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BackgroundInvestigating the role of late presenters (LP) on HIV-1 transmission is important, as they can contribute to the onward spread of HIV-1 virus in the long period before diagnosis, when they are not aware of their HIV statusObjectiveTo describe the clinical and socio-demographic characteristics of HIV-1 infected individuals followed in Europe, to characterize patients in clusters and to compare transmission clusters (TC) in LP vs non-late presenters (NLP) populations.MethodsClinical, socio-demographic and genotypic information from 38531 HIV-1 infected patients was collected from the EuResist Integrated Database (EIDB) between 1981 and 2019. Sequences were aligned using VIRULIGN. Maximum likelihood (ML) phylogenies were constructed using FastTree. Putative transmission clusters were identified using ClusterPicker v1.332. Statistical analyses were performed using R.Results32652 (84·7%) sequences were from subtype B, 3603 (9·4%) were from subtype G, and 2276 (5·9%) were from subtype A. The median age was 33 (IQR: 26·0-41·0) years old and 75·5% of patients were males. The main transmission route was through homosexual (MSM) contact (36·9%) and 86·4% were originated from Western Europe. Most patients were native (84·2%), 59·6% had a chronic infection, and 73·4% had acquired drug resistance (ADR). CD4 count and viral load at diagnosis (log10) presented a median of 341 cells/mm3, and of log10 4·3 copies/mL, respectively. 51·4% of patients were classified as LP and 21·6% patients were inside TCs. Most patients from subtype B (85·6%) were in clusters, compared to subtypes A (5·2%) and G (9·2%). Phylogenetic analyses showed consistent clustering of MSM individuals. In subtype A, patients in TCs were more frequently MSM patients and with a recent infection. For subtype B, patients in TCs were more frequently those with older age (≥ 56), MSM transmission route, originating from Western Europe, migrants, and with a recent infection. For subtype G, patients in TC were more frequently patients with recent infection and migrants. When analysing cluster size, we found that NLP more frequently belonged to large clusters (>8 patients) when compared to LP.ConclusionWhile late presentation is still a threat to HIV-1 transmission, LP individuals are more present either outside or in small clusters, indicating a limited role of late presentation to HIV-1 transmission.

OBJECTIVE: To investigate the dynamics of phylogenetic transmission clusters involving immigrants of Portuguese Speaking Countries living in Portugal. DESIGN/METHODS: We included genomic sequences, sociodemographic and clinical data from 772 HIV migrants followed in Portugal between 2001 and 2017. To reconstruct HIV-1 transmission clusters, we applied phylogenetic inference from 16 454 patients: 772 migrants, 2973 Portuguese and 12 709 global controls linked to demographic and clinical data. Transmission clusters were defined using: clusters with SH greater than 90% (phylogenetic support), genetic distance less than 3.5% and clusters that included greater than 66% of patients from one specific geographic origin compared with the total of sequences within the cluster. Logistic regression was performed to assess factors associated with clustering. RESULTS: Three hundred and six (39.6%) of migrants were included in transmission clusters. This proportion differed substantially by region of origin [Brazil 54% vs. Portuguese Speaking African Countries (PALOPs) 36%, P

BackgroundUndiagnosed HIV-1 patients still account for 25% of worldwide HIV patients. Studying late presenters for HIV care may help to identify characteristics of such patients.ObjectiveThe present study aims to identify factors associated with late presentation (LP) and late presentation with advanced disease (LPAD) based on a population of patients followed in a Portuguese hospital between 1984 and 2017.MethodsSociodemographic and clinical data from infected patients with HIV-1 aged 18 years and older, followed in Egas Moniz Hospital, in Portugal were collected.ResultsOf the 907 patients included in this study, 68.7% were males and the median age was 37 years (IQR 30-47). 459 patients (50.6%) were LP and, of these, 284 patients (61.9%) were LPAD. The LP population mostly originated from Portugal and Sub-Saharan Africa (64.4% and 28.8%; p=0.004) and the HIV exposure category mainly heterosexuals and MSM (57.0% and 24.9%; ppp=0.002) and origin from Sub-Saharan Africa (aOR 2.24; 1.44-3.50; pConclusionLate presentation is a major obstacle to halt the HIV epidemic. In this population, the majority of newly diagnosed HIV-infected individuals were late presenters. Our results characterize vulnerable populations that should be frequently tested for HIV.

PurposeThe CRIAS (Health trajectories of Immigrant Children in Amadora) cohort study was created to explore whether children exposed to a migratory process experience different health risks over time, including physical health, cognitive, socioemotional and behavioural challenges and different healthcare utilisation patterns.ParticipantsThe original CRIAS was set up to include 604 children born in 2015, of whom 50% were immigrants, and their parents. Recruitment of 420 children took place between June 2019 and March 2020 at age 4/5 years, with follow-up carried out at age 5/6 years, at age 6/7 years currently under way.Findings to dateBaseline data at age 4/5 years (2019–2020) suggested immigrant children to be more likely to belong to families with less income, compared with non-immigrant children. Being a first-generation immigrant child increased the odds of emotional and behavioural difficulties (adjusted OR 2.2; 95% CI: 1.06 to 4.76); more immigrant children required monitoring of items in the psychomotor development test (38.5% vs 28.3%). The prevalence of primary care utilisation was slightly higher among immigrant children (78.0% vs 73.8%), yet they received less health monitoring assessments for age 4 years. Utilisation of the hospital emergency department was higher among immigrants (53.2% vs 40.6%). Age 5 years follow-up (2020–2021) confirmed more immigrant children requiring monitoring of psychomotor development, compared with non-immigrant children (33.9% vs 21.6%). Economic inequalities exacerbated by post-COVID-19 pandemic confinement with parents of immigrant children 3.2 times more likely to have their household income decreased.Future plansFurther follow-up will take place at 8, 10, 12/13 and 15 years of age. Funds awarded by the National Science Foundation will allow 900 more children from four other Lisbon area municipalities to be included in the cohort (cohort-sequential design).

International audience; The Zika virus (ZIKV) disease caused a public health emergency of international concern that started in February 2016. The overall number of ZIKV-related cases increased until November 2016, after which it declined sharply. While the evaluation of the potential risk and impact of future arbovirus epidemics remains challenging, intensified surveillance efforts along with a scale-up of ZIKV whole-genome sequencing provide an opportunity to understand the patterns of genetic diversity, evolution, and spread of ZIKV. However, a classification system that reflects the true extent of ZIKV genetic variation is lacking. Our objective was to characterize ZIKV genetic diversity and phylodynamics, identify genomic footprints of differentiation patterns, and propose a dynamic classification system that reflects its divergence levels. We analysed a curated dataset of 762 publicly available sequences spanning the full-length coding region of ZIKV from across its geographical span and collected between 1947 and 2021. The definition of genetic groups was based on comprehensive evolutionary dynamics analyses, which included recombination and phylogenetic analyses, within- and between-group pairwise genetic distances comparison, detection of selective pressure, and clustering analyses. Evidence for potential recombination events was detected in a few sequences. However, we argue that these events are likely due to sequencing errors as proposed in previous studies. There was evidence of strong purifying selection, widespread across the genome, as also detected for other arboviruses. A total of 50 sites showed evidence of positive selection, and for a few of these sites, there was amino acid (AA) differentiation between genetic clusters. Two main genetic clusters were defined, ZA and ZB, which correspond to the already characterized ‘African’ and ‘Asian’ genotypes, respectively. Within ZB, two subgroups, ZB.1 and ZB.2, represent the Asiatic and the American (and Oceania) lineages, respectively. ZB.1 is further subdivided into ZB.1.0 (a basal Malaysia sequence sampled in the 1960s and a recent Indian sequence), ZB.1.1 (South-Eastern Asia, Southern Asia, and Micronesia sequences), and ZB.1.2 (very similar sequences from the outbreak in Singapore). ZB.2 is subdivided into ZB.2.0 (basal American sequences and the sequences from French Polynesia, the putative origin of South America introduction), ZB.2.1 (Central America), and ZB.2.2 (Caribbean and North America). This classification system does not use geographical references and is flexible to accommodate potential future lineages. It will be a helpful tool for studies that involve analyses of ZIKV genomic variation and its association with pathogenicity and serve as a starting point for the public health surveillance and response to on-going and future epidemics and to outbreaks that lead to the emergence of new variants.The Zika virus (ZIKV) disease caused a public health emergency of international concern that started in February 2016. The overall number of ZIKV-related cases increased until November 2016, after which it declined sharply. While the evaluation of the potential risk and impact of future arbovirus epidemics remains challenging, intensified surveillance efforts along with a scale-up of ZIKV whole-genome sequencing provide an opportunity to understand the patterns of genetic diversity, evolution, and spread of ZIKV. However, a classification system that reflects the true extent of ZIKV genetic variation is lacking. Our objective was to characterize ZIKV genetic diversity and phylodynamics, identify genomic footprints of differentiation patterns, and propose a dynamic classification system that reflects its divergence levels. We analysed a curated dataset of 762 publicly available sequences spanning the full-length coding region of ZIKV from across its geographical span and collected between 1947 and 2021. The definition of genetic groups was based on comprehensive evolutionary dynamics analyses, which included recombination and phylogenetic analyses, within- and between-group pairwise genetic distances comparison, detection of selective pressure, and clustering analyses. Evidence for potential recombination events was detected in a few sequences. However, we argue that these events are likely due to sequencing errors as proposed in previous studies. There was evidence of strong purifying selection, widespread across the genome, as also detected for other arboviruses. A total of 50 sites showed evidence of positive selection, and for a few of these sites, there was amino acid (AA) differentiation between genetic clusters. Two main genetic clusters were defined, ZA and ZB, which correspond to the already characterized ‘African’ and ‘Asian’ genotypes, respectively. Within ZB, two subgroups, ZB.1 and ZB.2, represent the Asiatic and the American (and Oceania) lineages, respectively. ZB.1 is further subdivided into ZB.1.0 (a basal Malaysia sequence sampled in the 1960s and a recent Indian sequence), ZB.1.1 (South-Eastern Asia, Southern Asia, and Micronesia sequences), and ZB.1.2 (very similar sequences from the outbreak in Singapore). ZB.2 is subdivided into ZB.2.0 (basal American sequences and the sequences from French Polynesia, the putative origin of South America introduction), ZB.2.1 (Central America), and ZB.2.2 (Caribbean and North America). This classification system does not use geographical references and is flexible to accommodate potential future lineages. It will be a helpful tool for studies that involve analyses of ZIKV genomic variation and its association with pathogenicity and serve as a starting point for the public health surveillance and response to on-going and future epidemics and to outbreaks that lead to the emergence of new variants.

To control the Human Immunodeficiency Virus (HIV) pandemic, the World Health Organization (WHO) set the 90-90-90 target to be reached by 2020. One major threat to those goals is late presentation, which is defined as an individual presenting a TCD4+ count lower than 350 cells/mm3 or an AIDS-defining event. The present study aims to identify determinants of late presentation in Europe based on the EuResist database with HIV-1 infected patients followed-up between 1981 and 2019. Our study includes clinical and socio-demographic information from 89851 HIV-1 infected patients. Statistical analysis was performed using RStudio and SPSS and a Bayesian network was constructed with the WEKA software to analyze the association between all variables. Among 89851 HIV-1 infected patients included in the analysis, the median age was 33 (IQR: 27.0–41.0) years and 74.4% were males. Of those, 28889 patients (50.4%) were late presenters. Older patients (&gt, 56), heterosexuals, patients originated from Africa and patients presenting with log VL &gt, 4.1 had a higher probability of being late presenters (p &lt, 0.001). Bayesian networks indicated VL, mode of transmission, age and recentness of infection as variables that were directly associated with LP. This study highlights the major determinants associated with late presentation in Europe. This study helps to direct prevention measures for this population.

The large diversity of new tick-borne phleboviruses, and the negative impacts of the virulent viruses on human/animal health have led to a growing interest in their analysis. In this report, new insights are brought out into the diversity of putative phleboviruses circulating in Portugal (both the continental territory and the islands of Sao Miguel, in the Azores, and Madeira), as well as in the Spanish western regions of Extremadura and Castilla and Leon. Phlebovirus sequences were frequently detected (L-segment) from both questing and feeding ticks, but especially in Rhipicephalus sanguineus sensu lato (s.l.) specimens. These sequences were detected in adult ticks, as well as nymphs and eggs, supporting the hypothesis of viral maintenance by vertical transmission. Though multiple genetic groups could be identified in phylogenetic trees (AnLuc, KarMa, RiPar virus 1, and Spanish group 1 and 2), all the sequences from Portugal and Spain shared common ancestry with other viral sequence obtained from samples collected over a large geographic coverage. Spatiotemporal analysis placed Middle-East as the geographic origin of the most recent common ancestor (MRCA) of all phleboviruses analysed in the present study. More recent viral transitions might include migrations from Spain to continental Portugal, and from there to the Portuguese Islands. Our findings suggest that the time of the MRCA of phleboviruses was dated around 225 years ago [95% HPD: 124–387 year before the last sampling date].

Schistosomiasis is a chronic neglected tropical disease saddling millions of people in the world, mainly children living in poor rural areas. Praziquantel (PZQ) is currently the only drug used for the treatment and control of this disease. However, the extensive use of this drug has brought concern about the emergence of PZQ-resistance/tolerance bySchistosoma mansoni. Studies ofSchistosomaspp. genome, transcriptome, and proteome are crucial to better understand this situation. In thisin vitrostudy, we compare the proteomes of aS. mansonivariant strain stably resistant to PZQ and isogenic to its fully susceptible parental counterpart, identifying proteins from male and female adult parasites of PZQ-resistant and PZQ-susceptible strains, exposed and not exposed to PZQ. A total of 60Schistosomaspp. proteins were identified, some of which present or absent in either strain, which may putatively be involved in the PZQ-resistance phenomenon. These proteins were present in adult parasites not exposed to PZQ, but some of them disappeared when these adult parasites were exposed to the drug. Understanding the development of PZQ-resistance inS. mansoniis crucial to prolong the efficacy of the current drug and develop markers for monitoring the potential emergence of drug resistance.

HIV and tuberculosis (TB) are among the global deadliest diseases. Migrant populations are particularly vulnerable to these infections. Yet, literature is still scarce on the epidemiology of HIV-TB co-infection among migrants. In this study, we characterized native and migrant HIV patients followed in Portuguese hospitals, who were diagnosed with TB, regarding their sociodemographic, clinical, and genomic characteristics. Among 67 patients with HIV and TB diagnoses, there were 24 migrants, most from sub-Saharan Africa. Most patients had CD4+ T cell counts below 350 cells/μL, and were diagnosed simultaneously for HIV and TB. When compared to natives, migrants presented a higher proportion of non-B HIV-1 infections. Patients infected with these non-B HIV-1 strains presented higher viral loads, which can have an important impact for the transmissibility and pathogenicity of both diseases. Future studies should investigate different HIV strains and consequences for TB and HIV transmission and disease outcomes, especially among vulnerable populations.

BackgroundTreatment for all recommendations has allowed access to antiretroviral (ARV) treatment to an increasing number of patients. This minimizes transmission of infection but can potentiate the risk for development of transmitted drug resistance (TDR) and acquired drug resistance (ADR).ObjectiveTo study the trends of TDR and ADR in patients followed in Portuguese hospitals between 2001 and 2017.Method11911 patients of the Portuguese REGA database were included. TDR was defined as the presence of one or more surveillance drug resistance mutations according to the WHO surveillance list. Phenotypic resistance to ARV was evaluated with Standford HIVdb v7.0. Patterns of TDR, ADR and prevalence of mutations over time were analysed with logistic regression.ResultsThe prevalence of TDR increased from 7.9% in 2003 to 13.1% in 2017 (pfor-trendfor-trend=0.002) and 2.9% to 8.9% (pfor-trendfor-trend=0.985). Paradoxically, the prevalence of ADR declined from 86.6% in 2001 to 51.0% in 2017 (pfor-trendfor-trendConclusionsWhile ADR is declining since 2001, TDR has been increasing, reaching a value of 13.1% by the end of 2017. It is urgent to develop public health programs to monitor levels and patterns of TDR in newly diagnosed patients.

Migration is associated with HIV-1 vulnerability. Objectives: To identify long-term trends in HIV-1 molecular epidemiology and antiretroviral drug resistance (ARV) among migrants followed up in Portugal Methods: 5177 patients were included between 2001 and 2017. Rega, Scuel, Comet, and jPHMM algorithms were used for subtyping. Transmitted drug resistance (TDR) and Acquired drug resistance (ADR) were defined as the presence of surveillance drug resistance mutations (SDRMs) and as mutations of the IAS-USA 2015 algorithm, respectively. Statistical analyses were performed. Results: HIV-1 subtypes infecting migrants were consistent with the ones prevailing in their countries of origin. Over time, overall TDR significantly increased and specifically for Non-nucleoside reverse transcriptase inhibitor (NNRTIs) andNucleoside reverse transcriptase inhibitor (NRTIs). TDR was higher in patients from Mozambique. Country of origin Mozambique and subtype B were independently associated with TDR. Overall, ADR significantly decreased over time and specifically for NRTIs and Protease Inhibitors (PIs). Age, subtype B, and viral load were independently associated with ADR. Conclusions: HIV-1 molecular epidemiology in migrants suggests high levels of connectivity with their country of origin. The increasing levels of TDR in migrants could indicate an increase also in their countries of origin, where more efficient surveillance should occur., SCOPUS: ar.j, info:eu-repo/semantics/published

Introduction: Treatment for All recommendations have allowed access to antiretroviral (ARV) treatment for an increasing number of patients. This minimizes the transmission of infection but can potentiate the risk of transmitted (TDR) and acquired drug resistance (ADR). Objective: To study the trends of TDR and ADR in patients followed up in Portuguese hospitals between 2001 and 2017. Methods: In total, 11,911 patients of the Portuguese REGA database were included. TDR was defined as the presence of one or more surveillance drug resistance mutation according to the WHO surveillance list. Genotypic resistance to ARV was evaluated with Stanford HIVdb v7.0. Patterns of TDR, ADR and the prevalence of mutations over time were analyzed using logistic regression. Results and Discussion: The prevalence of TDR increased from 7.9% in 2003 to 13.1% in 2017 (p &lt, 0.001). This was due to a significant increase in both resistance to nucleotide reverse transcriptase inhibitors (NRTIs) and non-nucleotide reverse transcriptase inhibitors (NNRTIs), from 5.6% to 6.7% (p = 0.002) and 2.9% to 8.9% (p &lt, 0.001), respectively. TDR was associated with infection with subtype B, and with lower viral load levels (p &lt, 0.05). The prevalence of ADR declined from 86.6% in 2001 to 51.0% in 2017 (p &lt, 0.001), caused by decreasing drug resistance to all antiretroviral (ARV) classes (p &lt, 0.001). Conclusions: While ADR has been decreasing since 2001, TDR has been increasing, reaching a value of 13.1% by the end of 2017. It is urgently necessary to develop public health programs to monitor the levels and patterns of TDR in newly diagnosed patients.

Background Tuberculosis (TB) is one of the main causes of death worldwide. In 2017, the proportion of TB cases among foreign-born individuals in Portugal was of 19%, and this proportion has been increasing. TB control among migrant populations largely depends on the role of the healthcare providers and their close contact with the patients. We aimed to explore the perspectives of primary care providers on the provision of TB care for migrant patients in Portugal. Methods An online survey and semi-structured interviews were conducted with healthcare providers from primary care settings in Portugal. Overall, 120 participated in the survey, and 17 were interviewed. Data from the survey and interviews were analysed using descriptive statistics and thematic analysis, respectively. Results Many participants (36.9%) considered that migrants arrive at an advanced stage of TB disease, and the main reason referred was migrants’ unawareness of the disease and its symptoms. Moreover, half (50.0%) of the providers considered that migrants frequently interrupt treatment, mainly due to their mobility, which causes difficulties in follow-up, and their social isolation that hampers social support. Overall, three main barriers for migrants’ access and use of TB care were mentioned: migrants’ low socioeconomic status, great bureaucracy required to register at services, and obstacles to benefit from social protection. Providers also suggested more training to improve their cultural competence and updating training on TB care. Conclusions Our study have shown social and economic factors that must be addressed to improve migrants’ access and use of TB care. In addition, increased health literacy for migrants and more training initiatives for providers can also contribute to improve TB care for migrants. Key messages Perspectives from primary care providers in Portugal highlighted social, economic, and administrative obstacles hampering the provision of TB care for migrants. With this work we expected to contribute with evidence to improve TB care for migrant patients in Portugal.

One of the cornerstones in combating the HIV pandemic is being able to assess the current state and evolution of local HIV epidemics. This remains a complex problem, as many HIV infected individuals remain unaware of their infection status, leading to parts of HIV epidemics being undiagnosed and under-reported. To that end, we firstly present a method to learn epidemiological parameters from phylogenetic trees, using approximate Bayesian computation (ABC). The epidemiological parameters learned as a result of applying ABC are subsequently used in epidemiological models that aim to simulate a specific epidemic. Secondly, we continue by describing the development of a tree statistic, rooted in coalescent theory, which we use to relate epidemiological parameters to a phylogenetic tree, by using the simulated epidemics. We show that the presented tree statistic enables differentiation of epidemiological parameters, while only relying on phylogenetic trees, thus enabling the construction of new methods to ascertain the epidemiological state of an HIV epidemic. By using genetic data to infer epidemic sizes, we expect to enhance understanding of the portions of the infected population in which diagnosis rates are low., Accepted at BNAIC 2019 (Benelux AI conference)

Aedes-borne arboviruses have spread globally with outbreaks of vast impact on human populations and health systems. The West African archipelago of Cape Verde had its first outbreak of Dengue in 2009, at the time the largest recorded in Africa, and was one of the few African countries affected by the Zika virus epidemic. Aedes aegypti was the mosquito vector involved in both outbreaks. We performed a phylogeographic and population genetics study of A. aegypti in Cape Verde in order to infer the geographic origin and evolutionary history of this mosquito. These results are discussed with respect to the implications for vector control and prevention of future outbreaks. Mosquitoes captured before and after the Dengue outbreak on the islands of Santiago, Brava, and Fogo were analyzed with two mitochondrial genes COI and ND4, 14 microsatellite loci and five kdr mutations. Genetic variability was comparable to other African populations. Our results suggest that A. aegypti invaded Cape Verde at the beginning of the Holocene from West Africa. Given the historic importance of Cape Verde in the transatlantic trade of the 16th-17th centuries, a possible contribution to the genetic pool of the founding populations in the New World cannot be fully discarded. However, contemporary gene flow with the Americas is likely to be infrequent. No kdr mutations associated with pyrethroid resistance were detected. The implications for vector control and prevention of future outbreaks are discussed. publishersversion published

Despite the Zika virus’ rapid spread throughout the Americas in 2015, its phylogenetic classification is limited to either an ‘African’ or ‘Asian’ genotype. This classification no longer reflects the present genetic diversity of circulating strains and their geographic reach. Using 414 publicly available Zika virus genomes from 40 different countries, we created alignments of different genomic fragments with high phylogenetic signal and used these to construct multiple maximum likelihood trees (IQTree). We observed groups of strains from three major geographic regions that consistently cluster into monophyletic clades: African (AF), Asian (AS) and American (AM), the latter of which cluster into four further sub-clades: Caribbean (C), South American (SA1 and SA2) and Central American (CA). The inter-genetic distances of these clades are all significantly greater than their intra-genetic distances (p=0.05). A decision demonstrates that only five nucleotide positions are needed to correctly classify 90% of our dataset into the newly described genotypes.

Viral pathogens causing global disease burdens are often characterized by high rates of evolutionary changes. The extensive viral diversity at baseline can shorten the time to escape from therapeutic or immune selective pressure and alter mutational pathways. The impact of genotypic background on the barrier to resistance can be difficult to capture, particularly for agents in experimental stages or that are recently approved or expanded into new patient populations. We developed an evolutionary model-based counting method to quickly quantify the population genetic potential to resistance and assess population differences. We demonstrate its applicability to HIV-1 integrase inhibitors, as their increasing use globally contrasts with limited availability of non-B subtype resistant sequence data and corresponding knowledge gap. A large sequence data set encompassing most prevailing HIV-1 subtypes and resistance-associated mutations of currently approved integrase inhibitors was investigated. A complex interplay between codon predominance, polymorphisms, and associated evolutionary costs resulted in a subtype-dependent varied genetic potential for 15 resistance mutations against integrase inhibitors. While we confirm the lower genetic barrier of subtype B for G140S, we convincingly discard a similar effect previously suggested for G140C. A supplementary analysis for HIV-1 reverse transcriptase inhibitors identified a lower genetic barrier for K65R in subtype C through differential codon usage not reported before. To aid evolutionary interpretations of genomic differences for antiviral strategies, we advanced existing counting methods with increased sensitivity to identify subtype dependencies of resistance emergence. Future applications include novel HIV-1 drug classes or vaccines, as well as other viral pathogens.

Viral pathogens causing global disease burdens are often characterised by high rates of evolutionary changes, facilitating escape from therapeutic or immune selective pressure. Extensive viral diversity at baseline can shorten the time to resistance emergence and alter mutational pathways, but the impact of genotypic background on the genetic barrier can be difficult to capture, in particular for antivirals in experimental stages, recently approved or expanded into new settings. We developed an evolutionary-based counting method to quantify the population genetic potential to resistance and assess differences between populations. We demonstrate its applicability to HIV-1 integrase inhibitors, as their increasing use globally contrasts with limited availability of non-B subtype resistant sequences and corresponding knowledge gap on drug resistance. A large sequence dataset encompassing most prevailing subtypes and resistance mutations of first- and second-generation inhibitors were investigated. A varying genetic potential for resistance across HIV-1 subtypes was detected for 15 mutations at 12 positions, with notably 140S in subtype B, while 140C was discarded to vary across subtypes. An additional analysis for HIV-1 reverse transcriptase inhibitors identified a higher potential for 65R in subtype C, on the basis of a differential codon usage not reported before. The evolutionary interpretation of genomic differences for antiviral treatment remains challenging. Our framework advances existing counting methods with an increased sensitivity that identified novel subtype dependencies as well as rejected previous statements. Future applications include novel HIV-1 drug classes as well as other viral pathogens.

In recent years, an increasing number of outbreaks of Dengue, Chikungunya and Zika viruses have been reported in Asia and the Americas. Monitoring virus genotype diversity is crucial to understand the emergence and spread of outbreaks, both aspects that are vital to develop effective prevention and treatment strategies. Hence, we developed an efficient method to classify virus sequences with respect to their species and sub-species (i.e. serotype and/or genotype). This tool provides an easy-to-use software implementation of this new method and was validated on a large dataset assessing the classification performance with respect to whole-genome sequences and partial-genome sequences. Available online: http://krisp.org.za/tools.php., SCOPUS: ar.j, info:eu-repo/semantics/published

We examined differences in sexual risk behaviors, HIV prevalence, and demographic characteristics between men who have sex with men (MSM) who visit different types of venues to meet sexual partners, and identified correlates of high-risk behaviors. A cross-sectional behavioral survey was conducted with a venue-based sample of 1011 MSM in Portugal. Overall, 36.3 % of MSM usually visit cruising venues to meet sexual partners (63.7 % only visit social gay venues). Cruising venues' visitors reported higher HIV prevalence (14.6 % [95 % CI 11-18 %] vs. 5.5 % [95 % CI 4-7 %]). Visiting cruising venues was more likely among those older, reporting high number of male sexual partners, group sex, and unprotected anal sex with a partner whose HIV status was unknown. Cruising venues play an important role in increasing risk of HIV transmission among MSM who frequent them. Venue-focused behavioral interventions that promote healthy sexual behaviors are needed.

Background: Tuberculosis (TB) is still a major global health problem. The increasing number of cases observed among foreign-born populations contrasts with the decreasing trends observed in later years in some high-income countries. Healthcare providers are key interveners in the control of TB and HIV-TB infections. In this study, we aimed to explore the perspectives of healthcare providers working in primary care in Portugal about the provision of TB care for migrant patients with TB or HIV-TB co-infection. Methods: We applied a mixed-methods approach using an online survey and semi-structured interviews with primary healthcare providers. A total of 120 Portuguese healthcare providers participated in the survey, and 17 were interviewed. Survey and interview data were analysed applying descriptive statistics and thematic analysis, respectively. Results: Migrants' lack of knowledge on TB disease and its symptoms was the main reason for advanced-stage presentation of cases. Their high mobility and social isolation affect adherence to treatment. The providers also listed several barriers to migrants' access and use of TB care. The most frequently referred were limited socioeconomic resources, complex bureaucracy at the point of access and registration for healthcare services, especially for undocumented migrants, and obstacles for social protection. Providers also advocated more training initiatives on migrants' health, social and cultural contexts, on HIV and TB integrated care, and on TB scientific update for general practitioners and nurses working at primary healthcare centres. Conclusions: Future efforts should provide measures to overcome social, economic and administrative obstacles to care for TB-infected migrants, and promote regular training initiatives for national healthcare providers in order to raise awareness and facilitate better care to culturally diverse populations with TB. This study was funded by the Portuguese Foundation for Science and Technology (Fundação para a Ciência e a Tecnologia - FCT, www.FCT.pt), through funds to the Global Health and Tropical Medicine Research Centre (GHTM - UID/Multi/04413/2013). AMT was supported by FCT grant PD/BD/ 105916/2014. ABA was supported by the Investigador FCT programme. info:eu-repo/semantics/publishedVersion

Summary Virus sequence data are an essential resource for reconstructing spatiotemporal dynamics of viral spread as well as to inform treatment and prevention strategies. However, the potential benefit of these applications critically depends on accurate and correctly annotated alignments of genetically heterogeneous data. VIRULIGN was built for fast codon-correct alignments of large datasets, with standardized and formalized genome annotation and various alignment export formats. Availability and implementation VIRULIGN is freely available at https://github.com/rega-cev/virulign as an open source software project. Supplementary information Supplementary data is available at Bioinformatics online.

A 4-year-old child born to an HIV-1 seronegative mother was diagnosed with HIV-1, the main risk factor being transmission from the child's father who was seroconverting at the time of the child's birth. In the context of a forensic investigation, we aimed to identify the source of infection of the child and date of the transmission event. Samples were collected from the father and child at two time points about 4 years after the child's birth. Partial segments of three HIV-1 genes (gag, pol, and env) were sequenced and maximum likelihood (ML) and Bayesian methods were used to determine direction and estimate date of transmission. Neutralizing antibodies were determined using a single cycle assay. Bayesian trees displayed a paraphyletic-monophyletic topology in all three genomic regions, with the father's host label at the root, which is consistent with father-to-son transmission. ML trees found similar topologies in gag and pol and a monophyletic-monophyletic topology in env. Analysis of the time of the most recent common ancestor of each HIV-1 gene population indicated that the child was infected shortly after the father. Consistent with the infection history, both father and son developed broad and potent HIV-specific neutralizing antibody responses. In conclusion, the direction of transmission implicated the father as the source of transmission. Transmission occurred during the seroconversion period when the father was unaware of the infection and was likely accidental. This case shows how genetic, phylogenetic, and serological data can contribute for the forensic investigation of HIV transmission. info:eu-repo/semantics/publishedVersion

BACKGROUND: Together with Treponema pallidum subspecies pertenue, Haemophilus ducreyi is a major cause of exudative cutaneous ulcers (CUs) in children. For H. ducreyi, both class I and class II strains, asymptomatic colonization, and environmental reservoirs have been found in endemic regions, but the epidemiology of this infection is unknown. METHODS: Based on published whole-genome sequences of H. ducreyi CU strains, a single-locus typing system was developed and applied to H. ducreyi–positive CU samples obtained prior to, 1 year after, and 2 years after the initiation of a mass drug administration campaign to eradicate CU on Lihir Island in Papua New Guinea. DNA from the CU samples was amplified with class I and class II dsrA-specific primers and sequenced; the samples were classified into dsrA types, which were geospatially mapped. Selection pressure analysis was performed on the dsrA sequences. RESULTS: Thirty-seven samples contained class I sequences, 27 contained class II sequences, and 13 contained both. There were 5 class I and 4 class II types circulating on the island; 3 types accounted for approximately 87% of the strains. The composition and geospatial distribution of the types varied little over time and there was no evidence of selection pressure. CONCLUSIONS: Multiple strains of H. ducreyi cause CU on an endemic island and coinfections are common. In contrast to recent findings with T. pallidum pertenue, strain composition is not affected by antibiotic pressure, consistent with environmental reservoirs of H. ducreyi. Such reservoirs must be addressed to achieve eradication of H. ducreyi.

In this report, an RT-PCR approach based on the use of degenerate primers allowed the identification of negeviruses in four different species of mosquitoes (Ochlerotatus caspius, Culex pipiens, Cx. theileri and Cx. univittatus) collected in southern Portugal. The genomes of two of these viruses, sequenced to full completion, were shown to encode all the proteins encoded by previously described negeviruses. One of these viruses induces exuberant cytopathic effect in insect cell culture, with no obvious signs of apoptosis induction, replicating very rapidly and allowing for the detection of viral genomes in the infected culture supernatant as soon as 4h post-infection. This virus was also shown to use a dsRNA intermediate, which was found to be fully formed and active 3h after infection. Phylogenetic analysis of two products encoded by the viral ORF1 placed both viruses among Negev virus cluster, in the recently proposed Nelorpivirus taxon.

Diversity of the founding population of Human Immunodeficiency Virus Type 1 (HIV-1) transmissions raises many important biological, clinical, and epidemiological issues. In up to 40% of sexual infections, there is clear evidence for multiple founding variants, which can influence the efficacy of putative prevention methods, and the reconstruction of epidemiologic histories. To infer who-infected-whom, and to compute the probability of alternative transmission scenarios while explicitly taking phylogenetic uncertainty into account, we created an approximate Bayesian computation (ABC) method based on a set of statistics measuring phylogenetic topology, branch lengths, and genetic diversity. We applied our method to a suspected heterosexual transmission case involving three individuals, showing a complex monophyletic-paraphyletic-polyphyletic phylogenetic topology. We detected that seven phylogenetic lineages had been transmitted between two of the individuals based on the available samples, implying that many more unsampled lineages had also been transmitted. Testing whether the lineages had been transmitted at one time or over some length of time suggested that an ongoing superinfection process over several years was most likely. While one individual was found unlinked to the other two, surprisingly, when evaluating two competing epidemiological priors, the donor of the two that did infect each other was not identified by the host root-label, and was also not the primary suspect in that transmission. This highlights that it is important to take epidemiological information into account when analyzing support for one transmission hypothesis over another, as results may be nonintuitive and sensitive to details about sampling dates relative to possible infection dates. Our study provides a formal inference framework to include information on infection and sampling times, and to investigate ancestral node-label states, transmission direction, transmitted genetic diversity, and frequency of transmission.

BACKGROUND: International human migration has been rapidly growing. Migrants coming from low and middle income countries continue to be considerably vulnerable and at higher risk for infectious diseases, namely HIV (Human Immunodeficiency Virus) and tuberculosis (TB). In Europe, the number of patients with HIV-TB co-infection has been increasing and migration could be one of the potential driving forces. OBJECTIVE: This systematic review aims to improve the understanding on the burden of HIV-TB co-infection among migrants in Europe and to assess whether these populations are particularly vulnerable to this co-infection compared to nationals. DESIGN: MEDLINE®, Web of Science® and Scopus® databases were searched from March to April 2016 using combinations of keywords. Titles and abstracts were screened and studies meeting the inclusion criteria proceeded for full-text revision. These articles were then selected for data extraction on the prevalence, incidence and mortality. RESULTS: The majority of HIV-TB prevalence data reported in the analysed studies, including extrapulmonary/disseminated TB forms, was higher among migrant vs. nationals, some of the studies even showing increasing trends over time. Additionally, while HIV-TB incidence rates have decreased among migrants and nationals, migrants are still at a higher risk for this co-infection. Migrants with HIV-TB co-infection were also more prone to unsuccessful treatment outcomes, death and drug resistant TB. However, contradicting results also showed lower mortality compared to nationals. CONCLUSIONS: Overall, a disproportionate vulnerability of migrants to acquire the HIV-TB co-infection was observed across studies. Such vulnerability has been associated to low socioeconomic status, poor living conditions and limited access to healthcare. Adequate social support, early detection, appropriate treatment, and adequate access to healthcare are key improvements to tackle HIV-TB co-infection among these populations. publishersversion published

21st International BioInformatics Workshop on Virus Evolution and Molecular Epidemiology In Slovenia, a small country in Central Europe, less than 1 per 1,000 inhabitants are estimated to be infected with HIV-1. As in most of the Central and Western European countries, the majority of patients diagnosed with HIV-1 are infected with subtype B. However, due to migration, other subtypes can become more prevalent in the country. The aim of this study was to determine HIV-1 subtypes circulating in Slovenia and to further examine the molecular epidemiology of subtype A. A total of 367 Slovenian HIV-1 sequences were included in the study, representing 58% of all patients diagnosed in Slovenia until the end of the year 2013. Subtype was assigned by employing different HIV subtyping tools coupled with Maximum likelihood phylogenetic analysis. The latter was performed to examine the molecular epidemiology of subtype A as well. Identified clusters of Slovenian subtype A sequences were further analyzed for the determination of the time of the most recent common ancestor (tMRCA) by using Monte Carlo Markov chain (MCMC) method available in BEAST 2.1.3 software. We determined the prevalence of subtype B to be 85.3%, while subtype A was the most prevalent non-B subtype found in 18 patients (4.9%), followed by CRF02_AG (2.4%), subtype C (1.1%), subtypes D, G and CRF01_AE (0.8% each) and subtypes F1 and CRF22_01A1 (0.3% each). Subtypes could not be assigned to 12 sequences (3.3%). The phylogenetic tree obtained by ML analysis of the subtype A and subtype A related recombinants revealed that Slovenian sequences were part of 6 major international clusters. Two clusters consisting only of Slovenian sequences were identified and thus additional MCMC analysis was employed. Results of a Slovenian cluster of 4 subtype A sequences showed a posterior probability value of 1 and a tMRCA between the years 1985 and 2008, with a mean in the year 2001. In conclusion, in a Central European country, where subtype B predominates, the second most common subtype was found to be subtype A. Non-B subtypes were observed in one out of seven patients in Slovenia, a fraction that is not negligible, thus proving importance of surveillance of HIV subtype diversity and corresponding molecular epidemiology of non-B subtypes. publishersversion published