A New and Efficient Enrichment Method for Metagenomic Sequencing of Monkeypox Virus

[Abstract] Background The methodology described in previous literature for Monkeypox virus (MPXV) sequencing shows low efficiency when using metagenomic approaches. The aim of the present study was to evaluate a new fine-tuned method for extraction and enrichment of genomic MPXV DNA using clinical samples and to compare it to a non-enrichment metagenomic approach. Results A new procedure that allows sample enrichment in MPXV DNA, avoiding wasting the sequencing capacity in human DNA, was designed. This procedure consisted of host DNA depletion using a saponin/NaCl combination treatment and DNase, together with high g-force centrifugations. After typical quality control, samples using the enrichment method contained around 96% of reads not classified as human DNA, while the non-enrichment protocol showed around 5-10%. When reads not belonging to Orthopoxvirus were removed, enriched samples kept about 50% of the original read counts, while non-enriched ones kept only 2-7%. Conclusions Results showed a very significant improvement in sequencing efficiency, increasing the number of reads belonging to MPXV, the depth of coverage and the trustworthiness of the consensus sequences. This, in turn, allows for more samples to be included in a single cartridge, reducing costs and time to diagnosis, which can be very important factors when dealing with a contagious disease.This work was supported by a grant from the SERGAS-Galician Healthcare Service (Program “Innova Saúde”) to GB, by CIBERINFEC and also by Instituto de Salud Carlos III (ISCIII) through the projects PI20/00413 to MP and PI21/00704 to G

Trends on epidemiological, virological, and clinical features among newly diagnosed HIV-1 persons in Northwest Spain over the last 10 years

[Abstract] To describe temporal trend and characteristics of newly HIV-diagnosed patients in a medical care area in Northwest Spain over the last 10 years. All newly diagnosed patients for HIV-infection from 2004 to 2013 at a reference medical care area in Northwest of Spain were identified. Epidemiological, virological, immunological, and clinical data, as well as HIV genotype and drug resistance information were recorded. A total of 565 newly HIV-diagnosed patients were identified. The number of new cases increased in the last 5 years (66 cases/year). Overall, 53.1% had a median CD4 counts < 350 cells/µl and 33.6% had an AIDS defining criteria. Non-B variants were found in 34.4% of patients being subtype F (25.8%) the most common non-B subtype. The rate of transmitted drug resistance (TDR) over the study period was 3.7%, but a decreased to 2.6% was observed in the last 5 years. The most prevalent TDR mutations were: T215 revertants (1.5%), K219QENR (1.2%), for NRTIs; K103N (1.9%), for NNRTIs; L90M (0.3%), for PIs. Overall, 73.2% of patients started antiretroviral treatment and 9.9% of patients died during follow-up. The number of newly HIV diagnosed patients increased since year 2009. There is a high prevalence of late diagnosis (53%) and 33% had an AIDS defining criteria. Interestingly, the most prevalent non-B subtype in our population was F (25.8%). These findings support the need to facilitate the access for HIV testing to reduce the rate of late HIV diagnosis, improve the clinical outcome and prevent HIV transmission.Instituto de Salud Carlos III; CP08/00214Instituto de Salud Carlos III; PI10/02166Instituto de Salud Carlos III; CM13/00328Instituto de Salud Carlos III; PI13/0226

Seroprevalence of HCV and HIV infections by year of birth in Spain: impact of US CDC and USPSTF recommendations for HCV and HIV testing

Instituto de Salud Carlos III; CP08/00214Instituto de Salud Carlos III; PI10/02166Instituto de Salud Carlos III; PI13/02266Instituto de Salud Carlos III; CM13/0032

Any impact of blips and low-level viraemia episodes among HIV-infected patients with sustained virological suppression on ART?

[Abstract] Objectives. The objective of this study was to evaluate the prevalence of blips and risk of virological failure (VF) among HIV-infected patients with sustained virological suppression (HIV-RNA <50 copies/mL) on ART. Methods. Newly diagnosed (2004–13) HIV-infected patients with sustained virological suppression on ART (minimum follow-up of 3 months) were identified. Risk of VF was evaluated according to different plasma HIV-RNA quantification values based on the limits of quantification/detection of current commercial assays (20 copies/mL). Kaplan–Meier and Cox proportional hazards models were used to compare the cumulative incidence of VF. Results. A total of 565 newly diagnosed HIV-infected patients were identified: 453 started ART and 354 achieved virological suppression. Prevalence of blips (isolated HIV-RNA ranging from 50 to 200 copies/mL) and VF (HIV-RNA ≥50 copies/mL) was 22.7% and 8.8%, respectively (mean follow-up of 42 months). Multivariate analysis identified differences between HIV-RNA values as an independent predictor of VF (P = 0.008); risk of VF was higher for patients with blips [HR 2.500 (95% CI 0.524–11.926)] and for those with at least three consecutive detected, but not quantified, HIV-RNA determinations (HIV-RNA 200 copies/mL [33.7% at 24 and 60 months versus <5% for other HIV-RNA values; HR 6.943 (0.728–66.261), P = 0.092]. Conclusions. Blips are frequent (22.7%) among HIV-infected patients with sustained virological suppression on ART. HIV patients with blips and at least three consecutive detected, but not quantified, HIV-RNA determinations (<20 copies/mL) had a higher risk of VF. These findings highlight the relevance of maintaining HIV-RNA levels below the limits of quantification of current assays (<20 copies/mL).Instituto de Salud Carlos III; CPII14/00014Instituto de Salud Carlos III; PI10/02166Instituto de Salud Carlos III; PI13/0226

Molecular characterization of HIV-1 infection in Northwest Spain (2009–2013): investigation of the subtype F outbreak

[Abstract] Background. HIV-1 subtype B is the predominant one in European regions several, while other subtypes and recombinants are also circulating with high prevalence. A sub-epidemic of subtype F with specific characteristics and low response to treatment has been recently identified in Galicia. In this study we investigated the characteristics of the HIV-1 subtype F sub-epidemic in A Coruña and Santiago de Compostela in Northwest Spain. Methods. 420 newly HIV-1 diagnosed patients during 2009–2013 were enrolled in this study. HIV-1 subtyping was carried out using automated subtyping tools and phylogenetic analysis. Molecular epidemiology investigation of subtypes B and F was performed by means of phylogenetic analysis using fast maximum likelihood. Phylodynamic analysis was performed using Bayesian method as implemented in BEAST v1.8. Results. Subtype B found to be the predominant (61.2% and 70.4%) followed by subtype F (25.6% and 12.0%) in both areas (A Coruña and Santiago de Compostela, respectively). The latter found to mainly spread among men having sex with men (MSM). The vast majority of subtype F lineages from both areas clustered monophyletically, while subtype B sequences clustered in several tree branches. The exponential growth of subtype F sub-epidemic dated back in 2008 by means of phylodynamic analysis. Most of new infections during 2009–2013 occurred within the subtype F transmission cluster. Conclusions. Subtype F circulates at high prevalence in A Coruña and Santiago de Compostela in Northwest Spain, suggesting that the HIV-1 epidemic in this region has distinct characteristics to the rest of Spain. Subtype F has being spreading among MSM and is currently the most actively spreading network. The single cluster spread of this local sub-epidemic might provide an explanation for the distinct characteristics and the low response to antiretroviral treatment

Incidencia, factores de riesgo e impacto pronóstico de la infección por citomegalovirus tras el trasplante cardiaco

[Abstract] Introduction and objectives. To assess the risk factors of CMV infection after heart transplant (HT) and its influence on long-term prognosis. Methods. We conducted a retrospective single-centre study of 222 H T recipients. Risk factors for CMV infection were identified by means of multivariable Cox´s regression. Kaplan-Meier analysis and Cox´s regression were used to assess the long-term prognostic impact of CMV infection during the first post-transplant year. Results. Donor-recipient CMV serologic matching (hazard ratio [HR] 1.92, 95% confidence interval [95% CI] 1.2–3.09, p = .007), recipient age (HR 1.02, 95% CI 1.00–1.1, p = .02), diabetes mellitus (HR 1.86, 95% CI 1.4–3.05, p = .01), pre- transplant circulatory support (HR 1.59, 95% CI 1.06–2.38, p = .03) and the use of tacrolimus (HR 1.64, 95% CI 1.13–2.36, p = .009) were independently associated with increased risk of CMV infection. CMV infection during the first year post-HT was not associated with worse transplant outcomes in terms of mortality, incidence of heart failure, cardiac allograft vasculopathy or acute rejection. Conclusions. CMV infection was not associated with impaired long-term prognosis after HT.[Resumen] Introducción y objetivos. Analizar el impacto pronóstico de la infección por Citomegalovirus (CMV) durante el primer año tras el trasplante cardiaco (TC) y describir factores de riesgo. Métodos. Se realizó un estudio retrospectivo unicéntrico incluyendo 222 receptores de TC. La identificación de factores de riesgo de infección por CMV se llevó a cabo mediante regresión multivariable de Cox. Mediante los métodos de Kaplan-Meier y Cox se analizó la influencia de la infección por CMV durante el primer año sobre la supervivencia e incidencia de eventos clínicos adversos en el seguimiento a largo plazo. Resultados. En el análisis multivariante, el estado serológico donante/receptor frente a CMV (hazard ratio [HR] 1,92, intervalo de confianza 95% [IC 95%] 1,2–3,09; p = 0007, la edad del receptor HR 1,02, IC 95% 1,00–1,1; p = 0,02), la diabetes (HR 1,86, IC 95% 1,4-3,05; p = 0,01), el soporte circulatorio mecánico (HR 1,59, IC 95% 1,06-2,38; p = 0,03) y el uso de tacrolimus (HR 1,64, IC 95% 1,13-2,36; p = 0009, resultaron predictores independientes de infección por CMV post-trasplante. No se detectó una influencia significativa de la infección por CMV durante el primer año post-trasplante sobre la mortalidad, la incidencia de insuficiencia cardiaca, enfermedad vascular del injerto o rechazo agudo. Conclusiones. La infección por CMV durante el primer año post-trasplante no se asoció a un peor pronóstico a largo plazo

Deep-Sequencing Reveals Broad Subtype-Specific HCV Resistance Mutations Associated with Treatment Failure

[Abstract] A percentage of hepatitis C virus (HCV)-infected patients fail direct acting antiviral (DAA)-based treatment regimens, often because of drug resistance-associated substitutions (RAS). The aim of this study was to characterize the resistance profile of a large cohort of patients failing DAA-based treatments, and investigate the relationship between HCV subtype and failure, as an aid to optimizing management of these patients. A new, standardized HCV-RAS testing protocol based on deep sequencing was designed and applied to 220 previously subtyped samples from patients failing DAA treatment, collected in 39 Spanish hospitals. The majority had received DAA-based interferon (IFN) α-free regimens; 79% had failed sofosbuvir-containing therapy. Genomic regions encoding the nonstructural protein (NS) 3, NS5A, and NS5B (DAA target regions) were analyzed using subtype-specific primers. Viral subtype distribution was as follows: genotype (G) 1, 62.7%; G3a, 21.4%; G4d, 12.3%; G2, 1.8%; and mixed infections 1.8%. Overall, 88.6% of patients carried at least 1 RAS, and 19% carried RAS at frequencies below 20% in the mutant spectrum. There were no differences in RAS selection between treatments with and without ribavirin. Regardless of the treatment received, each HCV subtype showed specific types of RAS. Of note, no RAS were detected in the target proteins of 18.6% of patients failing treatment, and 30.4% of patients had RAS in proteins that were not targets of the inhibitors they received. HCV patients failing DAA therapy showed a high diversity of RAS. Ribavirin use did not influence the type or number of RAS at failure. The subtype-specific pattern of RAS emergence underscores the importance of accurate HCV subtyping. The frequency of “extra-target” RAS suggests the need for RAS screening in all three DAA target regions.Ministerio de Economía y Empresa; IDI-20151125Ministerio de Ciencia, Innovación y Universidades; SAF SAF 2017-87846-