The prevalence of HIV-1 group M non-B subtypes has been increasing in Western Europe in recent years largely as a result of population movements . Specifically, in native Spaniards, the rate of non-B variants increased from 1.5% in 2000–2002 to 11.4% in 2007–2010, with the circulating recombinant form CRF02_AG (37%) being the most common non-B subtype in Spain . Historically, the HIV epidemic in Galicia, a coastal region situated in northwest Spain, has been characterized by a high diversity of HIV genetic forms presumably due to international population movements through this area .
Natural polymorphisms at positions associated with resistance are frequently found among reverse transcriptase and protease sequences from HIV non-B variants . Although the impact of these polymorphisms on treatment responses appears limited, recent studies have demonstrated their relevance in some HIV variants with specific antiretroviral agents [5,6]. Since the prevalence of circulating HIV variants is a dynamic phenomenon and new antiretroviral agents are continually being introduced into the therapeutic arsenal against HIV infection, this issue requires continuous monitoring.
Herein, we describe the characteristics of all newly diagnosed HIV-1 persons in the past 5 years at our institution in northwest Spain serving 501 526 citizens. Patient demographics (age, sex, and risk behavior), and laboratory (HIV-RNA, HIV subtypes, HIV drug resistance and CD4+ cell counts) and clinical parameters at the time of diagnosis were recorded. In addition, the response to antiretroviral therapy (ART) was retrospectively analyzed.
A total of 296 newly diagnosed HIV-1 patients were identified from 2009 to 2013 at our institution. HIV subtype could be determined in 230 patients. Non-B variants were found in 37.8% of patients with the following distribution: F (29.6%), C (2.6%), A (2.2%), CRF02_AG (1.7%), G (0.9%), K (0.4%), and CRF01_AE (0.4%). Subsequent analyses were performed comparing the two most prevalent subtypes seen in our population: subtype B (n = 143) and subtype F (n = 68).
Table 1 displays the main characteristics of this population at the time of diagnosis. Persons infected with subtype F were overwhelmingly male (97.1%) with a predominance of MSM compared to subtype B patients (81.8% vs. 45.8%, respectively; P < 0.001). Those infected with subtype F were also significantly younger than those infected with subtype B (36 vs. 39 years, respectively; P = 0.037). No differences were found with regard to nationality, with the majority in both groups being Spanish. Likewise, the percentages of subtype F versus B patients with an AIDS-defining illness (24.2% vs. 35%, respectively) or CD4+ cell counts below 350 cells/μl (42.6% vs. 50.3%, respectively) were similar. The mean CD4+ cell count was also comparable between the groups (384.04 vs. 378.03, respectively); however, HIV-RNA levels at the time of diagnosis were higher among subtype F patients compared to subtype B patients (5.3 vs. 4.9 log copies/ml, respectively; P = 0.002). The rate of transmitted drug resistance during the study period was 4.2%, and resistance was only found in subtype B sequences.
After diagnosis, 80.8% of subtype F and 72.7% of subtype B patients initiated ART. The rates of virological response after ART initiation were retrospectively evaluated comparing both groups of patients (Table 1). The mean time to initiation of ART was significantly shorter among patients infected with subtype F variants (10.53 vs. 17.60 months, respectively; P < 0.001). No differences were observed with regard to the composition of the initial ART regimen between subtype F and B patients: two nucleoside reverse transcriptase inhibitors (NRTI) + one non-nucleoside reverse transcriptase inhibitor (NNRTI) (55.4% vs.47.5%), two NRTI + one protease inhibitor (35.7% vs. 46.5%) and two NRTI + one integrase inhibitor (INI) (8.9% vs. 6.1%).
Virologic response, defined as achievement of HIV-RNA below 50 copies/ml, was assessed at 24, 48, and 96 weeks after ART initiation. Interestingly, the rates of virologic response were significantly lower among patients infected with subtype F variants compared to subtype B variants at weeks 24 (31% vs. 78.3%), 48 (51.7% vs. 85.2%), and 96 (61.1% vs. 94.3%). Importantly, the virologic response did not vary based on the composition of the ART regimen (e.g. protease inhibitor vs. NNRTI). However, similar mean increases in CD4+ cell counts 48 and 96 weeks after ART initiation were seen in both groups (Table 1).
In multivariate analysis, infection with a subtype F variant [odds ratio (OR) 14.8 (2.5–16.7), P < 0.001] and a high baseline plasma HIV-RNA level [OR 11.4 (1.7–7.9), P = 0.001] were independent predictors of a poor virologic response at 24 weeks after adjusting for ART initiation delay and baseline CD4+ cell count.
Subtype F accounts for less than 1% of HIV-1 infections worldwide, primarily being found in Africa (Congo) and South America (Brazil). In Europe, subtype F has an unusually high prevalence in Rumania (>70%) due to parenteral transmission in children during the late 1980s . Recently, subtype F HIV-1 infection has also been recognized among Italian heterosexual men . A rapid expansion of HIV-1 subtype F among MSM in Galicia was reported in newly diagnosed patients during 2010–2011 . Herein we confirm the continued spread of subtype F among MSM in northwest Spain and, for the first time, provide evidence of a suboptimal response to ART when compared with subtype B patients.
In regard to treatment response, some studies have highlighted the relevance of natural polymorphisms conferring resistance to specific antiretrovirals among ART-naïve individuals infected with non-B subtypes [5,6]. In this study, no significant differences were observed in the prevalence of major polymorphisms associated with resistance to antiretrovirals within reverse transcriptase or protease sequences from subtype F and B viruses. As expected, some differences in minor polymorphisms were found. The variant V106I, which is associated with low-level resistance to the NNRTI etravirine [10,11], was found more frequently among subtype F (compared to subtype B) sequences (83.8% vs. 3.5%, respectively; P < 0.001). Similarly, variants L10V, M36I, and L89M at positions associated with resistance to protease inhibitors were more frequent among subtype F (compared to subtype B) sequences (91.2% vs. 6.3%; 89.1% vs. 32.2%; and 91.2% and 8.4%, respectively; P < 0.001). The presence of these polymorphisms has been associated with a reduced susceptibility to the protease inhibitor tipranavir in subtype F specimens (fold-changes <2.7); however, no impact on darunavir susceptibility was found . Since none of the patients in this study were treated with etravirine or tipranavir-based regimens, the presence of polymorphisms associated with reduced susceptibility to these drugs does not account for the poor virologic response observed among subtype F patients. Therefore, the susceptibility of subtype F variants to antiretroviral agents currently used for the treatment of HIV infection needs to be assessed.
Of note, in the study published by Poveda et al. , subtype F possessed the highest replication capacity (performed by Phenosense assay; Monogram Biosciences, San Francisco, California, USA) among the HIV subtypes tested. This finding is consistent with the significantly higher baseline plasma HIV-RNA levels observed among subtype F patients in our population. However, in the multivariate analysis accounting for baseline viral load, subtype F infection retained a significant association with delayed virologic response.
Poor adherence to ART in the group of subtype F patients is another possible explanation for the lower rates of virologic response. Although a direct measurement of adherence is not available in this study, several studies have reported a higher degree of adherence to ART and HIV care among the MSM population than in other HIV-risk groups . Moreover, similar increases in CD4+ cell counts were seen after ART initiation in both groups; therefore, poor adherence among subtype F patients is an unlikely explanation for the poor virologic outcomes. Finally, there was no difference between the groups in terms of the number of different ART regimens initiated during the study period.
In summary, subtype F is the most prevalent non-B subtype among newly diagnosed HIV-1-infected persons in northwest Spain, with preferential transmission among MSM. For the first time, we have identified a significantly slower virologic response to ART among subtype F patients. Subtype F and a high baseline HIV-RNA were the major determinants of a poor virologic response to ART. Factors not associated with virologic response include: the timing of ART initiation, baseline CD4+ cell count, and the initial ART regimen (NNRTI vs. protease inhibitor). Given the potential clinical implications of these results, additional studies are warranted to identify the reasons for poor ART virologic response in subtype F patients.
Funding: The work was supported in part by grants from Fondo de Investigación Sanitaria (CP08/00214, PI10/02166, PI13/02266), and Fundación Profesor Novoa Santos, A. Coruña.
Author contributions: E.P. did the study design. B.P., M.G., A.M., A.C.-I., A.C., S.L.-C., I.R.-O., J.D.-P., and E.P. contributed with laboratory and clinical data. E.P. and S.P. did the statistical analysis. E.P., B.P., and D.W. wrote the manuscript. All authors revised and provided critical comments.
Conflicts of interest
There are no conflicts of interest.
1. Abecasis AB, Wensing AM, Paraskevis D, Vercauteren J, Theys K, Van de Vijver DA, et al. HIV-1 subtype distribution and its demographic determinants in newly diagnosed patients in Europe suggest highly compartmentalized epidemics
2. Treviño A, Soriano V, Rodríguez C, Arredondo M, Rivas P, Herrero-Mendoza D, 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
3. Thomson MM, Delgado E, Manjón N, Ocampo A, Villahermosa ML, Mariño A, et al. HIV-1 genetic diversity in Galicia Spain: BG intersubtype recombinant viruses circulating among injecting drug users
4. Kantor R, Katzenstein D, Efron B, Carvalho AP, Wynhoven B, Cane P, et al. Impact of HIV-1 subtype and antiretroviral therapy on protease and reverse transcriptase genotype: results of a global collaboration
. PLoS Med
5. Poveda E, de Mendoza C, Parkin N, Choe S, García-Gasco P, Corral A, Soriano V. Evidence for different susceptibility to tipranavir and darunavir in patients infected with distinct HIV-1 subtypes
6. Garrido C, Soriano V, Geretti AM, Zahonero N, Garcia S, Booth C, et al. Resistance associated mutations to dolutegravir (S/GSK1349572) in HIV-infected patients-impact of HIV subtypes and prior raltegravir experience
. Antiviral Res
7. Apetrei C, Necula A, Holm-Hansen C, Loussert-Ajaka I, Pandea I, Cozmei C, et al. HIV diversity in Romania
8. Lai A, Simonetti F, Zehender G, De Luca A, Micheli V, Meraviglia P, et al. HIV-1 subtype F1 epidemiological networks among Italian heterosexual males are associated with introduction events from South America
. PLoS One
9. Thomson MM, Fernández-García A, Delgado E, Vega Y, Díez-Fuertes F, Sanchez-Martínez M, et al. Rapid expansion of HIV-1 subtype F cluster of recent origin among men who have sex with men in Galicia
. Spain J Acquir Immune Defic Syndr
10. Poveda E, de Mendoza C, Pattery T, González MM, Villacian J, Soriano V. Phenotypic impact of resistance mutations on etravirine susceptibility in HIV patients with prior failure to non-nucleoside analogues
11. Vingerhoets J, Nijs S, Tambuyzer L, Hoogstoel A, Anderson D, Pichio G. Similar predictions of etravirine sensitivity regardless of genotypic testing method used: comparison of available scoring systems
. Antivir Ther
12. Giordano TP, Hartman C, Gilford AL, Backus LI, Morgan KO. Predictors of retention in HIV care among a national cohort of US veterans
. HIV Clin Trials