The HIV-1 group M subtype B is the predominant HIV-1 clade in Western Europe. However, the prevalence of HIV-1 non-B subtypes has been increasing in the last years across Western and Central Europe . This trend is also observed in Spain, with the circulating recombinant form 02_AG (CRF02_AG) being the most common circulating non-B subtype (38%) . Nonetheless, we have previously described in Galicia (Northwest of Spain) an unexpectedly high prevalence of subtype F with a rapid and preferential transmission among MSM [3,4].
Phylogenetic analysis identified the putative origin of this subtype F in Latin America and Brazil and established its introduction in Galicia approximately 24 years ago; however, its growth to epidemic proportion occurred in the last 8 years . Moreover, we also described that virological response rates to antiretroviral therapy (ART) were significantly lower among subtype F-infected patients compared with subtype B at weeks 24, 48, and 96 . Interestingly, subtype F and high baseline plasma HIV RNA levels were independently associated with virological response at 24 weeks. The reasons underlying these differences remain elusive.
In that study, most patients started ART based on nonnucleoside reverse transcriptase inhibitors (NNRTI) (51.4%) or protease inhibitors (41.1%). Only a small proportion of patients (7.5%) initiated an integrase inhibitor (INI)-based therapy. Considering the high efficacy and safety profile of INI, current HIV treatment guidelines have positioned this drug family as the preferred option for treatment-naïve patients [6–8]. In this context, it is interesting to evaluate whether this poor response to ART observed among subtype F compared with subtype B is also maintained with INI-based therapies.
Herein, we evaluate the virological response of simultaneous HIV-1 subtype F compared with subtype B-infected patients who initiated ART based on INI from January 2015 to February 2017 at our institution in Northwest Spain. Patient demographics (age, sex, and risk behaviour), laboratory (HIV RNA, HIV subtypes, and CD4+ cell counts), and clinical parameters at the time of diagnosis and treatment initiation were recorded. In addition, the response to ART was retrospectively analysed. A statistical analysis was performed using SPSS, version 19 (SPSS Inc., IBM Corp., Armonk, New York, USA).
A total of 153 newly diagnosed HIV-1 patients started ART from January 2015 to February 2017 at our institution. From them, 114 patients (74.5%) received an INI-based regimen and of these, HIV subtype could be determined in 103 patients. Non-B variants were found in 45.6% of patients with the following distribution: F (27.2%), CRF02_AG (5.7%), A (3.9%), CRF01_AE (2.9%), G (1.9%), CRF14_BG (1%), C (1%), D (1%) and CRF18_cpx (1%). Subsequent analyses were performed comparing the two most prevalent subtypes seen in our population who started ART with an INI-based regimen: subtype B (n = 56) and subtype F (n = 28). None of the baseline genotype sequences from subtype B or F harboured any substitution associated with resistance to INI or NRTIs.
Table 1 displays the main characteristics of this population at the time of diagnosis and ART initiation. There were baseline differences between patients infected with subtype B or F regarding the nationality, most of them White but with more Latinos with subtype B (19% vs. 0%, respectively, P = 0.013). Moreover, the rate of AIDS-defining diseases at ART initiation was higher for subtype F-infected patients (12.5% vs. 39.3%, respectively; P = 0.002). Mean CD4+ at diagnosis (532 vs. 295; P = 0.01) was lower in the subtype F group. Mean HIV RNA levels at diagnosis were significantly higher among subtype F compared with subtype B patients (4.8 vs. 5.3; P = 0.004). Not unexpectedly, the mean time to ART initiation was shorter among patients infected with subtype F variants (22.2 vs. 2.8 months; P = 0.003).
Virological response, defined as achievement of HIV RNA below 50 copies/ml, was retrospectively assessed at 12, 24, 36, and 48 weeks after ART initiation. Interestingly, the rates of virological response were lower among patients infected with subtype F variants compared with subtype B variants at weeks 12 (75% vs. 25%) and 24 (95% vs. 59.1%), but not thereafter. However, considering the definition used by the US Department of Health and Human Services for virological failure (HIV RNA >200 copies/ml) none patients infected with subtype F failed after 36 weeks of follow-up . Increases in CD4+ cell counts were similar between subtype B and F in the first weeks but significantly different at weeks 36 (703 vs. 302) and 48 (766 vs. 425) (Table 1).
In multivariate analysis, only infection with a subtype F variant [odds ratio 11.8 (95% confidence interval (CI) 1.1–119.9); P = 0.037] was recognized as an independent predictor of a poor virological response at 24 weeks after adjusting for viral load, CD4+ cell count, and AIDS-defining diseases at ART initiation. The corresponding odds ratio for baseline viral load, CD4+, and AIDS-defining diseases at ART initiation were: [3.4 (95% CI 0.6–19.0); P = 0.166], [0.9 (95% CI 0.9–1.0); P = 0.539]; [1.3 (95% CI 0.2–8.8; P = 0.767], respectively.
Previously, we have described an impaired treatment response among subtypes F compared to B subtypes at 24, 48, and even at 96 weeks, most of them exposed to NNRTI- or protease inhibitor-based regimens . Herein, we demonstrated that this poorer treatment response to ART among F subtypes has also been confirmed in 114 HIV-infected patients receiving an INI-based regimen in the last two years. However, in this case, the differences in the rates of patients achieving suppressed viremia between B and F subtype was only statistically significant at 12 and 24 weeks (75% vs. 25% and 95% vs. 59.1%, respectively), suggesting a slower virological response in subtype F. These findings might suggest that INI-based therapies could be more effective among subtype F infected patients than NNRTI or protease inhibitors. However, prospective and controlled studies are necessary to confirm this.
The reasons underlying these findings are still unknown. The presence of natural polymorphisms conferring resistance to specific antiretrovirals or poor adherence to ART in the group of subtype F patients might explain in part the lower rates of virological response. However, in our previous study, the presence of polymorphisms associated with reduced susceptibility to these drugs did not account for the poor virological response observed among subtype F patients . Moreover, considering pharmacy records (adjusted by missing visits), no differences were found in rates of adherence between subtype B and F patients in the present study (98.4% vs. 97.8%, respectively). Although lower adherence has been described among some ethnic minorities in the United States , no Latin origin was observed among subtype F-infected patients, therefore, these racial differences would not explain the poor response observed in subtype F. In more detail, all Latinos included in the study were infected by subtype B variants and came from Venezuela (27.4%), Colombia (18.2%), Republican Dominican (18.2%), Peru (18.2%), Cuba (9.0%), and 9.0% had an undetermined Latin origin. In addition, around half of the Latinos included in the study acquired the HIV infection in Spain.
Some studies have already described that natural polymorphisms in the integrase region that might influence the genetic barrier for the development of resistance or drive the selection of a specific resistance pathway were more frequent among HIV-1 non-B subtypes than in B subtypes [10,11]. However, there is no evidence to date that natural genetic variation within the HIV-1 integrase affects the clinical responses to current available INI [12–15]. As the overall prevalence of subtype F accounts for less than 1% of HIV-1 infections worldwide, it is likely to be under represented in clinical trials. Therefore, it will be necessary to assess the susceptibility of subtype F variants to currently used INI.
In the present study, no significant differences were observed in the prevalence of polymorphisms associated with resistance to INI within the integrase sequences analysed. Indeed, none of the integrase sequences from subtype F harboured any substitution associated with resistance to INI. Only two patients infected with subtype B harboured the T97A mutation. This is a polymorphic accessory mutation with minimal effects on INI susceptibility but in combination with other major resistance mutations (i.e. Y143C/R) reduces susceptibility to elvitegravir and raltegravir. Furthermore, 60.7% of subtype F study participants were treated with INI with low barrier against resistance development (raltegravir and elvitegravir) and we did not see any virological failure at 48 weeks or selection of resistance-associated substitutions.
Of note, an impaired CD4+ recovery was observed after 48 weeks of therapy, with significantly higher CD4+ cell counts observed among subtype B patients compared with F subtypes after 36 and 48 weeks (703.3 vs. 302.7 and 766.1 vs. 425.2, respectively). Subtype F study participants had significantly lower CD4+ cell counts at diagnosis and numerically lower nadir CD4+ cell counts and CD4+ cells at ART initiation. These two later did not achieve statistical significance probably because of the low number of study participants with subtype F. Indeed, a higher proportion of them had AIDS-defining diseases at ART initiation (12.5% vs. 39.9%, respectively; P = 0.002). These features, might explain in part this lower CD4+ recovery after ART initiation compared with B subtypes. Thus, subtype F study participants had a significantly more advanced HIV infection at diagnosis. If this reflects a more rapid progression of HIV infection with subtype F vs. B, or a later diagnosis in these study participants due to other unknown factors deserves further investigation. Similarly, the jeopardized CD4+ cell recovery could simply be a reflection of the lower CD4+ cell count at ART initiation, or alternatively could be intrinsically related to subtype F attributes or the slower virological response observed with INI.
These findings might have important implications in the prevention of HIV transmission. Several studies have demonstrated the high preventive value of achieving controlled HIV RNA levels (<50 copies/ml) in the initial treatment in HIV-infected patients [16–18]. Therefore, as ART is one of the most successful strategies to prevent HIV transmission, a delay in suppressing viremia once ART is initiated might increase the time at risk for HIV transmission.
Moreover, these data highlight specific features among subtype F HIV-infected patients related with a worse clinical prognosis (i.e. higher HIV RNA levels and AIDS-related events, lower CD4+ recovery after ART) compared with subtype B. Therefore, identifying specific epidemiological features of subtype F-infected study participants could help design specific public health strategies for their earlier targeted diagnosis. HIV subtype determination must be recommended for newly diagnosed HIV-infected individuals in our sanitary area for an optimization of their clinical follow-up. Indeed, specific strategies for prevention, diagnosis, and treatment for subtype F-infected patients would be desirable.
The molecular mechanism that might explain the higher viral load and the lower CD4+ cell counts observed among the subtype F HIV-infected population compared with subtype B merits further study. Differences regarding the replication capacity or fitness have been described among HIV-1 subtypes. Some studies have suggested that HIV-1 subtype F isolates may be slightly more fit than the majority of group M isolates. However, these studies are performed in small numbers of subtype F isolates, based on specific gene sequence or genetic factors (i.e. HIV-1 protease, long terminal repeat) and not on the entire viral genome. Moreover, immunological pressures and host environment may also play an important role in the evolution of viral subtypes that may ultimately impact on viral replication and pathogenesis [19–22]. Therefore, additional studies analysing the potential virological and immunological mechanisms that might explain the apparent increased pathogenicity of subtypes F compared with subtypes B are required.
The main limitations for this study are the existence of significant baseline differences in characteristics associated with poorer treatment response, the small sample size, and that it is a single-centre study which could introduce uncontrolled bias. Moreover, it might be a potential bias in regard to the selection of the study population as only those patients who started ART were included.
In summary, subtype F remains as the most prevalent non-B subtype among newly diagnosed HIV-1-infected persons in Northwest Spain. For the first time, we have identified a significant delay in the initial virological response and a poorer immune recovery associated with subtype F in patients initiating an INI-based regimen. Indeed, the presence of subtype F was identified as the major determinant of a poor virological response to INI-based regimens. These results might have implications for the prevention of HIV transmission, ART virological response, and immune recovery in this population. Therefore, additional prospective studies are warranted to identify the reasons underlying the later diagnosis vs. faster progression of this subtype as well as the delayed viroimmunological response with INI regimens to implement the best prevention and clinical strategies for the clinical follow-up of subtype F patients.
The work was supported in part by grants from Fondo de Investigación Sanitaria (CPII14/00014, PI10/02166, PI13/02266, CM13/00328, CM15/00233, PI16/02159), and Fundación Profesor Novoa Santos, A Coruña. We would like to thank to Biobank of A Coruña (SERGAS) for providing us the technical, ethical and legal advice necessary for the development of our research.
Conflicts of interest
There are no conflicts of interest.
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