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

Bracho, Maria Alma PhD*,†; Sentandreu, Vicente BSc; Alastrué, Ignacio MD§; Belda, Josefina MD, PhD; Juan, Amparo MD§; Fernández-García, Elisa MD, PhD; Santos, Concepción MD§; Zafra, Trinidad MD; Tasa, Teresa MD§; Colomina, Sonia MD; González-Candelas, Fernando PhD*,†

JAIDS Journal of Acquired Immune Deficiency Syndromes: March 1st, 2014 - Volume 65 - Issue 3 - p e130–e133
doi: 10.1097/01.qai.0000435602.73469.56
Letters to the Editor

*Unidad Mixta de Investigación en Genómica y Salud CSISP-UVEG/Institut Cavanilles-Universitat de València, Spain

CIBER de Epidemiologia y Salud Pública, València, Spain

Servei Central de Suport a la Investigació Experimental, Universitat de València, Spain

§Centre d’Informació i Prevención de la SIDA, Direcció General de Salut Pública, València, Spain

Centre d’Informació i Prevención de la SIDA, Direcció General de Salut Pública, Alacant, Spain

Supported by ISCIII-FIS Grant CP07/00078 (M.A.B.) from the Ministerio de Sanidad y Consumo, Grants AP-032/11 and GV/2011/008 from Generalitat Valenciana (M.A.B.), Grant BFU2008-03000 from Ministerio de Ciencia e Innovación, and Grant BFU2011-24112 from Ministerio de Economía y Competitividad (F.G-C., M.A.B.).

The authors have no conflicts of interest to disclose.

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 (]. 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.



To better understand the origin and relationships of the Spanish MSM isolates with worldwide variants, a BLAST search in the Los Alamos National Laboratory ( 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.

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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.

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1. Beyrer C, Baral SD, van Griensven F, et al.. Global epidemiology of HIV infection in men who have sex with men. Lancet. 2012;380:367–377.
2. Baggaley RF, White RG, Boily MC. HIV transmission risk through anal intercourse: systematic review, meta-analysis and implications for HIV prevention. Int J Epidemiol. 2010;39:1048–1063.
3. Li H, Bar KJ, Wang S, et al.. High multiplicity infection by HIV-1 in men who have sex with men. PLoS Pathog. 2010;6:e1000890.
4. Thomson MM, Fernandez-Garcia 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–e51.
5. Lewis F, Hughes GJ, Rambaut A, et al.. Episodic sexual transmission of HIV revealed by molecular phylodynamics. PLoS Med. 2008;5:e50.
6. Bezemer D, van Sighem A, Lukashov VV, et al.. Transmission networks of HIV-1 among men having sex with men in the Netherlands. AIDS. 2010;24:271–282.
7. Kouyos RD, von Wyl V, Yerly S, et al.. Molecular epidemiology reveals long-term changes in HIV type 1 subtype B transmission in Switzerland. J Infect Dis. 2010;201:1488–1497.
8. Zehender G, Ebranati E, Lai A, et al.. Population dynamics of HIV-1 subtype B in a cohort of men-having-sex-with-men in Rome, Italy. J Acquir Immune Defic Syndr. 2010;55:156–160.
9. Chalmet K, Staelens D, Blot S, et al.. Epidemiological study of phylogenetic transmission clusters in a local HIV-1 epidemic reveals distinct differences between subtype B and non-B infections. BMC Infect Dis. 2010;10:262.
10. Giuliani M, Montieri S, Palamara G, et al.. Non-B HIV type 1 subtypes among men who have sex with men in Rome, Italy. AIDS Res Hum Retroviruses. 2009;25:157–164.
11. Hemelaar J, Gouws E, Ghys PD, et al.. Global and regional distribution of HIV-1 genetic subtypes and recombinants in 2004. AIDS. 2006;20:W13–W23.
12. Hemelaar J, Gouws E, Ghys PD, et al.. Global trends in molecular epidemiology of HIV-1 during 2000-2007. AIDS. 2011;25:679–689.
13. Akrim M, Lemrabet S, Elharti E, et al.. HIV-1 subtype distribution in Morocco based on national sentinel surveillance data 2004-2005. AIDS Res Ther. 2012;9:5.
14. Chaix ML, Seng R, Frange P, et al.. Increasing HIV-1 non-B subtype primary infections in patients in France and effect of HIV subtypes on virological and immunological responses to cART. Clin Infect Dis. 2013;56:880–887.
15. Veras NM, Santoro MM, Gray RR, et al.. Molecular epidemiology of HIV type 1 CRF02_AG in Cameroon and African patients living in Italy. AIDS Res Hum Retroviruses. 2011;27:1173–1182.
16. Lospitao E, Alvarez A, Soriano V, et al.. HIV-1 subtypes in Spain: a retrospective analysis from 1995 to 2003. HIV Med. 2005;6:313–320.
17. de Felipe B, Perez-Romero P, Abad-Fernandez M, et al.. Prevalence and resistance mutations of non-B HIV-1 subtypes among immigrants in Southern Spain along the decade 2000-2010. Virol J. 2011;8:416.
18. Gonzalez-Alba JM, Holguin A, Garcia R, et al.. Molecular surveillance of HIV-1 in Madrid, Spain: a phylogeographic analysis. J Virol. 2011;85:10755–10763.
19. Yebra G, de Mulder M, Martin L, et al.. Most HIV type 1 non-B infections in the Spanish cohort of antiretroviral treatment-naive HIV-infected patients (CoRIS) are due to recombinant viruses. J Clin Microbiol. 2012;50:407–413.
20. van Harmelen J, Wood R, Lambrick M, et al.. An association between HIV-1 subtypes and mode of transmission in Cape Town, South Africa. AIDS. 1997;11:81–87.
21. Paraschiv S, Otelea D, Batan I, et al.. Molecular typing of the recently expanding subtype B HIV-1 epidemic in Romania: evidence for local spread among MSMs in Bucharest area. Infect Genet Evol. 2012;12:1052–1057.
22. de Oliveira T, Pillay D, Gifford RJ. The HIV-1 subtype C epidemic in South America is linked to the United Kingdom. PLoS One. 2010;5:e9311.
23. Balode D, Skar H, Mild M, et al.. Phylogenetic analysis of the Latvian HIV-1 epidemic. AIDS Res Hum Retroviruses. 2012;28:928–932.
24. Holguin A, Alvarez A, Soriano V. Heterogeneous nature of HIV-1 recombinants spreading in Spain. J Med Virol. 2005;75:374–380.
25. Bracho MA, Saludes V, Martro E, et al.. Complete genome of a European hepatitis C subtype 1g isolate: phylogenetic and genetic analyses. Virol J. 2008;5:72.
26. Kosakovsky Pond SL, Posada D, Stawiski E, et al.. An evolutionary model-based algorithm for accurate phylogenetic breakpoint mapping and subtype prediction in HIV-1. PLoS Comput Biol. 2009;5:e1000581.
27. Alcantara LC, Cassol S, Libin P, et al.. A standardized framework for accurate, high-throughput genotyping of recombinant and non-recombinant viral sequences. Nucleic Acids Res. 2009;37:W634–W642.
28. Garcia F, Perez-Cachafeiro S, Guillot V, et al.. Transmission of HIV drug resistance and non-B subtype distribution in the Spanish cohort of antiretroviral treatment naive HIV-infected individuals (CoRIS). Antiviral Res. 2011;91:150–153.
29. Trevino A, Soriano V, Rodriguez C, et al.. Changing rate of non-B subtypes and coinfection with hepatitis B/C viruses in newly diagnosed HIV type 1 individuals in Spain. AIDS Res Hum Retroviruses. 2011;27:633–638.
30. Holguin 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;6:327–334.
31. Abecasis AB, Wensing AM, Paraskevis D, et al.. HIV-1 subtype distribution and its demographic determinants in newly diagnosed patients in Europe suggest highly compartmentalized epidemics. Retrovirology. 2013;10:7.
32. ECDC/WHO. HIV/AIDS Surveillance in Europe 2009. Stockholm, Sweden: European Centre for Disease Prevention and Control; 2010.
33. Stamatakis A. RAxML-VI-HPC: maximum likelihood-based phylogenetic analyses with thousands of taxa and mixed models. Bioinformatics. 2006;22:2688–2690.
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