Emerging Trends in CRF02_AG Variants Transmission Among Men Who Have Sex With Men in Spain

To the Editors: Increasing incidence of HIV-1 infections among men who have sex with men (MSM) is being reported steadily in countries of different incomes in recent years.1 The HIV epidemics among MSM is characterized by a high transmission efficiency2 and high probability of infection with multiple variants.3 Phylodynamics studies have proved a relatively rapid spread and important clustering of infections within MSM transmission networks.4,5 In Western Europe, most reported HIV transmission clusters among MSM are subtype B5−9 although a few local clusters of non-B subtype have been reported recently.4,10 CRF02_AG is the fourth most common HIV strain, accounting for 5% and 8% of HIV global infections worldwide in the 2000–2004 and 2004–2007 periods, respectively.11,12 CRF02_AG is distributed mainly in West Africa and, to a less degree, in the Middle East and North Africa.12 Because of migrations from these endemic regions, increasing CRF02_AG prevalence has been reported recently in countries where subtype B epidemics dominates such as Morocco,13 France,14 Italy,15 and Spain.16–18 In fact, CRF02_AG is the second most prevalent strain currently circulating in Spain (2004–2008) accounting for about 5% of newly diagnosed individuals.19 Apart from being born in a sub-Saharan country, another feature largely associated with CRF02_AG infection in the immigrant population is heterosexual unprotected sex.18 Segregation of HIV-1 variants or subtypes by geographic area12 and transmission route20 has often been detected since the beginning of the epidemics, but after 30 years of transmissions, this initial scenario has experienced many changes as natives, migrants, and travelers exhibiting more than 1 risk behavior act as infection bridges among subtype-defined groups.21–23 This study describes the early and possibly progressive expansion of CRF02_AG infections among MSM in Eastern Spain supported by the emergence of multiple and independent CRF02_AG local transmission clusters among native Spanish, Latin American, and sub-Saharan MSM. This study was approved by the Ethics Committee of the institution [Centro Superior de Investigación en Salud Pública (CSISP)]. HIV-1 protease-reverse transcriptase sequences were obtained, between 2004 and 2012, from 762 newly HIV-1 diagnosed people in 2 first-line anonymous HIV counseling and testing centers in Alacant and València, Spain. Subject information included demographic data, route of HIV transmission, and clinical data. Plasma samples underwent viral RNA extraction followed by RT-PCR and direct sequencing using amplification and sequencing procedures, described elsewhere with minor modifications.24,25 Sequences include the complete protease region and the first 335 amino acids of the reverse transcriptase (1302 nucleotides). Baseline PR-RT sequences underwent subtyping, detection of resistance-associated mutations, and identification of transmission clusters. HIV-1 subtypes were obtained with subtype prediction programs [SCUEAL,26 REGA,27 and COMET (http://comet.retrovirology.lu/)]. From these 762 sequences derived from newly diagnosed individuals, 621 (81.5%) were subtype B, the predominant variant in Western Europe including Spain. The remaining 141 sequences (18.5%) were non-B subtypes, a proportion similar to that found in previous studies based in Spain (15.2%,28 19.4%,29 and 24%30). Among the non-B subtype sequences, the most frequent was CRF02_AG (36 sequences, 4.7% of total) followed by subtype G or BG recombinant forms (21, 2.8%), sub-subtype A1 (13, 1.7%), sub-subtype F1 (8, 1.1%), CRF12_BF (8, 1.1%), CRF06_cpx (2, 0.3%), CRF11_cpx (1, 0.1%), CRF19_cpx (1, 0.1%), CRF24_BG (1, 0.1%), and 36 sequences (4.7%) corresponding to different complex recombinants or unique recombinant forms. Five sequences of these 36 unique recombinant forms contained fragments of variable length of CRF02_AG, but because of their complex recombinant composition, they were not included in further analysis. Features associated to 36 CRF02_AG isolates detected during 2004–2012 were distributed unevenly. In the 2004–2008 period, 13 of 17 CRF02_AG isolates (77%) were associated to heterosexual sub-Saharans, and only 4 (23%) were associated to non-Africans: 1 Spanish intravenous drug user, 2 Latin Americans (1 MSM, 1 heterosexual), and 1 heterosexual Caribbean. In the 2009–2012 period, 8 of 19 CRF02_AG isolates (42%) were reported in heterosexual sub-Saharans, 9 (47%) were MSM (7 Spaniards, 1 Latin American, and 1 sub-Saharan), and 2 (11%) were non-African–non-MSM (1 Spanish heterosexual, 1 Portuguese intravenous drug user). From these 9 newly diagnosed infections in MSM during the 2009–2012 period, 2 were recent transmissions (<6 months since seroconversion), and 6 other infections had tested HIV negative in the previous 2 years. After examining the maximum likelihood phylogenetic tree built with the 36 CRF02_AG isolates (Fig. 1), some significant clusters were detected. Most relevantly, sequences from 6 native Spanish MSM were included in 2 highly supported independent MSM clusters and a highly supported MSM transmission pair. The mean intracluster patristic genetic distances were 0.002, 0.009, and 0.016 substitutions per nucleotide for the transmission pair and clusters A and B, respectively (Fig. 1), whereas the average patristic genetic distance of nonclustered CRF02_AG sequences obtained in this study was 0.073 substitutions per nucleotide. Interestingly, in each of these 3 CRF02_AG MSM groups, 1 of the isolates was associated to a non-Spanish MSM (1 Latin American, 1 sub-Saharan, and 1 Latin American, respectively). The 9 CRF02_AG MSM clustered isolates, along with 1 Spanish MSM ungrouped isolate, were all from individuals diagnosed between 2007 and 2012 and comprised all the CRF02_AG MSM isolates recorded in our study since 2004. A change in the transmission pathway of CRF02_AG variants seems to have occurred because no CRF02_AG isolate associated to MSM was detected during the period 2004–2006. The 8 CRF02_AG newly diagnosed individuals in this period were all heterosexual (3 men and 5 women). Apart from the MSM clusters, no additional clusters among the rest of CRF02_AG isolates obtained in this study were found, except the one formed by 2 heterosexual women from Ecuador.FIGURE 1: Maximum likelihood phylogenetic reconstruction for pol sequences of 36 CRF02_AG HIV-1 isolates from new diagnoses in València and Alacant (Spain) during 2004–2012. Phylogenetic tree was constructed by using RAxML33 with the general time-reversible model of nucleotide substitution, a gamma-distribution approximation for among-site rate heterogeneity, and bootstrap support for branches by performing 1000 replicates. Name of isolates obtained in this study contain accession number, either country or region of birth, risk of infection, and year of HIV diagnosis. Additional CRF02_AG sequences obtained from the HIV database through BLAST searches (in italics) contain accession number followed by country of sampling and year of HIV diagnosis. Name of reference sequences contain subtype or sub-subtype followed by their accession number. Country of birth is indicated with the 2-letter country code: EC, Ecuador; ES, Spain; IT, Italy; PT, Portugal. Region of origin is indicated as follows: CAR, Caribbean; LAM, Latin America; SSA, sub-Saharan Africa. Risk of infection is abbreviated as follows: HET, heterosexual; IDU, intravenous drug user; MSM, men that have sex with men. MSM isolates from this study are highlighted in bold. Scale bar indicates number of substitutions per nucleotide. Only bootstrap values higher than 70% are shown. Sequences obtained in this study are available under GenBank accession numbers HF567872, HF567874-HF567884, HF567886-HF567889, HF567891-HF567894, HF567897-HF567912.To better understand the origin and relationships of the Spanish MSM isolates with worldwide variants, a BLAST search in the Los Alamos National Laboratory (http://www.hiv.lanl.gov) was performed. Country of origin of the sequences that showed the highest identity scores with our CRF02_AG Spanish MSM were recorded, and those sequences that formed significant phylogenetic clusters with query sequences (data not shown) were subsequently included in the maximum likelihood phylogenetic analysis (Fig. 1). The nonclustered Spanish MSM CRF02_AG isolate formed a high-supported transmission pair with a retrieved isolate sampled in 2007 in Spain. These 2 sequences presented the highest identity scores with deposited sub-Saharan sequences (data not shown). A similar result was obtained when using the sequences from the CRF02_AG MSM transmission pair as query sequences. Conversely, the BLAST searches with clusters A and B retrieved non-African sequences with the highest identity scores. Specifically, cluster A was included in a highly supported cluster with 4 Italian isolates diagnosed in 2005 and 2007, with 2 of them from MSM.10 Similarly, sequences from cluster B were included in a highly supported cluster that contained 2 sequences sampled in Ecuador before 2001, 1 sequence from an Ecuadorian immigrant sampled in Spain in 2005 and 6 sequences corresponding to newly diagnosed individuals during 2005–2007 in Spain, with no available information about transmission route or country of origin. These enlarged clusters of closely related isolates sampled in Italy (cluster A) and Ecuador and Spain (cluster B) would place the local transmission of CRF02_AG variants among MSM in a context of national and international transmission networks. All these results taken together indicate that the majority of circulating CRF02_AG isolates still represent separate introductions through sub-Saharan and, to a less degree, Latin American migration, as previously reported in Spain.19 In contrast to recent relatively large-scale studies examining HIV-1 subtypes distribution among native and immigrants in Madrid,18 Spain19, and Europe,31 this region-wide study shows that the current features associated to CRF02_AG in newly diagnosed individuals seem to start changing by breaking the strong social clustering of CRF02_AG variants commonly associated to sub-Saharan heterosexuals.18 In this study, this incipient shift has been detected in 3 independent local CRF02_AG MSM transmission groups probably reflecting multiple intersection events between the second most prevalent HIV-1 clade in Spain19 and the most prevalent transmission risk (ie, unprotected sex among MSM) associated to newly diagnosed individuals in Western countries1 including Spain.32 In conclusion, this study supports the first detailed report in Spain of an infection bridge among subtype-defined groups in which CRF02_AG variants, commonly described as infecting sub-Saharan heterosexual immigrants, have been found in incipient, multicultural MSM transmission networks. Moreover, a detailed characterization of local MSM transmission clusters provides a valuable information to be implemented in the design of prevention measures against HIV infection among MSM, one of the most concerning priorities in current public health policies in Western countries. ACKNOWLEDGMENTS The authors thank the personnel at the Centre d'Informació i Prevenció de la SIDA (CIPS) d'Alacant i València, Universitat de València and CSISP for technical assistance, and Dr Manoli Torres for her helpful comments on this study.