotection was highest in younger than 14-years old. Protective antibodies against influenza B were highest in those older than 65 years old, especially for B/Yamagata lineage, 33.3% (95% CI: 25.7–41.9). Women showed a high seroprevalence to influenza, although without statistical significance, when compared to men. A significant decreasing trend in seroprotection from north to south regions of Portugal mainland was observed. Conclusions: Our results emphasize that low seroprotection increases the risk of influenza infection in the following winter season. Seroepidemiological studies can inform policy makers on the need for vaccination and additional preventive measures. This work was supported by the National Institute of Health Doutor Ricardo Jorge, IP Lisbon, Portugal. info:eu-repo/semantics/acceptedVersion

ResumoO vírus da hepatite D (VHD) é um vírus satélite, que necessita do vírus da hepatite B (VHB) para a sua replicação. Na maioria dos casos, o VHD suprime a replicação do VHB e, nestas circunstâncias, está recomendado o tratamento com Interferão. Nos doentes em que não se observa resposta virológica após o 1.° ano de terapêutica, o seu prolongamento pode aumentar a taxa de resposta virológica sustentada.Caso clínicoHomem, 42 anos, com hepatite crónica B, anti-HBe positivo e baixa carga viral. Apresentava aminotransferases aumentadas que persistiram, mesmo após negativação do ADN-VHB. A positividade da fração IgM do AcVHD conduziu ao diagnóstico de co-infecção VHB-VHD. Foi tratado com PegInterfeão α-2a durante 102 semanas. Observou-se normalização das aminotransferases à 33.ª semana, negativação da IgM do AcVHD à 88.ª semana e resposta virológica sustentada com perda do AgHB.ComentáriosEste caso ilustra a importância de se manter o tratamento nos doentes com infecção VHD até resposta virológica, uma vez que a cura do VHD pode acompanhar-se da cura da infecção VHB.AbstractHepatitis D virus (HDV) is a satellite virus which needs hepatitis B virus (HBV) for its replication. In most cases, HDV suppresses HBV replication and in these circumstances the treatment should be with Interferon. In patients that have no virological response after the 1th year of therapy, continuing it will possible increase the virological response and the loss of the HBs antigen.Case report42 years old man with chronic HBV, anti-HBe positive and low HBV viral load. He had increased transaminases which had persisted even after HBV-DNA negativation. We performed antibody anti-HDV that came positive and treated the patient with PegInterferon α-2a during 102 weeks. We assist to normalization of the transaminases at week 33 and negativation of IgM-HVD at week 88. At the end of the treatment RNA-HDV was negative and the patient lost HBs antigen that persisted over the next sixth months.CommentsThis case illustrates the importance of maintaining treatment until HDV virological response since the cure of the HDV may be accompanied by the cure of HBV infection.

Em colaboração com o Laboratório de Doenças Evitáveis pela Vacinação (DDI), com o Centro Hospitalar de Setúbal e com o Centro Hospitalar Lisboa Central Abstract of the presentation: Journal of Clinical Virology 82S (2016) S1–S142. (Abstract no: 289) Background: Enterovirus D68 (EV-D68) was first isolated in 1962, and since then associated with respiratory illness. The report of severe respiratory and neurological disease including deaths associated to EV-D68 in United States and Canada during August 2014 highlighted the need of epidemiological information regarding EV-D68 circulation. In Europe information was scarce, available only for few countries. In Portugal there was no data available and was critical to know the epidemiology of EV-D68, especially in children hospitalized with severe respiratory or neurological disease. This study aims to identify EV-D68 in Enterovirus positive respiratory samples in children under 18 with clinical diagnosis of severe respiratory infection or neurological illness. Methods: During 2015/16 winter season, between November/2015 and March/2016, 29 EV positive cases were reported to the National Influenza and Other Respiratory Virus Reference Laboratory (NIC) by two hospitals located in Lisbon and Setubal districts. EV diagnosis was performed in hospitals by biomolecular methods using commercial kits (real time multiplex-PCR, FTD Respiratory pathogens 21 and CLART Pneumovir, Genomica, respectively). EV-D68 was diagnosed by an in house real-time PCR [1]. Virus isolation in RD cell line and phylogenentic analysis of the VP1/VP3 genomic regions will enable the identification of genetic groups in circulation. All samples were irreversibly anonymized. Demographic and clinical data were collected. Results: EV-D68 was confirmed in 20 respiratory samples previously positive for EV (69%; 20/29). Samples were collected from children with age ranging from 2 months to 6 years old, both genders (9 female; 11 male) with diagnosis of severe respiratory or neurological illness. Eighteen cases were hospitalized (90%; 18/20). Bronchiolitis and pneumonia were the most frequently reported diagnosis, corresponding to 70% (14/20). Two cases have neurologic diagnosis. EV-D68 was identified throughout all study period with the higher number of positive cases detected during January 2016, in week 3. Virus isolation and genetic characterization are under way with expected results in virus phylogeny and evaluation on similarity with recent circulating strains in United States, Canada and European countries. Conclusions: EV-D68 was detected in a high positive rate (69%) among EV positive cases. This positive rate of EV-D68 was higher compared to the positivity rate of 10,2 %, calculated in a European study during 2014 [2]. This finding could be linked to the selection of severe and hospitalized patients in present study, highlighting the involvement of EV-D68 with severe respiratory disease in children. The identification of EV-D68 is also crucial in respiratory samples in children with clinical diagnosis of neurological illness. This study is the first attempt to describe the prevalence of EV-D68 in severe paediatric cases, in Portugal. The strength of EV-D68 surveillance in paediatric and adult population at the national level will be important to understand the epidemiology of EV-D68, age-related susceptibility and association with disease severity. info:eu-repo/semantics/publishedVersion