Continued propagation of the CRF19_cpx variant among HIV-positive MSM patients in Spain

Continued propagation of the CRF19_cpx variant among HIV-positive MSM patients in Spain Abstract Objectives The HIV-1 CRF19_cpx genetic form has been recently associated with greater pathogenicity. We used CoRIS, a national cohort of 31 reference hospitals in Spain, to investigate the current epidemiological situation of this variant in Spain. Patients and methods We analysed 4734 naive HIV-1-positive patients diagnosed during the 2007–15 period with an available pol gene sequence in the CoRIS resistance database. HIV-1 CRF19_cpx was ascribed through REGA3.0 and confirmed by a phylogenetic analysis. We analysed the presence of the transmission clusters of HIV-1 CRF19_cpx by maximum likelihood [with the randomized accelerated maximum likelihood (RAxML) program] and the time to the most recent common ancestor using Bayesian inference (BEAST, v. 1.7.5). Results Nineteen patients were infected with CRF19_cpx: all were male, they had a mean age of 42.9 years (95% CI: 36.4–52.5 years), the majority were MSM [n = 18 (95%)] and of Spanish nationality [n = 16 (84.2%)] and they had high CD4+ T cell counts (∼415 cells/mm3). Fifteen patients were grouped into four different transmission clusters: two clusters (two patients each) grouped the patients from Valencia and another cluster grouped one patient from Madrid and another from Seville. We found a larger cluster that grouped nine patients from southern Spain (Malaga and Seville), of which six presented mutation G190A. We estimated the origin of all the transmission clusters to take place between 2009 and 2010. Conclusions We demonstrate that this variant has spread in Spain in recent years among young HIV-positive MSM and we note a recent expansion in southern Spain in patients who carry mutation G190A. We alert healthcare managers to enhance preventive measures to prevent the continuous spread of HIV-1 CRF19_cpx. Introduction The HIV-1 CRF19_cpx genetic form (A, D, G intersubtype circulating recombinant form), described for the first time in Cuba in 2005,1 has been associated with great pathogenicity: patients are diagnosed with high viral loads, high RANTES chemokine expression and rapidly progress to AIDS.2 One characteristic of this genetic form is its failure to spread widely to other countries: ∼80% of the sequences included in the Los Alamos HIV sequence database (LANL) come from Cuba and the prevalence for Spain is <1%.3,4 Nonetheless, the recent finding of several transmission networks in eastern Spain (Valencia) in which HIV-positive male patients are infected with the HIV-1 CRF19_cpx genetic form indicates that this genetic form is likely to expand in the coming years.5 The Cohort of the Spanish Network of AIDS Research (CoRIS)6 is a national prospective multicentre cohort formed exclusively of naive HIV-positive patients from 31 reference Spanish hospitals. Over time, CoRIS has offered a valuable source of clinical, demographic and virological information to study the prevalence of different genetic forms of HIV-1,3,4 and has enabled epidemiological monitoring of resistance to different first-line antiretroviral drugs.4,7,8 Phylogenetic studies applied to large series of sequences are essential to monitor the impact of migration on a given local epidemiology,9–12 and to establish the origin and diversification of certain transmission networks.12–14 In this study, we used several phylogenetic analysis tools to investigate the current epidemiological situation of the HIV-1 CRF19_cpx variant in Spain. Patients and methods Study population The analysis included 4734 partial pol gene sequences [protease (PR) codons 4–99; reverse transcriptase (RT) codons 38–247] of all the newly HIV-1 diagnosed patients during the 2007–15 period retrieved in CoRIS. Viral sequences were generated from the earliest sample available before ART initiation (baseline) and were linked to demographic (risk group, age, gender, country of origin and attending hospital of origin), clinical (CD4+ T cell count) and virological (viral load) information. Ethics All subjects provided written informed consent to participate. The study was reviewed and approved by the institutional review boards of all the participating centres. HIV-1 subtype assignment A preliminary search for the HIV-1 CRF19_cpx genetic forms was done using the REGA 3.0 subtyping tool (http://dbpartners.stanford.edu: 8080/RegaSubtyping/stanford-hiv/typingtool/). All these variants were definitely confirmed by a phylogenetic analysis, which was performed by maximum likelihood (ML) analysis with the randomized accelerated ML (RAxML) program, accessible via the CIPRES Science Gateway.15 For this analysis, a general time-reversible (GTR) model with γ-distributed rate heterogeneity across sites and 1000 bootstrap iterations was employed. A representative data set of HIV-1 sequences group M, pure subtypes (A–K) and recombinant forms (at least four representative sequences of each pure subtype and the CRF currently available from the analysis) from LANL was taken as a reference. The HIV-1 CRF19_cpx classification was considered consistent if the query sequence was included with the corresponding reference sequences in a monophyletic cluster supported by high bootstrap values (>70%).16 Analysis of the phylogenetic relationship among the patients infected with HIV-1 CRF19_cpx In order to characterize the phylogenetic relationship between the sequences found in the HIV-1 CRF19_cpx genetic form in Spain, these sequences were analysed with all the sequences available in LANL (start 2279, end, 3290; HXB2 nucleotide position) (last accessed December 2016): 162 sequences [Cuba (n = 152), Spain (n = 5), UK (n = 1), USA (n = 3), Greece (n = 1)]. As the breakpoints in the pol region were lacking for the HIV-1 CRF19_cpx genetic form (subtype D in pol), a consistent data set of 100 HIV-1 subtype D sequences retrieved from LANL was added. All the sequences were aligned through ClustalW17 and were manually edited. Given the convergent evolution of the antiretroviral drug resistance mutations in the phylogenetic analysis,18 all the drug resistance mutations were removed (drug resistance mutation present in at least two patients) that were associated with the codons with major resistance in PR (30, 32, 33, 46, 48, 48, 50, 54, 76, 82, 84, 88 and 90) and RT (41, 62, 65, 67, 69, 70, 74, 75, 77, 100, 103, 106, 108, 115, 116, 151, 181, 184, 188, 190, 210, 215, 219, 225 and 236). A preliminary analysis was run using ML in RAxML with the best-fitting model of nucleotide substitution obtained through FindModel (at http://www.hiv.lanl.gov/content/sequence/findmodel/findmodel.html). The best-fitting model with the lowest Akaike’s information criterion (AIC) in Find Model was the GTR model with γ-distributed rate heterogeneity across sites using 1000 bootstrap iterations. We investigated the presence of associations between the HIV-1 CRF19_cpx Spanish sequences from the CoRIS cohort with related nodes by taking bootstrap values ≥90% as significantly reliable node supports. To confirm the transmission networks that grouped the sequences from the CoRIS cohort, a Bayesian Markov Chain Monte Carlo (MCMC) approach was applied to all the HIV-1 CRF19_cpx (n = 181) genetic forms, as implemented in v. 1.7.5 of BEAST.19 The Shapiro–Rambaut–Drummond–2006 (SRD06) substitution model was used, together with a relaxed uncorrelated log-normal clock (UCLN).20 A comparison was made among the different demographic models (Bayesian Skyline Plot, Constant, Exponential and Logistic) using Tracer v. 1.6 and the best model was chosen according to the lowest AIC through MCMC (AICM). Each MCMC approach was run for 250 million states by sampling every 50 000. Only traces with an effective sample size (ESS) of >200 were accepted in v. 1.6 of TRACER after excluding an initial 10% (http://tree.bio.ed.ac.uk/software/tracer/). These previously defined transmission networks (through ML) were confirmed by Bayesian inference using posterior probability (pp) values >0.9 as the thresholds. Finally, the most recent common ancestor for these clusters was estimated. Analysis of antiretroviral drug resistance mutations We analysed drug resistance mutations with the HIVseq program, available from the HIV Drug Resistance Database of Stanford University (https://hivdb.stanford.edu/hivseq/by-sequences/) and the WHO surveillance drug resistance mutation list (last updated in 2009 by Bennett et al.21). Results Nineteen patients (0.4%) were infected with the CRF19_cpx variant during the period 2007–15. The demographic, clinical and virological characteristics of these patients are shown in Table 1. Table 1. Demographic, clinical and virological characteristics of the patients infected with the CRF19_cpx variant in Spain during the period 2007–15 Patient ID  Gender  Age (years)  Risk practice  Year of diagnosis  Country of origin  City of origin  CD4+ T cell count (cells/mm3)  Viral load (log10 copies/mL)  Drug resistance mutations  ART  1  M  36  MSM  2013  Spain  Malaga  161  5.1  WT  FTC/TDF/RPV  2  M  29  MSM  2014  Spain  Seville  500  5.6  WT  unk  3  M  31  MSM  2014  Spain  Malaga  491  4.5  G190A    4  M  62  MSM  2014  Spain  Malaga  390  2.4  G190A    5  M  34  MSM  2014  Spain  Malaga  155  5.5  G190A    6  M  64  MSM  2010  Cuba  Madrid  428  4.9  WT  FTC/TDF/RPV  7  M  40  MSM  2010  Spain  Valencia  241  6.0  WT  FTC/TDF/RPV  8  M  27  MSM  2013  Spain  Malaga  407  3.6  G190A  FTC/TDF/RPV  9  M  42  MSM  2011  Spain  Madrid  665  5.1  WT  FTC/TDF/RPV  10  M  36  MSM  2013  Cuba  Barcelona  731  4.3  WT  FTC/TDF/RPV  11  M  70  MSM  2010  Spain  Valencia  251  5.8  M46MI  FTC/TDF/RPV  12  M  71  MSM  2011  Spain  Valencia  435  5.2  WT    13  M  58  HTX  2009  Cuba  Barcelona  33  5.5  D67G, T15C, K219E  unk  14  M  40  MSM  2010  Spain  Valencia  323  4.6  WT  FTC/TDF/RPV  15  M  30  MSM  2011  Spain  Canaries  439  4.9  WT  FTC/TDF/RPV  16  M  47  MSM  2015  Spain  Seville  525  5.9  WT  unk  17  M  37  MSM  2015  Spain  Seville  905  2.8  WT    18  M  35  MSM  2015  Spain  Malaga  207  4.2  G190A  unk  19  M  27  MSM  2015  Spain  Malaga  612  5.3  G190A    Patient ID  Gender  Age (years)  Risk practice  Year of diagnosis  Country of origin  City of origin  CD4+ T cell count (cells/mm3)  Viral load (log10 copies/mL)  Drug resistance mutations  ART  1  M  36  MSM  2013  Spain  Malaga  161  5.1  WT  FTC/TDF/RPV  2  M  29  MSM  2014  Spain  Seville  500  5.6  WT  unk  3  M  31  MSM  2014  Spain  Malaga  491  4.5  G190A    4  M  62  MSM  2014  Spain  Malaga  390  2.4  G190A    5  M  34  MSM  2014  Spain  Malaga  155  5.5  G190A    6  M  64  MSM  2010  Cuba  Madrid  428  4.9  WT  FTC/TDF/RPV  7  M  40  MSM  2010  Spain  Valencia  241  6.0  WT  FTC/TDF/RPV  8  M  27  MSM  2013  Spain  Malaga  407  3.6  G190A  FTC/TDF/RPV  9  M  42  MSM  2011  Spain  Madrid  665  5.1  WT  FTC/TDF/RPV  10  M  36  MSM  2013  Cuba  Barcelona  731  4.3  WT  FTC/TDF/RPV  11  M  70  MSM  2010  Spain  Valencia  251  5.8  M46MI  FTC/TDF/RPV  12  M  71  MSM  2011  Spain  Valencia  435  5.2  WT    13  M  58  HTX  2009  Cuba  Barcelona  33  5.5  D67G, T15C, K219E  unk  14  M  40  MSM  2010  Spain  Valencia  323  4.6  WT  FTC/TDF/RPV  15  M  30  MSM  2011  Spain  Canaries  439  4.9  WT  FTC/TDF/RPV  16  M  47  MSM  2015  Spain  Seville  525  5.9  WT  unk  17  M  37  MSM  2015  Spain  Seville  905  2.8  WT    18  M  35  MSM  2015  Spain  Malaga  207  4.2  G190A  unk  19  M  27  MSM  2015  Spain  Malaga  612  5.3  G190A    M, male, F, female; HTX, heterosexual; unk, treatment unknown; FTC, emtricitabine; TDF, tenofovir disoproxil fumarate; RPV, rilpivirine. All the patients were male, their mean age was 42.9 years (95% CI: 36.4–52.5 years), the majority were MSM [n = 18 (95%)] and of Spanish nationality [n = 16 (84.2%)] and they were from different areas [Malaga (n = 7), Valencia (n = 4), Seville (n = 3), Madrid (n = 2), Barcelona (n = 2) and the Canary Islands (n = 1)]. They had a median CD4+ T cell count of 415.7 cells/mm3 (95% CI: 317.7–513.7 cells/mm3) and a median viral load of 5.1 log10 copies/mL (IQR: 4.4–5.5 log10 copies/mL). The ML phylogenetic tree (Figure 1) demonstrated that 15 of the 19 patients (78.9%) were grouped into four different transmission clusters. Table 2 provides their demographic, clinical and virological characteristics. Three transmission clusters formed by two patients were found: two clusters grouped the patients from Valencia (clusters II and III) and another cluster related one patient from Seville and another from Madrid (cluster IV). We found one larger-sized transmission cluster (cluster I) that grouped the nine patients from southern Spain: seven from Malaga and two from Seville. In this cluster, six of the patients harboured mutation G190A in the RT gene. Finally, we found four unrelated patients, three of Cuban nationality, whose sequences grouped with other sequences from Cuba. Table 2. Demographic, clinical and virological data for the HIV-1 CRF19_cpx patients included in the transmission networks found in Spain Cluster ID  n  Sampling interval  Risk practice  Country of origin  City of origin  Viral load (log10 copies/mL), median (IQR)  CD4+ T cell count (cells/mm3), median (IQR)  Cluster I  9  2013–15  MSM  Spain  Malaga–Seville  4.4 (3.6–5.2)  407 (207–525)  Cluster II  2  2010  MSM  Spain  Valencia  5.5 (5.7–5.9)  262 (282–303)  Cluster III  2  2010–11  MSM  Spain  Valencia  5.5 (5.6–5.7)  343 (297–389)  Cluster IV  2  2011–14  MSM  Spain  Madrid–Seville  5.4 (5.3–5.5)  541.3 (583–624)  Cluster ID  n  Sampling interval  Risk practice  Country of origin  City of origin  Viral load (log10 copies/mL), median (IQR)  CD4+ T cell count (cells/mm3), median (IQR)  Cluster I  9  2013–15  MSM  Spain  Malaga–Seville  4.4 (3.6–5.2)  407 (207–525)  Cluster II  2  2010  MSM  Spain  Valencia  5.5 (5.7–5.9)  262 (282–303)  Cluster III  2  2010–11  MSM  Spain  Valencia  5.5 (5.6–5.7)  343 (297–389)  Cluster IV  2  2011–14  MSM  Spain  Madrid–Seville  5.4 (5.3–5.5)  541.3 (583–624)  Figure 1. View largeDownload slide ML tree of the CRF19_cpx HIV-1 pol sequences sampled in Spain (n = 19) with all the available HIV-1 CRF19_cpx pol sequences retrieved from the Los Alamos HIV sequence database (n = 162). Collapsed branches refer to a random data set of the 100 HIV-1 subtype D pol reference sequences, which was used as an outgroup. The tree was constructed according to the GTR + Gamma model of nucleotide substitution using RAxML. Branches are drawn on a scale with the bar at the bottom, which represents a 0.05 nucleotide substitution per site. Red branches indicate the CRF19_cpx sequences of the patients sampled in Spain (CoRIS cohort). The city of the patients sampled in Spain is represented on the branch tips (with the same colour code in the map). The statistically well-supported nodes (bootstrap values ≥90%) are indicated by an asterisk. Figure 1. View largeDownload slide ML tree of the CRF19_cpx HIV-1 pol sequences sampled in Spain (n = 19) with all the available HIV-1 CRF19_cpx pol sequences retrieved from the Los Alamos HIV sequence database (n = 162). Collapsed branches refer to a random data set of the 100 HIV-1 subtype D pol reference sequences, which was used as an outgroup. The tree was constructed according to the GTR + Gamma model of nucleotide substitution using RAxML. Branches are drawn on a scale with the bar at the bottom, which represents a 0.05 nucleotide substitution per site. Red branches indicate the CRF19_cpx sequences of the patients sampled in Spain (CoRIS cohort). The city of the patients sampled in Spain is represented on the branch tips (with the same colour code in the map). The statistically well-supported nodes (bootstrap values ≥90%) are indicated by an asterisk. Table 3 shows the time to the most recent common ancestor obtained for the various transmission clusters using different demographic growth models. Bayesian inference analysis confirmed the presence of these transmission clusters (pp > 0.9). Table 3. Most recent common ancestor dates (95% HPD) for the transmission clusters found in Spanish patients for the HIV-1 CRF19_cpx genetic form using different demographic growth models Cluster ID  Demographic growth models (AICM ± SE)   Bayesian skyline plot (29 570.7 ± 0.941)  constanta (29 552.033 ± 0.74)  exponential (29 570.892 ± 0.696)  logistic (29 570.97 ± 0.569)  Cluster I  2010.5 (2008.8–2011.6)  2010.2 (2006.7–2010.7)  2007.1 (2004.8–2009.5)  2008.5 (2005.9–2010.4)  Cluster II  2009.4 (2009–200.9)  2009 (2008.5–2010)  2009.7 (2008.1–2010)  2009.9 (2008.5–2010)  Cluster III  2009.7 (2009.1–2009.9)  2009.8 (2008.7–2010)  2009.2 (2008.3–2010)  2009.8 (2008.5–2010)  Cluster IV  2009.3 (2008.3–2010.9)  2009.8 (2007.8–2010.9)  2010.2 (2007.2–2010.9)  2008.5 (2007.8–2011)  Cluster ID  Demographic growth models (AICM ± SE)   Bayesian skyline plot (29 570.7 ± 0.941)  constanta (29 552.033 ± 0.74)  exponential (29 570.892 ± 0.696)  logistic (29 570.97 ± 0.569)  Cluster I  2010.5 (2008.8–2011.6)  2010.2 (2006.7–2010.7)  2007.1 (2004.8–2009.5)  2008.5 (2005.9–2010.4)  Cluster II  2009.4 (2009–200.9)  2009 (2008.5–2010)  2009.7 (2008.1–2010)  2009.9 (2008.5–2010)  Cluster III  2009.7 (2009.1–2009.9)  2009.8 (2008.7–2010)  2009.2 (2008.3–2010)  2009.8 (2008.5–2010)  Cluster IV  2009.3 (2008.3–2010.9)  2009.8 (2007.8–2010.9)  2010.2 (2007.2–2010.9)  2008.5 (2007.8–2011)  SE, standard error; HPD, high posterior density. a Demographic growth model with the best fit. We found that the constant demographic growth model obtained a better fit (with a lower AICM value). Figure 2 represents the maximum clade credibility tree used when this model was applied. Figure 2 shows that the different transmission networks originated in a short time interval (2009–10). In 2009 we found the origin of three main transmission networks, located mainly in eastern Spain (Valencia) and in Seville–Madrid: cluster II in 2009 (95% CI: 2008.5–2010), cluster III in 2009.8 (95% CI: 2008.7–2010) and cluster IV in 2009.8 (95% CI: 2007.8–2010.9). Finally at around 2010.2 (95% CI: 2006.7–2010.7), another cluster of HIV-positive patients appeared from southern Spain (cluster I). Figure 2. View largeDownload slide Maximum clade credibility tree generated by the Bayesian inference method using the constant-growth model, as implemented in v. 1.7.5 of BEAST, with all the HIV-1 CRF19_cpx pol sequences (n = 181). Branches were drawn for the country of origin of the sequences. All the Spanish clusters from CoRIS with highly supported nodes (pp > 0.9) are highlighted in blue and defined on a timescale. The sequences are named by the CoRIS ID number or the GenBank accession numbers in other Spanish sequences. Figure 2. View largeDownload slide Maximum clade credibility tree generated by the Bayesian inference method using the constant-growth model, as implemented in v. 1.7.5 of BEAST, with all the HIV-1 CRF19_cpx pol sequences (n = 181). Branches were drawn for the country of origin of the sequences. All the Spanish clusters from CoRIS with highly supported nodes (pp > 0.9) are highlighted in blue and defined on a timescale. The sequences are named by the CoRIS ID number or the GenBank accession numbers in other Spanish sequences. To date, none of the patients presents AIDS symptoms. Thirteen of the 19 patients started ART, of whom nine were treated with an NNRTI, rilpivirine, and with two nucleoside RT analogues: emtricitabine and tenofovir disoproxil fumarate. The treated patients progressed favourably to show undetectable viral loads. Discussion We demonstrate that HIV-1 variant CRF19_cpx continues to progressively extend between Spanish MSM from different areas of Spain and that most of these patients present high CD4+ T cell levels, which suggests that many of these patients could present acute or recent infection. According to the latest follow-up, now available in CoRIS, none of the patients infected with this variant has progressed to AIDS. All the patients who started treatment have successfully been suppressed. CRF19_cpx infections in Spain have either been restricted mainly to patients from Cuba or have eventually been reported in Spanish native patients infected from Cubans.3,4,22,23 Only five HIV-1 CRF19_cpx sequences retrieved in Spain have been reported to the LANL database throughout 2002–14 and these were phylogenetically related mainly to the viruses that circulate in Cuba. Only the last two sequences included in LANL in 2014 originated from two young Spanish MSM patients. This finding suggests a change in CRF19_cpx epidemiology in Spain and indicates that this variant has definitely been introduced into Spain and has been propagating in the young MSM population there in the recent years. Our data agree with those presented by Patiño Galindo et al.5 in the Valencian community, who found a similar origin for the first transmission networks at the end of 2010. We also demonstrate the expansion of this variant in southern Spain; in this case the variant is associated with mutation G190A, which confers on most of these patients a high level of resistance to nevirapine and an intermediate level of resistance to efavirenz. Nevertheless, all the patients who started treatment have been successfully suppressed with first-line regimes that were not based on these antiretroviral drugs. Fortunately, none of the HIV-infected patients with the CRF19_cpx genetic form displayed AIDS symptoms at the time of the last follow-up, which might be closely related to diagnosis and early ART. Although these data must be interpreted cautiously, since it has been previously demonstrated that patients infected with this genetic form exhibit a greater pathogenicity,2 our data do not support this observation. The spread of a new genetic form via a transmission cluster in MSM has previously been found to be directly related to its higher prevalence in a given area,24–26 or even to a higher proportion of patients who exhibit resistance to antiretroviral drugs.27 It is therefore extremely important to undertake collaborative efforts to perform continuous epidemiological monitoring to forecast future outbreaks of CRF19_cpx and other new HIV variants. Our study has several limitations. Firstly, we have analysed the CoRIS population, which may not be completely representative of the Spanish epidemic. Secondly, not all of the CoRIS participating centres contributed FASTA sequences for our analysis; fortunately, however, although the whole Spanish territory is not covered, we have previously shown28 the representativeness of CoRIS and our sequence data set for the analysis of transmitted resistance and subtype introduction. Finally, we have only analysed data up to 2015, and transmission may have continued during 2016. This is a common limitation to such studies as ‘real-time’ data are very difficult, if not impossible, to collect from cohort studies. In summary, by using phylogenetic and phylodynamic analyses, we have demonstrated the recent introduction and propagation of the HIV-1 CRF19_cpx variant in Spain among young HIV-positive MSM and its recent expansion in southern Spain via people carrying the G190A mutation in RT. These findings may serve to alert healthcare managers to enhance preventive measures in order to prevent the continuous expansion of HIV-1 CRF19_cpx, and generally of HIV-1 in MSM, in Spain. Finally, we were unable to demonstrate greater pathogenicity of HIV-1 CRF19_cpx compared with other HIV-1 variants. Acknowledgements This study would not have been possible without the collaboration of all the patients, medical and nursery staff and data mangers who have taken part in the project. Centres and researchers involved in CoRIS Executive committee: Santiago Moreno, Julia del Amo, David Dalmau, Maria Luisa Navarro, Maria Isabel González, Jose Luis Blanco, Federico Garcia, Rafael Rubio, Jose Antonio Iribarren, Félix Gutiérrez, Francesc Vidal, Juan Berenguer, Juan González. Fieldwork, data management and analysis: Paz Sobrino, Victoria Hernando, Belén Alejos, Débora Álvarez, Inma Jarrín, Cristina Moreno. BioBanK HIV: M Ángeles Muñoz-Fernández, Isabel García-Merino, Coral Gómez Rico, Jorge Gallego de la Fuente y Almudena García Torre. Participating centres:Hospital General Universitario de Alicante (Alicante): Joaquín Portilla, Esperanza Merino, Sergio Reus, Vicente Boix, Livia Giner, Carmen Gadea, Irene Portilla, Maria Pampliega, Marcos Díez, Juan Carlos Rodríguez, Jose Sánchez-Payá. Hospital Universitari de Bellvitge (Hospitalet de Llobregat): Daniel Podzamczer, Elena Ferrerm Arkaitz Imaz, Evan Van Den Eyncle, Silvana Di Yacovo, Maria Sumoy. Hospital Universitario de Canarias (Santa Cruz de Tenerife): Juan Luis Gómez, Jehovana Hernández, María Remedios Alemán, María del Mar Alonso, María Inmaculada Hernández, Felicitas Díaz-Flores, Dácil García, Ricardo Pelazas. Hospital Universitario Central de Asturias (Oviedo): Victor Asensi, Eulalia Valle, José Antonio Cartón. Hospital Clínico San Carlos (Madrid): Vicente Estrada Pérez, Maria Jesus Téllez Molina, Jorge Vergas García, Elisa Pérez-Cecila Carrera. Hospital Doce de Octubre (Madrid): Rafael Rubio, Federico Pulido, Otilia Bisbal, Mariano Matarranz, Maria Lagarde, Rafael Rubio-Martín, Asunción Hernando, Laura Bermejo y Lourdes Dominguez. Hospital Universitario Donostia (San Sebastián): José Antonio Iribarren, Julio Arrizabalaga, María José Aramburu, Xabier Camino, Francisco Rodríguez-Arrondo, Miguel Ángel von Wichmann, Lidia Pascual Tomé, Miguel Ángel Goenaga, Ma Jesús Bustinduy, Harkaitz Azkune Galparsoro. Maialen Ibarguren, Mirian Aguado, Maitane Umerez. Hospital General Universitario de Elche (Elche): Félix Gutiérrez, Mar Masiá, Cristina López, Sergio Padilla, Andrés Navarro, Fernando Montolio, Catalina Robledano, Joan Gregori Colomé, Araceli Adsuar, Rafael Pascual, Federico Carlos, Maravillas Martinez, Jara Llenas García, Marta Fernández, Elena García. Hospital Germans Trías i Pujol (Badalona): Roberto Muga, Jordi Tor, Arantza Sanvisens. Hospital General Universitario Gregorio Marañón (Madrid): Juan Berenguer, Juan Carlos López Bernaldo de Quirós, Pilar Miralles, Isabel Gutiérrez, Margarita Ramírez, Belén Padilla, Paloma Gijón, Ana Carrero, Teresa Aldamiz-Echevarría, Francisco Tejerina, Francisco Jose Parras, Pascual Balsalobre, Cristina Diez. Hospital Universitari de Tarragona Joan XXIII, IISPV, Universitat Rovira i Virgili (Tarragona): Francesc Vidal, Joaquín Peraire, Consuelo Viladés, Sergio Veloso, Montserrat Vargas, Miguel López-Dupla, Montserrat Olona, Alba Aguilar, Joan Josep Sirvent, Verónica Alba, Olga Calavia. Hospital Universitario La Fe (Valencia): Marta Montero, José Lacruz, Marino Blanes, Eva Calabuig, Sandra Cuellar, José López, Miguel Salavert. Hospital Universitario La Paz/IdiPaz (Madrid): Juan González, Ignacio Bernardino de la Serna, José Ramón Arribas, María Luisa Montes, Jose Ma Peña, Blanca Arribas, Juan Miguel Castro, Fco Javier Zamora, Ignacio Pérez, Miriam Estébanez, Silvia García, Marta Díaz, Natalia Stella Alcáriz, Jesús Mingorance, Dolores Montero, Alicia González, Maria Isabel de José. Hospital de la Princesa (Madrid): Ignacio de los Santos, Jesús Sanz, Ana Salas, Cristina Sarriá, Ana Gómez Berrocal, Lucio Garcia-Fraile. Hospital San Pedro-CIBIR (Logroño): José Antonio Oteo, José Ramón Blanco, Valvanera Ibarra, Luis Metola, Mercedes Sanz, Laura Pérez-Martínez. Hospital Universitario Miguel Servet (Zaragoza): Ascensión Pascual, Carlos Ramos, Piedad Arazo, Desiré Gil. Hospital Universitari Mutua de Terrassa (Terrassa): David Dalmau, Angels Jaén, Mireia Cairó, Daniel Irigoyen, Queralt Jordano, Mariona Xercavins, Javier Martinez-Lacasa, Pablo Velli, Roser Font, Montse Sanmartí, Laura Ibáñez. Complejo Hospitalario de Navarra (Pamplona): María Rivero, Marina Itziar Casado, Jorge Alberto Díaz, Javier Uriz, Jesús Repáraz, Carmen Irigoyen, María Jesús Arraiza. Hospital Parc Taulí (Sabadell): Ferrán Segura, María José Amengual, Gemma Navarro, Montserrat Sala, Manuel Cervantes, Valentín Pineda, Victor Segura, Marta Navarro, Esperanza Antón, Ma Merce Nogueras. Hospital Ramón y Cajal (Madrid): Santiago Moreno, José Luis Casado, Fernando Dronda, Ana Moreno, María Jesús Pérez Elías, Dolores López, Carolina Gutiérrez, Nadia Madrid, Angel Lamas, Paloma Martí, Alberto de Diaz, Sergio Serrrano, Lucas Donat. Hospital Reina Sofía (Murcia): Alfredo Cano, Enrique Bernal, Ángeles Muñoz. Hospital San Cecilio (Granada): Federico García, José Hernández, Alejandro Peña, Leopoldo Muñoz, Jorge Parra, Marta Alvarez, Natalia Chueca, Vicente Guillot, David Vinuesa, Jose Angel Fernández. Centro Sanitario Sandoval (Madrid): Jorge Del Romero, Carmen Rodríguez, Teresa Puerta, Juan Carlos Carrió, Mar Vera, Juan Ballesteros. Hospital de la Santa Creu i Sant Pau (Barcelona): Pere Domingo, Ma Antonia Sambeat, Karuna Lamarca, Gracia Mateo, Mar Gutiérrez, Irene Fernández. Hospital Universitario Santiago de Compostela (Santiago de Compostela): Antonio Antela, Elena Losada. Hospital Son Espases (Palma de Mallorca): Melchor Riera, Maria Peñaranda, Maria Leyes, Ma Angels Ribas, Antoni A Campins, Carmen Vidal, Leire Gil, Francisco Fanjul, Carmen Marinescu. Hospital Universitari Vall d’Hebron (Barcelona): Esteban Ribera. Hospital Virgen de la Victoria (Málaga): Jesús Santos, Manuel Márquez, Isabel Viciana, Rosario Palacios, Isabel Pérez, Carmen Maria González. Hospital Universitario Virgen del Rocío (Sevilla): Pompeyo Viciana, Manuel Leal, Luis Fernando López-Cortés, Nuria Espinosa. Hospital Universitario de Basurto (Bilbao): Josefa Muñoz, Miren Zuriñe Zubero, Josu Mirena Baraia-Etxaburu, Sofía Ibarra, Oscar Ferrero, Josefina López de Munain, Ma Mar Cámara. Iñigo López, Mireia de la Peña. Hospital Universitario Infanta Sofía (San Sebastián de los Reyes): Inés Suárez-García, Eduardo Malmierca. Hospital Universitario Costa del Sol (Marbella): Julián Olalla, Alfonso del Arco, Javier De la Torre, José Luis Prada, Zaira Caracuel. Hospital del Poniente (El Ejido): Ana Maria Lopez-Lirola, Ana Belén Lozano, Elisa Fernández, Inés Pérez, Juan Manuel Fernández. Hospital Universitario Santa Lucia (Cartagena): Onofre Juan Martínez, Francisco Jesús Vera, Lorena Martínez, Josefina García, Begoña Alcaraz, Amaya Jimeno. INIBIC-Complejo Hospitalario Universitario de A Coruña (A Coruña): Eva Poveda, Berta Pernas, Álvaro Mena, Marta Grandal, Ángeles Castro, José D. Pedreira. Hospital Clínico Universitario Virgen de la Arrixaca (Murcia): Carlos Galera, Helena Albendin, Asunción Iborra, Antonio Moreno, Maria Angeles Campillo, Asunción vidal. Hospital Marina Baixa (Villajoyosa): Concha Amador, Francisco Pasquau, Javier Ena, Concha Benito, Vicenta Fenoll. Complejo Hospitalario de Jaén (Jaén): Mohamed Omar Mohamed-Balghata, Maria Amparo Gómez. Hospital San Agustín de Aviles (Avilés): Miguel Alberto de Zarraga, Maria Eugenia Rivas. Fundación Jiménez Diaz (Madrid): Miguel Górgolas. Funding CoRIS is supported by the Instituto de Salud Carlos III through the Red Temática de Investigación Cooperativa en Sida (RD06/006, RD12/0017/0018 and RD16CIII/0002/0006) as part of the Plan Nacional R+D+I and is cofinanced by ISCIII-Subdirección General de Evaluación and Fondo Europeo de Desarrollo Regional (FEDER).  J. A. F.-C. and A. B. P. are supported by grant RD12/0017/006. F. G. has received a research extension grant from the Programa de Intensificación de la Actividad de Investigación del Servicio Andaluz de Salud. Transparency declarations None to declare. References 1 Casado G, Thomson MM, Sierra M et al.   Identification of a novel HIV-1 circulating ADG intersubtype recombinant form (CRF19_cpx) in Cuba. J Acquir Immune Defic Syndr  2005; 40: 532– 7. Google Scholar CrossRef Search ADS PubMed  2 Kouri V, Khouri R, Alemán Y et al.   CRF19_cpx is an evolutionary fit HIV-1 variant strongly associated with rapid progression to AIDS in Cuba. EBioMedicine  2015; 2: 244– 54. Google Scholar CrossRef Search ADS PubMed  3 Yebra G, de Mulder M, Martín L et al.   Most HIV type 1 non-B infections in the Spanish cohort of antiretroviral treatment-naïve HIV-infected patients (CoRIS) are due to recombinant viruses. J Clin Microbiol  2012; 50: 407– 13. Google Scholar CrossRef Search ADS PubMed  4 García F, Pérez-Cachafeiro S, Guillot V et al.   Transmission of HIV drug resistance and non-B subtype distribution in the Spanish cohort of antiretroviral treatment naïve HIV-infected individuals (CoRIS). Antiviral Res  2011; 91: 150– 3. Google Scholar CrossRef Search ADS PubMed  5 Patiño Galindo JA, Torres-Puente M, Gimeno C et al.   Expansion of the CRF19_cpx variant in Spain. J Clin Virol  2015; 69: 146– 9. Google Scholar CrossRef Search ADS PubMed  6 Caro-Murillo AM, Castilla J, Pérez-Hoyos S et al.   Cohorte RIS de pacientes con infección por VIH sin tratamiento antirretroviral previo (CoRIS): metodología y primeros resultados. Enfermedades Infecc Microbiol Clínica  2007; 25: 23– 31. Google Scholar CrossRef Search ADS   7 Monge S, Guillot V, Alvarez M et al.   Clinically relevant transmitted drug resistance to first line antiretroviral drugs and implications for recommendations. PLoS One  2014; 9: e90710. Google Scholar CrossRef Search ADS PubMed  8 Monge S, Guillot V, Alvarez M et al.   Analysis of transmitted drug resistance in Spain in the years 2007-2010 documents a decline in mutations to the non-nucleoside drug class. Clin Microbiol Infect  2012; 18: E485– 90. Google Scholar CrossRef Search ADS PubMed  9 González-Alba JM, Holguín Á, Garcia R et al.   Molecular surveillance of HIV-1 in Madrid, Spain: a phylogeographic analysis. J Virol  2011; 85: 10755– 63. Google Scholar CrossRef Search ADS PubMed  10 Ciccozzi M, Lai A, Ebranati E et al.   Phylogeographic reconstruction of HIV type 1B in Montenegro and the Balkan region. AIDS Res Hum Retroviruses  2012; 28: 1280– 4. Google Scholar CrossRef Search ADS PubMed  11 Yebra G, Kalish ML, Leigh Brown AJ. Reconstructing the HIV-1 CRF02_AG and CRF06_cpx epidemics in Burkina Faso and West Africa using early samples. Infect Genet Evol  2016; 46: 209– 18. Google Scholar CrossRef Search ADS PubMed  12 Delatorre E, Bello G. Phylodynamics of the HIV-1 epidemic in Cuba. PLoS One  2013; 8: e72448. Google Scholar CrossRef Search ADS PubMed  13 Bello G, Afonso JM, Morgado MG. Phylodynamics of HIV-1 subtype F1 in Angola, Brazil and Romania. Infect Genet Evol  2012; 12: 1079– 86. Google Scholar CrossRef Search ADS PubMed  14 Patiño-Galindo JA, Thomson MM, Pérez-Álvarez L et al.   Transmission dynamics of HIV-1 subtype B in the Basque country, Spain. Infect Genet Evol  2016; 40: 91– 7. Google Scholar CrossRef Search ADS PubMed  15 Miller MA, Pfeiffer W, Schwartz T. Creating the CIPRES Science Gateway for Inference of Large Phylogenetic Trees . New Orleans, LA, USA: Gateway Computing Environments Workshop (GCE), 2010. 16 Hillis DM, Bull JJ. An empirical test of bootstrapping as a method for assessing confidence in phylogenetic analysis. Syst Biol  1993; 4: 182– 92. Google Scholar CrossRef Search ADS   17 Larkin MA, Blackshields G, Brown NP et al.   Clustal W and Clustal X version 2.0. Bioinformatics  2007; 23: 2947– 8. Google Scholar CrossRef Search ADS PubMed  18 Lewis F, Hughes GJ, Rambaut A et al.   Episodic sexual transmission of HIV revealed by molecular phylodynamics. PLoS Med  2008; 5: e50. Google Scholar CrossRef Search ADS PubMed  19 Drummond AJ, Suchard MA, Xie D et al.   Bayesian phylogenetics with BEAUti and the BEAST 1.7. Mol Biol Evol  2012; 29: 1969– 73. Google Scholar CrossRef Search ADS PubMed  20 Drummond AJ, Ho SYW, Phillips MJ et al.   Relaxed phylogenetics and dating with confidence. PLoS Biol  2006; 4: e88. Google Scholar CrossRef Search ADS PubMed  21 Bennett DE, Camacho RJ, Otelea D et al.   Drug resistance mutations for surveillance of transmitted HIV-1 drug-resistance. PLoS One  2009; 4: e4724. Google Scholar CrossRef Search ADS PubMed  22 Holguín A, de Mulder M, Yebra G et al.   Increase of non-B subtypes and recombinants among newly diagnosed HIV-1 native Spaniards and immigrants in Spain. Curr HIV Res  2008; 6: 327– 34. Google Scholar CrossRef Search ADS PubMed  23 Yebra G, Delgado R, Pulido F et al.   Different trends of transmitted HIV-1 drug resistance in Madrid, Spain, among risk groups in the last decade. Arch Virol  2014; 159: 1079– 87. Google Scholar CrossRef Search ADS PubMed  24 Paraskevis D, Kostaki E, Beloukas A et al.   Molecular characterization of HIV-1 infection in Northwest Spain (2009-2013): investigation of the subtype F outbreak. Infect Genet Evol  2015; 30: 96– 101. Google Scholar CrossRef Search ADS PubMed  25 Pernas B, Grandal M, Mena A et al.   High prevalence of subtype F in newly diagnosed HIV-1 persons in northwest Spain and evidence for impaired treatment response. AIDS  2014; 28: 1837– 40. Google Scholar CrossRef Search ADS PubMed  26 Thomson MM, Fernández-García A, Delgado E et al.   Rapid expansion of a HIV-1 subtype F cluster of recent origin among men who have sex with men in Galicia, Spain. J Acquir Immune Defic Syndr  2012; 59: e49– 51. Google Scholar CrossRef Search ADS PubMed  27 Vega Y, Delgado E, Fernández-García A et al.   Epidemiological surveillance of HIV-1 transmitted drug resistance in Spain in 2004-2012: relevance of transmission clusters in the propagation of resistance mutations. PLoS One  2015; 10: e0125699. Google Scholar CrossRef Search ADS PubMed  28 Monge S, Díez M, Alvarez M et al.   Use of cohort data to estimate national prevalence of transmitted drug resistance to antiretroviral drugs in Spain (2007–2012). Clin Microbiol Infect  2015; 21: 105.e1. Google Scholar CrossRef Search ADS   © The Author(s) 2018. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please email: journals.permissions@oup.com. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Antimicrobial Chemotherapy Oxford University Press

Continued propagation of the CRF19_cpx variant among HIV-positive MSM patients in Spain

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Oxford University Press
ISSN
0305-7453
eISSN
1460-2091
D.O.I.
10.1093/jac/dkx474
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Abstract

Abstract Objectives The HIV-1 CRF19_cpx genetic form has been recently associated with greater pathogenicity. We used CoRIS, a national cohort of 31 reference hospitals in Spain, to investigate the current epidemiological situation of this variant in Spain. Patients and methods We analysed 4734 naive HIV-1-positive patients diagnosed during the 2007–15 period with an available pol gene sequence in the CoRIS resistance database. HIV-1 CRF19_cpx was ascribed through REGA3.0 and confirmed by a phylogenetic analysis. We analysed the presence of the transmission clusters of HIV-1 CRF19_cpx by maximum likelihood [with the randomized accelerated maximum likelihood (RAxML) program] and the time to the most recent common ancestor using Bayesian inference (BEAST, v. 1.7.5). Results Nineteen patients were infected with CRF19_cpx: all were male, they had a mean age of 42.9 years (95% CI: 36.4–52.5 years), the majority were MSM [n = 18 (95%)] and of Spanish nationality [n = 16 (84.2%)] and they had high CD4+ T cell counts (∼415 cells/mm3). Fifteen patients were grouped into four different transmission clusters: two clusters (two patients each) grouped the patients from Valencia and another cluster grouped one patient from Madrid and another from Seville. We found a larger cluster that grouped nine patients from southern Spain (Malaga and Seville), of which six presented mutation G190A. We estimated the origin of all the transmission clusters to take place between 2009 and 2010. Conclusions We demonstrate that this variant has spread in Spain in recent years among young HIV-positive MSM and we note a recent expansion in southern Spain in patients who carry mutation G190A. We alert healthcare managers to enhance preventive measures to prevent the continuous spread of HIV-1 CRF19_cpx. Introduction The HIV-1 CRF19_cpx genetic form (A, D, G intersubtype circulating recombinant form), described for the first time in Cuba in 2005,1 has been associated with great pathogenicity: patients are diagnosed with high viral loads, high RANTES chemokine expression and rapidly progress to AIDS.2 One characteristic of this genetic form is its failure to spread widely to other countries: ∼80% of the sequences included in the Los Alamos HIV sequence database (LANL) come from Cuba and the prevalence for Spain is <1%.3,4 Nonetheless, the recent finding of several transmission networks in eastern Spain (Valencia) in which HIV-positive male patients are infected with the HIV-1 CRF19_cpx genetic form indicates that this genetic form is likely to expand in the coming years.5 The Cohort of the Spanish Network of AIDS Research (CoRIS)6 is a national prospective multicentre cohort formed exclusively of naive HIV-positive patients from 31 reference Spanish hospitals. Over time, CoRIS has offered a valuable source of clinical, demographic and virological information to study the prevalence of different genetic forms of HIV-1,3,4 and has enabled epidemiological monitoring of resistance to different first-line antiretroviral drugs.4,7,8 Phylogenetic studies applied to large series of sequences are essential to monitor the impact of migration on a given local epidemiology,9–12 and to establish the origin and diversification of certain transmission networks.12–14 In this study, we used several phylogenetic analysis tools to investigate the current epidemiological situation of the HIV-1 CRF19_cpx variant in Spain. Patients and methods Study population The analysis included 4734 partial pol gene sequences [protease (PR) codons 4–99; reverse transcriptase (RT) codons 38–247] of all the newly HIV-1 diagnosed patients during the 2007–15 period retrieved in CoRIS. Viral sequences were generated from the earliest sample available before ART initiation (baseline) and were linked to demographic (risk group, age, gender, country of origin and attending hospital of origin), clinical (CD4+ T cell count) and virological (viral load) information. Ethics All subjects provided written informed consent to participate. The study was reviewed and approved by the institutional review boards of all the participating centres. HIV-1 subtype assignment A preliminary search for the HIV-1 CRF19_cpx genetic forms was done using the REGA 3.0 subtyping tool (http://dbpartners.stanford.edu: 8080/RegaSubtyping/stanford-hiv/typingtool/). All these variants were definitely confirmed by a phylogenetic analysis, which was performed by maximum likelihood (ML) analysis with the randomized accelerated ML (RAxML) program, accessible via the CIPRES Science Gateway.15 For this analysis, a general time-reversible (GTR) model with γ-distributed rate heterogeneity across sites and 1000 bootstrap iterations was employed. A representative data set of HIV-1 sequences group M, pure subtypes (A–K) and recombinant forms (at least four representative sequences of each pure subtype and the CRF currently available from the analysis) from LANL was taken as a reference. The HIV-1 CRF19_cpx classification was considered consistent if the query sequence was included with the corresponding reference sequences in a monophyletic cluster supported by high bootstrap values (>70%).16 Analysis of the phylogenetic relationship among the patients infected with HIV-1 CRF19_cpx In order to characterize the phylogenetic relationship between the sequences found in the HIV-1 CRF19_cpx genetic form in Spain, these sequences were analysed with all the sequences available in LANL (start 2279, end, 3290; HXB2 nucleotide position) (last accessed December 2016): 162 sequences [Cuba (n = 152), Spain (n = 5), UK (n = 1), USA (n = 3), Greece (n = 1)]. As the breakpoints in the pol region were lacking for the HIV-1 CRF19_cpx genetic form (subtype D in pol), a consistent data set of 100 HIV-1 subtype D sequences retrieved from LANL was added. All the sequences were aligned through ClustalW17 and were manually edited. Given the convergent evolution of the antiretroviral drug resistance mutations in the phylogenetic analysis,18 all the drug resistance mutations were removed (drug resistance mutation present in at least two patients) that were associated with the codons with major resistance in PR (30, 32, 33, 46, 48, 48, 50, 54, 76, 82, 84, 88 and 90) and RT (41, 62, 65, 67, 69, 70, 74, 75, 77, 100, 103, 106, 108, 115, 116, 151, 181, 184, 188, 190, 210, 215, 219, 225 and 236). A preliminary analysis was run using ML in RAxML with the best-fitting model of nucleotide substitution obtained through FindModel (at http://www.hiv.lanl.gov/content/sequence/findmodel/findmodel.html). The best-fitting model with the lowest Akaike’s information criterion (AIC) in Find Model was the GTR model with γ-distributed rate heterogeneity across sites using 1000 bootstrap iterations. We investigated the presence of associations between the HIV-1 CRF19_cpx Spanish sequences from the CoRIS cohort with related nodes by taking bootstrap values ≥90% as significantly reliable node supports. To confirm the transmission networks that grouped the sequences from the CoRIS cohort, a Bayesian Markov Chain Monte Carlo (MCMC) approach was applied to all the HIV-1 CRF19_cpx (n = 181) genetic forms, as implemented in v. 1.7.5 of BEAST.19 The Shapiro–Rambaut–Drummond–2006 (SRD06) substitution model was used, together with a relaxed uncorrelated log-normal clock (UCLN).20 A comparison was made among the different demographic models (Bayesian Skyline Plot, Constant, Exponential and Logistic) using Tracer v. 1.6 and the best model was chosen according to the lowest AIC through MCMC (AICM). Each MCMC approach was run for 250 million states by sampling every 50 000. Only traces with an effective sample size (ESS) of >200 were accepted in v. 1.6 of TRACER after excluding an initial 10% (http://tree.bio.ed.ac.uk/software/tracer/). These previously defined transmission networks (through ML) were confirmed by Bayesian inference using posterior probability (pp) values >0.9 as the thresholds. Finally, the most recent common ancestor for these clusters was estimated. Analysis of antiretroviral drug resistance mutations We analysed drug resistance mutations with the HIVseq program, available from the HIV Drug Resistance Database of Stanford University (https://hivdb.stanford.edu/hivseq/by-sequences/) and the WHO surveillance drug resistance mutation list (last updated in 2009 by Bennett et al.21). Results Nineteen patients (0.4%) were infected with the CRF19_cpx variant during the period 2007–15. The demographic, clinical and virological characteristics of these patients are shown in Table 1. Table 1. Demographic, clinical and virological characteristics of the patients infected with the CRF19_cpx variant in Spain during the period 2007–15 Patient ID  Gender  Age (years)  Risk practice  Year of diagnosis  Country of origin  City of origin  CD4+ T cell count (cells/mm3)  Viral load (log10 copies/mL)  Drug resistance mutations  ART  1  M  36  MSM  2013  Spain  Malaga  161  5.1  WT  FTC/TDF/RPV  2  M  29  MSM  2014  Spain  Seville  500  5.6  WT  unk  3  M  31  MSM  2014  Spain  Malaga  491  4.5  G190A    4  M  62  MSM  2014  Spain  Malaga  390  2.4  G190A    5  M  34  MSM  2014  Spain  Malaga  155  5.5  G190A    6  M  64  MSM  2010  Cuba  Madrid  428  4.9  WT  FTC/TDF/RPV  7  M  40  MSM  2010  Spain  Valencia  241  6.0  WT  FTC/TDF/RPV  8  M  27  MSM  2013  Spain  Malaga  407  3.6  G190A  FTC/TDF/RPV  9  M  42  MSM  2011  Spain  Madrid  665  5.1  WT  FTC/TDF/RPV  10  M  36  MSM  2013  Cuba  Barcelona  731  4.3  WT  FTC/TDF/RPV  11  M  70  MSM  2010  Spain  Valencia  251  5.8  M46MI  FTC/TDF/RPV  12  M  71  MSM  2011  Spain  Valencia  435  5.2  WT    13  M  58  HTX  2009  Cuba  Barcelona  33  5.5  D67G, T15C, K219E  unk  14  M  40  MSM  2010  Spain  Valencia  323  4.6  WT  FTC/TDF/RPV  15  M  30  MSM  2011  Spain  Canaries  439  4.9  WT  FTC/TDF/RPV  16  M  47  MSM  2015  Spain  Seville  525  5.9  WT  unk  17  M  37  MSM  2015  Spain  Seville  905  2.8  WT    18  M  35  MSM  2015  Spain  Malaga  207  4.2  G190A  unk  19  M  27  MSM  2015  Spain  Malaga  612  5.3  G190A    Patient ID  Gender  Age (years)  Risk practice  Year of diagnosis  Country of origin  City of origin  CD4+ T cell count (cells/mm3)  Viral load (log10 copies/mL)  Drug resistance mutations  ART  1  M  36  MSM  2013  Spain  Malaga  161  5.1  WT  FTC/TDF/RPV  2  M  29  MSM  2014  Spain  Seville  500  5.6  WT  unk  3  M  31  MSM  2014  Spain  Malaga  491  4.5  G190A    4  M  62  MSM  2014  Spain  Malaga  390  2.4  G190A    5  M  34  MSM  2014  Spain  Malaga  155  5.5  G190A    6  M  64  MSM  2010  Cuba  Madrid  428  4.9  WT  FTC/TDF/RPV  7  M  40  MSM  2010  Spain  Valencia  241  6.0  WT  FTC/TDF/RPV  8  M  27  MSM  2013  Spain  Malaga  407  3.6  G190A  FTC/TDF/RPV  9  M  42  MSM  2011  Spain  Madrid  665  5.1  WT  FTC/TDF/RPV  10  M  36  MSM  2013  Cuba  Barcelona  731  4.3  WT  FTC/TDF/RPV  11  M  70  MSM  2010  Spain  Valencia  251  5.8  M46MI  FTC/TDF/RPV  12  M  71  MSM  2011  Spain  Valencia  435  5.2  WT    13  M  58  HTX  2009  Cuba  Barcelona  33  5.5  D67G, T15C, K219E  unk  14  M  40  MSM  2010  Spain  Valencia  323  4.6  WT  FTC/TDF/RPV  15  M  30  MSM  2011  Spain  Canaries  439  4.9  WT  FTC/TDF/RPV  16  M  47  MSM  2015  Spain  Seville  525  5.9  WT  unk  17  M  37  MSM  2015  Spain  Seville  905  2.8  WT    18  M  35  MSM  2015  Spain  Malaga  207  4.2  G190A  unk  19  M  27  MSM  2015  Spain  Malaga  612  5.3  G190A    M, male, F, female; HTX, heterosexual; unk, treatment unknown; FTC, emtricitabine; TDF, tenofovir disoproxil fumarate; RPV, rilpivirine. All the patients were male, their mean age was 42.9 years (95% CI: 36.4–52.5 years), the majority were MSM [n = 18 (95%)] and of Spanish nationality [n = 16 (84.2%)] and they were from different areas [Malaga (n = 7), Valencia (n = 4), Seville (n = 3), Madrid (n = 2), Barcelona (n = 2) and the Canary Islands (n = 1)]. They had a median CD4+ T cell count of 415.7 cells/mm3 (95% CI: 317.7–513.7 cells/mm3) and a median viral load of 5.1 log10 copies/mL (IQR: 4.4–5.5 log10 copies/mL). The ML phylogenetic tree (Figure 1) demonstrated that 15 of the 19 patients (78.9%) were grouped into four different transmission clusters. Table 2 provides their demographic, clinical and virological characteristics. Three transmission clusters formed by two patients were found: two clusters grouped the patients from Valencia (clusters II and III) and another cluster related one patient from Seville and another from Madrid (cluster IV). We found one larger-sized transmission cluster (cluster I) that grouped the nine patients from southern Spain: seven from Malaga and two from Seville. In this cluster, six of the patients harboured mutation G190A in the RT gene. Finally, we found four unrelated patients, three of Cuban nationality, whose sequences grouped with other sequences from Cuba. Table 2. Demographic, clinical and virological data for the HIV-1 CRF19_cpx patients included in the transmission networks found in Spain Cluster ID  n  Sampling interval  Risk practice  Country of origin  City of origin  Viral load (log10 copies/mL), median (IQR)  CD4+ T cell count (cells/mm3), median (IQR)  Cluster I  9  2013–15  MSM  Spain  Malaga–Seville  4.4 (3.6–5.2)  407 (207–525)  Cluster II  2  2010  MSM  Spain  Valencia  5.5 (5.7–5.9)  262 (282–303)  Cluster III  2  2010–11  MSM  Spain  Valencia  5.5 (5.6–5.7)  343 (297–389)  Cluster IV  2  2011–14  MSM  Spain  Madrid–Seville  5.4 (5.3–5.5)  541.3 (583–624)  Cluster ID  n  Sampling interval  Risk practice  Country of origin  City of origin  Viral load (log10 copies/mL), median (IQR)  CD4+ T cell count (cells/mm3), median (IQR)  Cluster I  9  2013–15  MSM  Spain  Malaga–Seville  4.4 (3.6–5.2)  407 (207–525)  Cluster II  2  2010  MSM  Spain  Valencia  5.5 (5.7–5.9)  262 (282–303)  Cluster III  2  2010–11  MSM  Spain  Valencia  5.5 (5.6–5.7)  343 (297–389)  Cluster IV  2  2011–14  MSM  Spain  Madrid–Seville  5.4 (5.3–5.5)  541.3 (583–624)  Figure 1. View largeDownload slide ML tree of the CRF19_cpx HIV-1 pol sequences sampled in Spain (n = 19) with all the available HIV-1 CRF19_cpx pol sequences retrieved from the Los Alamos HIV sequence database (n = 162). Collapsed branches refer to a random data set of the 100 HIV-1 subtype D pol reference sequences, which was used as an outgroup. The tree was constructed according to the GTR + Gamma model of nucleotide substitution using RAxML. Branches are drawn on a scale with the bar at the bottom, which represents a 0.05 nucleotide substitution per site. Red branches indicate the CRF19_cpx sequences of the patients sampled in Spain (CoRIS cohort). The city of the patients sampled in Spain is represented on the branch tips (with the same colour code in the map). The statistically well-supported nodes (bootstrap values ≥90%) are indicated by an asterisk. Figure 1. View largeDownload slide ML tree of the CRF19_cpx HIV-1 pol sequences sampled in Spain (n = 19) with all the available HIV-1 CRF19_cpx pol sequences retrieved from the Los Alamos HIV sequence database (n = 162). Collapsed branches refer to a random data set of the 100 HIV-1 subtype D pol reference sequences, which was used as an outgroup. The tree was constructed according to the GTR + Gamma model of nucleotide substitution using RAxML. Branches are drawn on a scale with the bar at the bottom, which represents a 0.05 nucleotide substitution per site. Red branches indicate the CRF19_cpx sequences of the patients sampled in Spain (CoRIS cohort). The city of the patients sampled in Spain is represented on the branch tips (with the same colour code in the map). The statistically well-supported nodes (bootstrap values ≥90%) are indicated by an asterisk. Table 3 shows the time to the most recent common ancestor obtained for the various transmission clusters using different demographic growth models. Bayesian inference analysis confirmed the presence of these transmission clusters (pp > 0.9). Table 3. Most recent common ancestor dates (95% HPD) for the transmission clusters found in Spanish patients for the HIV-1 CRF19_cpx genetic form using different demographic growth models Cluster ID  Demographic growth models (AICM ± SE)   Bayesian skyline plot (29 570.7 ± 0.941)  constanta (29 552.033 ± 0.74)  exponential (29 570.892 ± 0.696)  logistic (29 570.97 ± 0.569)  Cluster I  2010.5 (2008.8–2011.6)  2010.2 (2006.7–2010.7)  2007.1 (2004.8–2009.5)  2008.5 (2005.9–2010.4)  Cluster II  2009.4 (2009–200.9)  2009 (2008.5–2010)  2009.7 (2008.1–2010)  2009.9 (2008.5–2010)  Cluster III  2009.7 (2009.1–2009.9)  2009.8 (2008.7–2010)  2009.2 (2008.3–2010)  2009.8 (2008.5–2010)  Cluster IV  2009.3 (2008.3–2010.9)  2009.8 (2007.8–2010.9)  2010.2 (2007.2–2010.9)  2008.5 (2007.8–2011)  Cluster ID  Demographic growth models (AICM ± SE)   Bayesian skyline plot (29 570.7 ± 0.941)  constanta (29 552.033 ± 0.74)  exponential (29 570.892 ± 0.696)  logistic (29 570.97 ± 0.569)  Cluster I  2010.5 (2008.8–2011.6)  2010.2 (2006.7–2010.7)  2007.1 (2004.8–2009.5)  2008.5 (2005.9–2010.4)  Cluster II  2009.4 (2009–200.9)  2009 (2008.5–2010)  2009.7 (2008.1–2010)  2009.9 (2008.5–2010)  Cluster III  2009.7 (2009.1–2009.9)  2009.8 (2008.7–2010)  2009.2 (2008.3–2010)  2009.8 (2008.5–2010)  Cluster IV  2009.3 (2008.3–2010.9)  2009.8 (2007.8–2010.9)  2010.2 (2007.2–2010.9)  2008.5 (2007.8–2011)  SE, standard error; HPD, high posterior density. a Demographic growth model with the best fit. We found that the constant demographic growth model obtained a better fit (with a lower AICM value). Figure 2 represents the maximum clade credibility tree used when this model was applied. Figure 2 shows that the different transmission networks originated in a short time interval (2009–10). In 2009 we found the origin of three main transmission networks, located mainly in eastern Spain (Valencia) and in Seville–Madrid: cluster II in 2009 (95% CI: 2008.5–2010), cluster III in 2009.8 (95% CI: 2008.7–2010) and cluster IV in 2009.8 (95% CI: 2007.8–2010.9). Finally at around 2010.2 (95% CI: 2006.7–2010.7), another cluster of HIV-positive patients appeared from southern Spain (cluster I). Figure 2. View largeDownload slide Maximum clade credibility tree generated by the Bayesian inference method using the constant-growth model, as implemented in v. 1.7.5 of BEAST, with all the HIV-1 CRF19_cpx pol sequences (n = 181). Branches were drawn for the country of origin of the sequences. All the Spanish clusters from CoRIS with highly supported nodes (pp > 0.9) are highlighted in blue and defined on a timescale. The sequences are named by the CoRIS ID number or the GenBank accession numbers in other Spanish sequences. Figure 2. View largeDownload slide Maximum clade credibility tree generated by the Bayesian inference method using the constant-growth model, as implemented in v. 1.7.5 of BEAST, with all the HIV-1 CRF19_cpx pol sequences (n = 181). Branches were drawn for the country of origin of the sequences. All the Spanish clusters from CoRIS with highly supported nodes (pp > 0.9) are highlighted in blue and defined on a timescale. The sequences are named by the CoRIS ID number or the GenBank accession numbers in other Spanish sequences. To date, none of the patients presents AIDS symptoms. Thirteen of the 19 patients started ART, of whom nine were treated with an NNRTI, rilpivirine, and with two nucleoside RT analogues: emtricitabine and tenofovir disoproxil fumarate. The treated patients progressed favourably to show undetectable viral loads. Discussion We demonstrate that HIV-1 variant CRF19_cpx continues to progressively extend between Spanish MSM from different areas of Spain and that most of these patients present high CD4+ T cell levels, which suggests that many of these patients could present acute or recent infection. According to the latest follow-up, now available in CoRIS, none of the patients infected with this variant has progressed to AIDS. All the patients who started treatment have successfully been suppressed. CRF19_cpx infections in Spain have either been restricted mainly to patients from Cuba or have eventually been reported in Spanish native patients infected from Cubans.3,4,22,23 Only five HIV-1 CRF19_cpx sequences retrieved in Spain have been reported to the LANL database throughout 2002–14 and these were phylogenetically related mainly to the viruses that circulate in Cuba. Only the last two sequences included in LANL in 2014 originated from two young Spanish MSM patients. This finding suggests a change in CRF19_cpx epidemiology in Spain and indicates that this variant has definitely been introduced into Spain and has been propagating in the young MSM population there in the recent years. Our data agree with those presented by Patiño Galindo et al.5 in the Valencian community, who found a similar origin for the first transmission networks at the end of 2010. We also demonstrate the expansion of this variant in southern Spain; in this case the variant is associated with mutation G190A, which confers on most of these patients a high level of resistance to nevirapine and an intermediate level of resistance to efavirenz. Nevertheless, all the patients who started treatment have been successfully suppressed with first-line regimes that were not based on these antiretroviral drugs. Fortunately, none of the HIV-infected patients with the CRF19_cpx genetic form displayed AIDS symptoms at the time of the last follow-up, which might be closely related to diagnosis and early ART. Although these data must be interpreted cautiously, since it has been previously demonstrated that patients infected with this genetic form exhibit a greater pathogenicity,2 our data do not support this observation. The spread of a new genetic form via a transmission cluster in MSM has previously been found to be directly related to its higher prevalence in a given area,24–26 or even to a higher proportion of patients who exhibit resistance to antiretroviral drugs.27 It is therefore extremely important to undertake collaborative efforts to perform continuous epidemiological monitoring to forecast future outbreaks of CRF19_cpx and other new HIV variants. Our study has several limitations. Firstly, we have analysed the CoRIS population, which may not be completely representative of the Spanish epidemic. Secondly, not all of the CoRIS participating centres contributed FASTA sequences for our analysis; fortunately, however, although the whole Spanish territory is not covered, we have previously shown28 the representativeness of CoRIS and our sequence data set for the analysis of transmitted resistance and subtype introduction. Finally, we have only analysed data up to 2015, and transmission may have continued during 2016. This is a common limitation to such studies as ‘real-time’ data are very difficult, if not impossible, to collect from cohort studies. In summary, by using phylogenetic and phylodynamic analyses, we have demonstrated the recent introduction and propagation of the HIV-1 CRF19_cpx variant in Spain among young HIV-positive MSM and its recent expansion in southern Spain via people carrying the G190A mutation in RT. These findings may serve to alert healthcare managers to enhance preventive measures in order to prevent the continuous expansion of HIV-1 CRF19_cpx, and generally of HIV-1 in MSM, in Spain. Finally, we were unable to demonstrate greater pathogenicity of HIV-1 CRF19_cpx compared with other HIV-1 variants. Acknowledgements This study would not have been possible without the collaboration of all the patients, medical and nursery staff and data mangers who have taken part in the project. Centres and researchers involved in CoRIS Executive committee: Santiago Moreno, Julia del Amo, David Dalmau, Maria Luisa Navarro, Maria Isabel González, Jose Luis Blanco, Federico Garcia, Rafael Rubio, Jose Antonio Iribarren, Félix Gutiérrez, Francesc Vidal, Juan Berenguer, Juan González. Fieldwork, data management and analysis: Paz Sobrino, Victoria Hernando, Belén Alejos, Débora Álvarez, Inma Jarrín, Cristina Moreno. BioBanK HIV: M Ángeles Muñoz-Fernández, Isabel García-Merino, Coral Gómez Rico, Jorge Gallego de la Fuente y Almudena García Torre. Participating centres:Hospital General Universitario de Alicante (Alicante): Joaquín Portilla, Esperanza Merino, Sergio Reus, Vicente Boix, Livia Giner, Carmen Gadea, Irene Portilla, Maria Pampliega, Marcos Díez, Juan Carlos Rodríguez, Jose Sánchez-Payá. Hospital Universitari de Bellvitge (Hospitalet de Llobregat): Daniel Podzamczer, Elena Ferrerm Arkaitz Imaz, Evan Van Den Eyncle, Silvana Di Yacovo, Maria Sumoy. Hospital Universitario de Canarias (Santa Cruz de Tenerife): Juan Luis Gómez, Jehovana Hernández, María Remedios Alemán, María del Mar Alonso, María Inmaculada Hernández, Felicitas Díaz-Flores, Dácil García, Ricardo Pelazas. Hospital Universitario Central de Asturias (Oviedo): Victor Asensi, Eulalia Valle, José Antonio Cartón. Hospital Clínico San Carlos (Madrid): Vicente Estrada Pérez, Maria Jesus Téllez Molina, Jorge Vergas García, Elisa Pérez-Cecila Carrera. Hospital Doce de Octubre (Madrid): Rafael Rubio, Federico Pulido, Otilia Bisbal, Mariano Matarranz, Maria Lagarde, Rafael Rubio-Martín, Asunción Hernando, Laura Bermejo y Lourdes Dominguez. Hospital Universitario Donostia (San Sebastián): José Antonio Iribarren, Julio Arrizabalaga, María José Aramburu, Xabier Camino, Francisco Rodríguez-Arrondo, Miguel Ángel von Wichmann, Lidia Pascual Tomé, Miguel Ángel Goenaga, Ma Jesús Bustinduy, Harkaitz Azkune Galparsoro. Maialen Ibarguren, Mirian Aguado, Maitane Umerez. Hospital General Universitario de Elche (Elche): Félix Gutiérrez, Mar Masiá, Cristina López, Sergio Padilla, Andrés Navarro, Fernando Montolio, Catalina Robledano, Joan Gregori Colomé, Araceli Adsuar, Rafael Pascual, Federico Carlos, Maravillas Martinez, Jara Llenas García, Marta Fernández, Elena García. Hospital Germans Trías i Pujol (Badalona): Roberto Muga, Jordi Tor, Arantza Sanvisens. Hospital General Universitario Gregorio Marañón (Madrid): Juan Berenguer, Juan Carlos López Bernaldo de Quirós, Pilar Miralles, Isabel Gutiérrez, Margarita Ramírez, Belén Padilla, Paloma Gijón, Ana Carrero, Teresa Aldamiz-Echevarría, Francisco Tejerina, Francisco Jose Parras, Pascual Balsalobre, Cristina Diez. Hospital Universitari de Tarragona Joan XXIII, IISPV, Universitat Rovira i Virgili (Tarragona): Francesc Vidal, Joaquín Peraire, Consuelo Viladés, Sergio Veloso, Montserrat Vargas, Miguel López-Dupla, Montserrat Olona, Alba Aguilar, Joan Josep Sirvent, Verónica Alba, Olga Calavia. Hospital Universitario La Fe (Valencia): Marta Montero, José Lacruz, Marino Blanes, Eva Calabuig, Sandra Cuellar, José López, Miguel Salavert. Hospital Universitario La Paz/IdiPaz (Madrid): Juan González, Ignacio Bernardino de la Serna, José Ramón Arribas, María Luisa Montes, Jose Ma Peña, Blanca Arribas, Juan Miguel Castro, Fco Javier Zamora, Ignacio Pérez, Miriam Estébanez, Silvia García, Marta Díaz, Natalia Stella Alcáriz, Jesús Mingorance, Dolores Montero, Alicia González, Maria Isabel de José. Hospital de la Princesa (Madrid): Ignacio de los Santos, Jesús Sanz, Ana Salas, Cristina Sarriá, Ana Gómez Berrocal, Lucio Garcia-Fraile. Hospital San Pedro-CIBIR (Logroño): José Antonio Oteo, José Ramón Blanco, Valvanera Ibarra, Luis Metola, Mercedes Sanz, Laura Pérez-Martínez. Hospital Universitario Miguel Servet (Zaragoza): Ascensión Pascual, Carlos Ramos, Piedad Arazo, Desiré Gil. Hospital Universitari Mutua de Terrassa (Terrassa): David Dalmau, Angels Jaén, Mireia Cairó, Daniel Irigoyen, Queralt Jordano, Mariona Xercavins, Javier Martinez-Lacasa, Pablo Velli, Roser Font, Montse Sanmartí, Laura Ibáñez. Complejo Hospitalario de Navarra (Pamplona): María Rivero, Marina Itziar Casado, Jorge Alberto Díaz, Javier Uriz, Jesús Repáraz, Carmen Irigoyen, María Jesús Arraiza. Hospital Parc Taulí (Sabadell): Ferrán Segura, María José Amengual, Gemma Navarro, Montserrat Sala, Manuel Cervantes, Valentín Pineda, Victor Segura, Marta Navarro, Esperanza Antón, Ma Merce Nogueras. Hospital Ramón y Cajal (Madrid): Santiago Moreno, José Luis Casado, Fernando Dronda, Ana Moreno, María Jesús Pérez Elías, Dolores López, Carolina Gutiérrez, Nadia Madrid, Angel Lamas, Paloma Martí, Alberto de Diaz, Sergio Serrrano, Lucas Donat. Hospital Reina Sofía (Murcia): Alfredo Cano, Enrique Bernal, Ángeles Muñoz. Hospital San Cecilio (Granada): Federico García, José Hernández, Alejandro Peña, Leopoldo Muñoz, Jorge Parra, Marta Alvarez, Natalia Chueca, Vicente Guillot, David Vinuesa, Jose Angel Fernández. Centro Sanitario Sandoval (Madrid): Jorge Del Romero, Carmen Rodríguez, Teresa Puerta, Juan Carlos Carrió, Mar Vera, Juan Ballesteros. Hospital de la Santa Creu i Sant Pau (Barcelona): Pere Domingo, Ma Antonia Sambeat, Karuna Lamarca, Gracia Mateo, Mar Gutiérrez, Irene Fernández. Hospital Universitario Santiago de Compostela (Santiago de Compostela): Antonio Antela, Elena Losada. Hospital Son Espases (Palma de Mallorca): Melchor Riera, Maria Peñaranda, Maria Leyes, Ma Angels Ribas, Antoni A Campins, Carmen Vidal, Leire Gil, Francisco Fanjul, Carmen Marinescu. Hospital Universitari Vall d’Hebron (Barcelona): Esteban Ribera. Hospital Virgen de la Victoria (Málaga): Jesús Santos, Manuel Márquez, Isabel Viciana, Rosario Palacios, Isabel Pérez, Carmen Maria González. Hospital Universitario Virgen del Rocío (Sevilla): Pompeyo Viciana, Manuel Leal, Luis Fernando López-Cortés, Nuria Espinosa. Hospital Universitario de Basurto (Bilbao): Josefa Muñoz, Miren Zuriñe Zubero, Josu Mirena Baraia-Etxaburu, Sofía Ibarra, Oscar Ferrero, Josefina López de Munain, Ma Mar Cámara. Iñigo López, Mireia de la Peña. Hospital Universitario Infanta Sofía (San Sebastián de los Reyes): Inés Suárez-García, Eduardo Malmierca. Hospital Universitario Costa del Sol (Marbella): Julián Olalla, Alfonso del Arco, Javier De la Torre, José Luis Prada, Zaira Caracuel. Hospital del Poniente (El Ejido): Ana Maria Lopez-Lirola, Ana Belén Lozano, Elisa Fernández, Inés Pérez, Juan Manuel Fernández. Hospital Universitario Santa Lucia (Cartagena): Onofre Juan Martínez, Francisco Jesús Vera, Lorena Martínez, Josefina García, Begoña Alcaraz, Amaya Jimeno. INIBIC-Complejo Hospitalario Universitario de A Coruña (A Coruña): Eva Poveda, Berta Pernas, Álvaro Mena, Marta Grandal, Ángeles Castro, José D. Pedreira. Hospital Clínico Universitario Virgen de la Arrixaca (Murcia): Carlos Galera, Helena Albendin, Asunción Iborra, Antonio Moreno, Maria Angeles Campillo, Asunción vidal. Hospital Marina Baixa (Villajoyosa): Concha Amador, Francisco Pasquau, Javier Ena, Concha Benito, Vicenta Fenoll. Complejo Hospitalario de Jaén (Jaén): Mohamed Omar Mohamed-Balghata, Maria Amparo Gómez. Hospital San Agustín de Aviles (Avilés): Miguel Alberto de Zarraga, Maria Eugenia Rivas. Fundación Jiménez Diaz (Madrid): Miguel Górgolas. Funding CoRIS is supported by the Instituto de Salud Carlos III through the Red Temática de Investigación Cooperativa en Sida (RD06/006, RD12/0017/0018 and RD16CIII/0002/0006) as part of the Plan Nacional R+D+I and is cofinanced by ISCIII-Subdirección General de Evaluación and Fondo Europeo de Desarrollo Regional (FEDER).  J. A. F.-C. and A. B. P. are supported by grant RD12/0017/006. F. 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Journal of Antimicrobial ChemotherapyOxford University Press

Published: Apr 1, 2018

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