The HIV-1 epidemic is still one of the major public health problems in Spain. Around 3,500–4,000 new diagnoses (NDs) of HIV-1 infection are reported every year, with an estimated incidence of NDs of 7.5 per 100,000 population in 2019 (HIV STI and Hepatitis Surveillance Unit, 2020). A decreasing trend in the incidence of NDs has been observed since 2010, although it is still higher than the average rate found in the EU/EEA (European Centre for Disease Prevention and Control and WHO Regional Office for Europe, 2020). In 2019, most (56%) of the NDs were diagnosed among men who had sex with men (MSM) and 36% of all reported NDs were in people born outside of Spain (HIV STI and Hepatitis Surveillance Unit, 2020).

Molecular epidemiology is an important tool for describing the HIV-1 epidemic (Brenner et al., 2013; Paraskevis et al., 2016; Wertheim et al., 2017; Board et al., 2020). Individuals whose transmission is related group phylogenetically in clades named transmission clusters (TCs). Due to the high genetic variability of HIV-1, phylogenetic analysis allows to reconstruct transmission events through the identified TCs and infer the history of the HIV-1 epidemic (Hué et al., 2004, 2005).

National databases of protease and reverse transcriptase (Pr-RT) sequences, primarily obtained for antiretroviral drug resistance testing, contain valuable data about HIV-1 expansion and have been used in molecular epidemiology studies (Ragonnet-Cronin et al., 2016a; Parczewski et al., 2017; Oster et al., 2018; Petersen et al., 2018; Verhofstede et al., 2018; Pineda-Peña et al., 2019; Fabeni et al., 2021). The phylogenetic analyses performed in these studies combined with clinical and epidemiological data of the patients provide relevant public health information for the implementation of control measures and for monitoring the HIV-1 epidemic (Brenner et al., 2013; Vasylyeva et al., 2016, 2020; Paraskevis et al., 2019; Board et al., 2020; Campbell et al., 2020).

Spanish clinical guidelines recommend to perform a genotypic drug resistance test before starting antiretroviral therapy (ART) in all HIV-1-diagnosed patients and in ART-failing cases [AIDS study group (GESIDA) of the Spanish Society of Infectious Diseases and Clinical Microbiology and the National AIDS Plan, 2020]. Pr-RT sequences obtained for these tests have been analyzed in different molecular epidemiology studies, describing the genetic features of the HIV-1 epidemic in different regions of Spain (Thomson et al., 2001; Holguín et al., 2007; Cuevas et al., 2009a,b; González-Alba et al., 2011; Yebra et al., 2013; Pérez-Parra et al., 2015, 2016; Patiño-Galindo et al., 2016, 2017b; González-Domenech et al., 2020). The phylogenetic studies have also allowed the identification of large TCs among MSM which are actively growing in Spain (Delgado et al., 2015, 2019; Patiño-Galindo et al., 2017a; González-Domenech et al., 2018), as well as transmitted drug resistance mutations which are spreading in TCs (Cuevas et al., 2009b; Vega et al., 2015; Viciana et al., 2016; González-Domenech et al., 2018, 2020), highlighting the value of molecular epidemiology as a tool for HIV surveillance.

The Basque Country and Galicia are two regions located in northern Spain, comparable in terms of population and HIV-1 diagnosis rates. During 2013–2018, the mean resident population and rates of new HIV diagnoses were estimated in 2,2 million and 6,9 per 100,000 inhabitants for the Basque Country and 2,7 million and 5,6 per 100,000 inhabitants for Galicia (HIV STI and Hepatitis Surveillance Unit, 2020; Instituto Nacional de Estadistica, 2021). In this study, we analyzed the frequency of grouping in TCs among patients diagnosed in 2013–2018 in these northern Spanish regions, identifying the different features associated with TCs in the HIV-1 epidemics in these regions.

The phylogenetic analysis of sequences from almost 12,000 HIV-1-infected patients from our database has allowed us to investigate the transmission events occurring during the 2013–2018 period in two regions of northern Spain, Galicia and Basque Country, identifying TCs and their associated factors. Our ND cohort had a good representativeness, corresponding to 64% of the NDs reported to the SINIVIH from these regions during that period, especially in the Basque Country where it reached 86%.

Our analysis has assigned 51% of the NDs from Galicia and Basque Country to 162 different TCs, indicating that TCs are playing an important role in the spread of HIV-1 infections in both regions. There is no consensus on the criteria for defining a TC, as these should depend on the aim of the study (Hassan et al., 2017). We considered only TCs including at least 50% Spaniards and at least four documented infections, because they may be more epidemiologically relevant to describe HIV-1 strains spreading in an area. Smaller clusters comprising two (“pairs”) or three (“triplets”) individuals are much more numerous than larger ones (Hughes et al., 2009; Brown et al., 2011; Yebra et al., 2013) and in some studies are distinguished from larger clusters (Hoenigl et al., 2016; Fabeni et al., 2020). They could represent sexual couples with or without a second relationship by one of the members of the couple, and their epidemiologic relevance is uncertain. The inclusion of these requirements in the TC definition has allowed us to better identify the structure and factors associated to HIV-1 strains whose transmission is established in Spain.

Transmission clusters were found associated with male gender and MSM. In fact, almost all the largest TCs (≥30 patients) identified in our study were associated with MSM. HIV-1 molecular epidemiological studies in different countries, independently of the methodology used, have found a similar association (Parczewski et al., 2017; Wertheim et al., 2017; Ramirez et al., 2021). Also, studies in other Spanish regions have found this association of TCs with MSM (Pérez-Parra et al., 2015; González-Domenech et al., 2020). These findings indicate that MSM TCs are playing a main role in spreading HIV-1 infections and are consistent with the fact that MSM is the main frequent mode of HIV-1 transmission in Spain since the early 2000s (Nuñez et al., 2018).

In Spain, 36% of HIV-1 infections diagnosed in 2019 were in migrants (HIV STI and Hepatitis Surveillance Unit, 2020). Migrants play an important role in HIV-1 epidemic spread, contributing to bridging between networks from different geographic areas (Grossman et al., 2015; Su et al., 2018; Grabowski et al., 2020). In this study, we have examined the frequency of membership in Spanish TCs of migrants from different geographic areas. We have found that Latin Americans and Africans showed lower odds of belonging to TCs than Spaniards. However, there were important differences between these migrant populations, as 35% of Latin Americans were included in TCs, compared to only 1.2% of Sub-Saharan Africans, suggesting that HIV-1 transmission between Latin Americans and Spaniards is relatively common, presumably derived from cultural and linguistic affinities (Osorno-González de León et al., 2021). Similarly to our results, a low percentage of Sub-Saharan Africans in TCs have been also reported in Italy (Fabeni et al., 2020), Belgium (Verhofstede et al., 2018), and Madrid, Spain (Yebra et al., 2013), while in Israel, cross-ethnic HIV-1 spread from Israeli-born individuals to Ethiopian-born immigrants was rare (Grossman et al., 2015). In our study, TCs are mainly MSM-driven. In contrast, we have found high frequencies of females (52%) and heterosexuals (88%) in Sub-Saharan Africans, which, in addition to the likely HIV-1 acquisition in their countries of origin, can explain the low proportion of individuals in this migrant population belonging to TCs. Indeed, we have observed that Sub-Saharan Africans are infected frequently with genetic forms circulating in Africa.

Determining the origin of HIV-1 infections in the migrant population would allow proper allocation of resources for preventive measures (Fakoya et al., 2015; Álvarez del Arco et al., 2017). Previous studies based on models using clinical and behavioral data estimated high levels of post-migration HIV acquisition in Europe (Desgrées-du-Loâ et al., 2015; Álvarez del Arco et al., 2017), up to 71% among migrants in Spain (Álvarez del Arco et al., 2017). This figure could be an overestimate due to biases in data collection and failure to consider infections acquired by migrants when visiting their countries, as migrants’ mobility is associated with increased risk for HIV acquisition (Fenton et al., 2001; Dias et al., 2020). Phylogenetic analyses can provide additional information on the geographical area of HIV-1 acquisition. In our study, the lower percentage of foreigners found in TCs could be due to infections with virus strains which are not circulating in Spain, suggestive of imported infections, which would be especially frequent in Sub-Saharan Africans. Further studies using a combined phylogenetic and epidemiological approach can address the accuracy of the origin of the HIV-1 infections in the migrant populations in Spain.

With regard to the role of immigration on the origin of HIV-1 TCs in Spain, it is also interesting to note that 21% TCs identified in this study are of non-subtype B genetic forms, with their ancestry showing some correlation with the predominant geographic origins of the immigrant population in Spain. Thus, the fact that 16 TCs derive from genetic forms circulating in Latin America and Africa may correlate to the high number of immigrants from these geographic areas living in Spain, representing around 36 and 18%, respectively, of the approximately 7.3 million immigrants (Instituto Nacional de Estadistica, 2021). Similarly, the expansion of a large F1 subsubtype cluster of Romanian origin (F1_3) in Spain (Delgado et al., 2019) may relate to the fact that among European immigrants in Spain, Romanians are the most numerous, representing around 8% of all immigrants (Instituto Nacional de Estadistica, 2021).

Subtype B was the most represented genetic form associated to TCs. Only subtype F infections (76% of NDs in TCs) showed a higher frequency than subtype B infections (56% of NDs in TCs) of belonging to TCs. This was mainly due to the presence of two large TCs of F1 subsubtype, designated F1_1 (205 individuals) and F1_3 (36 individuals), which have spread among MSM mainly in Galicia and Basque Country, respectively (Delgado et al., 2015, 2019). Interestingly, two other large non-subtype B TCs associated with MSM, of CRF02_AG and A1 subsubtype, are also present in these regions (Delgado et al., 2019). In Western Europe, the HIV-1 epidemic among MSM is dominated by subtype B. However, the epidemic among MSM in Spain is becoming increasingly diverse through the expansion of multiple non-subtype B TCs, comprising or related to viruses circulating in other countries (González-Domenech et al., 2018; Delgado et al., 2019). This phenomenon has been also documented in other European countries (Dauwe et al., 2015; Ragonnet-Cronin et al., 2016b; Verhofstede et al., 2018; Fabeni et al., 2019; Ramirez et al., 2021), where viruses introduced from abroad have expanded successfully among the MSM population.

In general, there was not a clear temporal trend in the proportion of patients belonging to TC, although in Galicia our data show an increase in the proportion of patients in TCs in the last years. However, trends are difficult to evaluate due to the presence of fast-growing TCs in some periods.

A different distribution of TCs is responsible for the spread of HIV-1 in Galicia and the Basque Country. Large MSM TCs have spread locally in both regions and the majority of the identified TCs were associated with this population group. However, we found a higher frequency of TCs associated with PWID or heterosexual transmission in Galicia. Galician patients have 1.5 higher odds of belonging to TCs than individuals from the Basque Country. This can be related to the low percentage of migrants in Galicia, who have a reduced association to TCs, and the high number of patients belonging to the large F1_1 TC, currently comprising 205 individuals, which has successfully spread in Galicia and other Spanish regions (Delgado et al., 2015), even in other European countries (Delgado et al., 2015; Vinken et al., 2019). We have found an association of young patients (age range 20–29 years) to TCs in Galicia, similarly to the findings in other studies (Brenner et al., 2017; Dennis et al., 2018; Verhofstede et al., 2018; Fabeni et al., 2019; Paraskevis et al., 2019; González-Domenech et al., 2020), but not in the Basque Country. Public health interventions for reducing high risk behavior and control measures in this age group in Galicia should be implemented to decrease HIV-1 transmissions in the region. Moreover, our results highlight that even regions with comparable demographic features can show a different HIV-1 epidemic pattern, likely due to specific social and cultural differences at the regional level.

In this study, we have shown that MSM TCs are a keystone of the HIV-1 epidemic at regional and probably also at the country level, according to the propagation of the large MSM TCs identified in this study in different regions of Spain. Consequently, reinforcement of public health measures for preventing HIV-1 infections in MSM, such as behavioral interventions to reduce risky practices, pre-exposure prophylaxis (Grant et al., 2010; McCormack et al., 2016; Riddell et al., 2018), and early diagnosis and treatment of HIV-1 infections (European Centre for Disease Prevention and Control, 2015; United Nations Population Fund Global Forum on MSM & HIV United Nations Development Programme World Health Organization United States Agency for International Development World Bank, 2015) are recommended.

Implementation of preventive public health measures targeting MSM could reduce indirectly the spread of HIV-1 in Spain, which is mainly MSM-driven, in other population groups. National surveillance data in the United States suggest that infections among heterosexual women predominantly originate from MSM (Oster et al., 2015). Moreover, the presence of female and self-declared heterosexual male individuals in MSM-associated TCs is frequent (Hué et al., 2014; Esbjörnsson et al., 2016; Ragonnet-Cronin et al., 2018; Verhofstede et al., 2018), as we have also observed in the large TCs in our study (Table 3). Mixed demographics in TCs allows the spread of HIV-1 infections between different population groups, as we have seen in the expansion of MSM-associated TCs to heterosexuals in Spain (Delgado et al., 2019), indicating the importance of monitoring the evolution of TCs for implementing proper control measures in each population group. Studies to assess the contribution of MSMs to HIV-1 transmission to other population groups are needed in Spain.

The high representativeness of our cohort, especially in the Basque Country, suggests that the largest and most spread TCs in these regions have been identified, which supports the conclusions of the study. However, our data may have some limitations as some of the NDs from Basque Country and Galicia during the studied period were not analyzed and not all the TCs propagating in Spain were detected in our phylogenetic analysis.

Our study has provided valuable data on the HIV-1 epidemic in Spain, identifying specific features at geographical and population levels for tailoring more specific public health interventions. The implementation of a molecular HIV-1 surveillance system to monitor the evolution of the epidemic in Spain could allow to promptly detect rapidly expanding TCs needing urgent investigation and the implementation of additional public health measures to prevent their spread (Centers for Disease Control and Prevention, 2018), as well as to readjust the control measures in place according to the evolution of the TCs.

Basque Country: Hospital Universitario de Araba, Vitoria: Andrés Canut-Blasco, José Joaquín Portu, Carmen Gómez-González; Hospital Universitario de Basurto, Bilbao: Josefa Muñoz, Mª Carmen Nieto, María Zuriñe Zubero, Silvia Hernáez-Crespo, Estibaliz Ugalde; Hospital Universitario de Cruces, Barakaldo: Luis Elorduy, Elena Bereciartua, Leyre López Soria; Hospital de Galdakao: Mª José López de Goicoechea, José Mayo; Hospital Universitario Donostia: Gustavo Cilla, Julio Arrizabalaga, José Antonio Iribarren, Mª Jesús Bustinduy, Mª Julia Echevarría. Mª Yolanda Salicio, David Grandioso. Galicia: Área sanitaria de Ferrol: Ana Mariño, Patricia Ordóñez, Hortensia Álvarez, Nieves Valcarce; Complejo Hospitalario Universitario de A Coruña: Ángeles Cañizares, Mª Ángeles Castro. Hospital Universitario Lucus Augusti, Lugo: Ramón Rabuñal-Rey, María José García-País, Mª José Gude-González, Pilar Alonso-García, Antonio Moreno-Flores; Complejo Hospitalario Universitario de Ourense: Juan García Costa, Ricardo Fernández-Rodríguez, Raúl Rodríguez-Pérez, Jorge Guitián, María Dolores Díaz-López, María Genoveva Naval-Calviño; Complejo Hospitalario Universitario de Vigo: Celia Miralles, Antonio Ocampo, Sonia Pérez-Castro, Jorge Julio Cabrera; Complejo Hospitalario Universitario de Pontevedra: Julio Díz-Arén, Matilde Trigo, Mª Ángeles Pallarés. Navarra: Complejo Hospitalario de Navarra, Pamplona: Carmen Ezpeleta Baquedano, Carmen Martín Salas, Irati Arregui García, María Gracia Ruiz de Alda. Madrid: Centro Sanitario Sandoval, Madrid: Jorge del Romero, Carmen Rodríguez, Mar Vera, Óskar Ayerdi, Eva Orviz; Hospital Universitario de Fuenlabrada: María Isabel García-Arata, Santiago Prieto-Menchero; Hospital Clínico Universitario San Carlos, Madrid: Esther Culebras, Icíar Rodríguez-Avial; Hospital Universitario Fundación Jiménez Díaz, Madrid: Raquel Téllez-Pérez, Olalla Calabia-González, Alfonso Cabello-Úbeda, Miguel Górgolas Hernández-Mora; Hospital Universitario Severo Ochoa, Leganés: Sara María Quevedo, Lucía Puente, Manuel del Álamo; Hospital Fundación Alcorcón, Madrid: Carolina Campelo Gutiérrez, María José Goyanes Galán; Castilla y León: Hospital Clínico Universitario de Valladolid: Carmen Hinojosa, Carlos Dueñas, Begoña Monteagudo, Edita Sánchez; Hospital Río Hortega, Valladolid: Jessica Abadía, Belén Lorenzo Vidal; Hospital Virgen de la Concha, Zamora: Teresa Martín-Domínguez, Rosa Martínez-González. La Rioja: Hospital San Pedro, Logroño: José Ramón Blanco, Miriam Blasco. Aragón: Hospital Universitario Miguel Servet, Zaragoza: Ana María Martínez-Sapiña, Piedad Arazo. Castilla-La Mancha: Hospital Universitario de Guadalajara: Alejandro González Praetorius, Complejo Hospitalario de Toledo: César Gómez-Hernando, José Largo-Pau; Comunitat Valenciana: Hospital Universitari Sant Joan d’Alacant: Fernando Buñuel, Ana Infante.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found at: https://www.ncbi.nlm.nih.gov/genbank/, KT276255, KT276260, KT276263-KT276264, KT276266-KT276267, KT276270-KT276271, KU685562-KU685565, KU685567, KU685569-KU685575, KU685577, KU685581-KU685582, KU685586, KU685588-KU685590, KU685592, KX534325, KX534329, KX534331, KY465968, KY496624, KY514084, KY989950, MF999250-MF999256, MF999258-MF999261, MK177721-MK177733, MK177735-MK177752, MK177754-MK177757, MK177761-MK177772, MK177783-MK17785, MK177787-MK177790, MK177792, MK177795-MK177796, MK177798-MK177799, MK177801-MK177803, MK177805-MK177807, MK298150, MT436242, MT436244, MT436246-MT436247, MT436249-MT436250, MW344920-MW344921, MW584217-MW584224, OK011532, OK011542, OK011545-OK011546, OK011549, OK148912, OK148914, OK148917-OK148919, OK148921, OK148941-OK148942, OK148953-OK148957, OK148961, OK148965, OK148968-OK148972, OK148974, OL982314-OL982315, and OM914651-OM914711.

The studies involving human participants were reviewed and approved by Bioethics and Animal Well-being Committee of Instituto de Salud Carlos III. Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.

HG, ED, and MT conceived the study and supervised the experimental work. ED, HG, MT, and JC-G processed sequences and performed phylogenetic analyses. HG and ED performed data curation. HG, AD, and LG performed statistical analyses. SB, MS, VM, and EG-B performed experimental work. The members of the Spanish Group for the Study of New HIV Diagnoses recruited patients and obtained epidemiological and clinical data. HG wrote the manuscript draft, with contributions to the text by AD, MT, and ED. All authors contributed to the article and approved the submitted version.

This work was funded through Acción Estratégica en Salud Intramural (AESI), Instituto de Salud Carlos III, Project “Estudios sobre vigilancia epidemiológica molecular del VIH-1 en España,” PI16CIII/00033 and Project “Epidemiología molecular del VIH-1 en España y su utilidad para investigaciones biológicas y en vacunas“PI19CIII/0042; Red de Investigación en SIDA (RIS), Instituto de Salud Carlos III, Subdirección General de Evaluación y Fondo Europeo de Desarrollo Regional (FEDER), Plan Nacional ICDCI, project RD16ISCIII/0002/0004; and scientific agreements with Consellería de Sanidade, Government of Galicia (MVI 1004/16), and Osakidetza-Servicio Vasco de Salud, Government of Basque Country (MVI 1001/16).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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