Filogeografia y filodinamica de los linajes de VHC genotipo 1a en Espana

Introducción y objetivo: España se caracteriza por una elevada prevalencia de VHC genotipo 1a, lo que se traduce en un mayor potencial impacto de el polimorfismo Q80K sobre la actividad de simeprevir. En este estudio analizamos la epidemia de HCV 1a en España, en comparación a la de otros países, centrándonos en los movimientos migratorios de los virus HCV 1a con Q80K. Métodos: Hemos analizado 188 aislados de VHC genotipo 1a, estudiando la región NS3. Estos aislados se georeferenciaron junto a secuencias disponibles en diferentes bases de datos procedentes de diversas localizaciones a nivel mundial. Para conocer los patrones de transmisión se utilizaron métodos de inferencia Bayesiana espacio-temporal, utilizando métodos que permiten establecer el patrón de migración en función a la direccionalidad de los aislados. Resultados: Los aislados de HCV1a se separaron en dos clados, caracterizándose el clado I por un cluster de aislados que contenían la mayoría de los aislados con Q80K. La elevada prevalencia de Q80K en este clado se asoció con un efecto fundador de la epidemia de Q80K en Estados Unidos. Este país, junto a los otros de Europa Occidental constituyen el principal origen de los linajes de HCV 1a con Q80K que circulan en España. Asimismo, hemos observado circulación bidireccional entre España y otros países europeos. Sin embargo, no hemos detectado diferencias en los movimientos migratorios de los dos clados de aislados de HCV 1a. Conclusión: La epidemia de HCV1a en España se ha impulsado principalmente desde linajes procedentes de Estados Unidos, y en menor medida mediante circulación en Europa. De forma similar, hemos detectado flujos migratorios entre España y otros países de Europa. Estos flujos migratorios son los que con mayor probabilidad utilizarán los linajes de Q80K para transmitirse en España.status: publishe

Frequent de novo generation of HCV3a resistance-associated substitutions in Spain

Background: HCV subtype 3a, responsible for approximately 17% of HCV infections in Spain, remains a difficult-to-treat genotype despite the availability of highly effective treatments based on direct-acting antivirals. Current treatment regimens often combine a NS5A inhibitor with NS5B inhibitor (sofosbuvir). Resistance-associated substitutions (RASs) can have a profound impact on treatment response, especially in cirrhotic patients, with NS5A variant Y93H of particular interest due to its substantial fold-decrease in susceptibility to all NS5A inhibitors. For this reason, it is of interest to evaluate the virus epidemic history for patterns that can be of public health relevance. Methods: We combine publicly available with newly generated HCV3a NS5A and NS5B sequence data to elucidate the international HCV3a migration network with a focus on the role of Spain. Bayesian phylogenetic inference methods were used to estimate the epidemiological relations between the sampled virus lineages and to reconstruct the historical transmission patterns. Migration rates between locations were inferred using a discrete phylogeographic model in which rates from and to locations can differ. Results: There were no clear associations between the sample’s origin and amino acid usage patterns for NS5B RASs S282T, C316N/Y and V321A and for NS5A RASs M28T/V and L31M/V, while Q30L and Y93H appear overrepresented in Pakistanian (p=0.009) and Spanish strains respectively (p=0.052). Reconstruction of ancestral sequences shows that the Y93H RAS is usually de novo generated on external branches, dispersed over the whole phylogeny. Thus there is no founder effect for Y93H, as opposed to what is seen for HCV1a NS3 variant Q80K. The strengths and intensities of migration links between locations vary between the NS5A and NS5B datasets. Spain acts as a sink for HCV3a in both datasets but while most HCV3a import into Spain originates from Germany according to the NS5A data, the NS5B data point towards UK as the main source. Virus movements from Spain are usually towards other European countries (in particular to Portugal and Germany) and English-speaking countries (the so-called Anglosphere, which encompasses the Australia, Canada, India, Pakistan, the UK and the USA). The inconsistencies in the dominant origin location of HCV3a migration into Spain across datasets point out that each genomic region represents a different sample from the epidemic, and its combined phylogeographical analyses create a complementary picture of relevant migration patterns. This illustrates the usefulness of incorporating data from multiple genomic regions, the added value of longer genomic regions, and the need for broader sampling strategies. Conclusions: Spain can become an important 'host-spot' region of Y93H dissemination in the future, due to frequent de novo generation of this NS5A variant. Furthermore, while the inferred higher-level migration patterns are robust to the available sampling for a genomic sub-region, the details of the migration links between Spain and other locations vary by dataset. Our results indicate a need for the analyses of larger genomic regions, and a worldwide sampling of the HCV3a epidemic to more reliably infer the most important sources of HCV3a in Spain.status: publishe

Relapse or reinfection of hepatitis C after direct acting antiviral treatment: unraveled by phylogenetic analysis. Results from the Spanish GEHEP-004 cohort

Background: Despite high response rates associated to DAA treatment, no protective immunity is acquired, so patients that are cured after treatment can be infected with a new HCV strain, and therefore may be responsible for further transmission. Consequently, viral eradication may be hampered by high reinfection and transmission rates among patients with persistent risk behaviour. Distinguishing between virological relapse and reinfection is crucial to determine the true efficacy of current therapies and to define the most appropriate retreatment if needed. Methods: The GEHEP-004 cohort includes approximately 300 patients failing to different DAA regimens from 42 Spanish centers. For 53 patients treated between 2014 and 2016, the virus was sampled at two time points, before start of therapy and at time of failure. Sequencing was performed for two or three regions (NS3 – NS5A – NS5B), depending on the DAA regimen administered. For each taxon, the ten most similar sequences were retrieved from public databases by the use of BLAST. Concatenated alignments were used to infer phylogenetic trees by neighbour-joining and maximum-likelihood algorithms, with the GTR gamma model and 1000 bootstrap replicates. When comparing strains before and after treatment in one patient, evidence of reinfection was defined as a difference in HCV genotype or subtype, or as a significantly different clustering in distant clades in the tree. Evidence of relapse was defined as significant clustering in the same clade, while no conclusion was drawn when clades were supported with a bootstrap <70%. Simplot was used to detect recombination. Results: Genotype assignment by phylogenetic analysis revealed nine discordant cases (17.0%) with commercial assays at genotype and subtype level, while no recombinants were identified. At baseline, 41.5% of patients were determined to be infected with HCV1a, followed by HCV1b (24.5%), HCV4 (18.9%) and HCV3a (15.1%). Overall, 60.4% was co-infected with HIV. The large majority of patients for which the transmission route of infection was known, was classified as people who inject drugs (PWID) (78.6%), often co-infected with HIV (27/33) and half of them infected with HCV1a. Sexual transmission was observed in seven cases, of which five in HIV-positive men who have sex with men (MSM). Due to poor phylogenetic signal of single fragments, conclusions were only drawn for concatenated alignments. Overall, five patients were reinfected with a different HCV strain (4 PWID + 1 MSM), of which three with a different HCV genotype or subtype, and four co-infected with HIV. Virological relapse was defined for 44 patients, while no conclusion could be drawn for four patients. Conclusions: In our cohort, the majority of patients experienced a virological relapse. Almost 10% were reinfected, most of them PWID and HIV co-infected. Since about half of those reinfected, showed the same subtype as at baseline, phylogenetics is needed, not only to determine the correct HCV genotype, but also to distinguish between relapse and reinfection. Of note, phylogenetic analysis can only result in confident conclusions when long genomic stretches with sufficient phylogenetic signal are available, stressing the need to perform full-genome sequencing or to concatenate multiple regions.status: accepte

Inferring the migration routes of hepatitis C virus subtype 1a lineages identifies a need for pan-European prevention strategies

Background and Aims: Despite treatment response rates of nearly 100% for the majority of hepatitis C virus (HCV) infected patients, the virus can only be eradicated on a global scale through the combined effort of preventing new infections and curing diagnosed cases. As HCV1a constitutes a large part of recent infections, investigating by which migration routes it spreads, is of public health relevance. Method: HCV1a genetic data from two viral proteins, NS3 (n=2514) and NS5A (n=1957), was used to reconstruct the virus migration pathways through time and space on a global and European level. Data from thirteen European countries was available, including nine (Belgium, Germany, Ireland, Italy, Poland, Portugal, Russia, Spain and the United Kingdom) for which 1825 sequences were newly generated. These and publicly available spatiotemporally annotated virus genetic data were used to infer the migration rates between geographical locations with a fast and scalable Bayesian approach. To counter the impact of potential sampling biases on the phylogeographic reconstructions, the among-location exchange rate matrix was shared between the gene datasets. Results: Inferring migration pathways for both genes reveals extensive movements on a supra-continental scale, showing a dominant role for the United States in seeding HCV1a into Europe (74.6% of all migration links), as well as considerable migration within Europe. As only a small minority of outgoing migration events from Europe is directed to the United States (<5%), this suggests a scenario of one-way traffic to Europe. Within Europe, the migration network becomes increasingly complex and diffuse over time. Preliminary analyses to identify source-sink relations within Europe suggest that Germany functions as a hub for the spread of HCV1a lineages in Europe, exporting mainly viral strains to France, Italy, the United Kingdom and Spain. Conclusion: Molecular epidemiology of the European HCV1a epidemic illustrates complex patterns of human migrations that are increasing in complexity over time. The presence of a pan-European migration network impairs the efficacy of national-based intervention programs. In turn, this indicates that a supra-national coordinated approach is needed to more quickly and more efficiently curb the HCV1a epidemic. Furthermore, similar analyses for HCV3a and HCV4, both additional important sources of new infections, are needed to support evidence-based European prevention strategies.status: accepte