HIV/AIDS remains one of the most significant chronic transmissible diseases globally, affecting approximately 39 million people. In 2022, around 63,000 people died from HIV-1 infection, and at least 1.3 million new cases were reported annually, with the majority occurring in individuals over the age of 15 [1]. The introduction of antiretroviral therapy (ART) has greatly improved the management of HIV/AIDS cases by treating all diagnosed individuals, regardless of viral load or HIV/AIDS stage, improving access to care, and significantly reducing mortality and comorbidities in at least 51% [2]. New HIV cases have risen significantly worldwide in recent years (1.3 million), and 14% of people living with HIV (PLHIV) remain unaware of their status. Only 76% of diagnosed individuals have access to ART. This contributes to treatment delays, as late intervention often coincides with advanced HIV/AIDS, making treatment less effective and increasing the risk of opportunistic infections, complications, and viral transmission [3].

The World Health Organization (WHO) and the Joint United Nations Programme on HIV/AIDS (UNAIDS) proposed the 95-95-95 strategy, which aims to ensure that by 2025, 95% of people living with HIV know their status, 95% of those diagnosed receive antiretroviral therapy (ART), and 95% of those on ART achieve undetectable viral loads [4]. While significant progress has been made, this goal has challenges that still need attention. Critical factors have been identified as challenges, including achieving undetectable viral loads and managing poor clinical outcomes and complications such as co-infections. These setbacks are often driven by factors such as patient adherence issues, high rates of treatment discontinuation due to social challenges, poor retention in care, and ART resistance [5,6]. ART resistance is particularly linked to the HIV-1 viral ability to replicate and, in the presence of selective drug pressure, generate drug resistance mutations (DRMs), which can compromise the efficacy of ART, leading to poor therapeutic responses and treatment failure [7].

At least 26% of HIV/AIDS patients who start antiretroviral therapy (ART) carry HIV-1 strains resistant to first-line treatments, especially to Non-Nucleoside Reverse Transcriptase Inhibitors (NNRTIs) that are considered the backbone of ART. Several strategies have been implemented to address this issue, such as avoiding NNRTIs when the prevalence of drug resistance mutations (DRMs) exceeds 10%, strengthening adherence support, and improving patient retention in care [8]. The World Health Organization (WHO) has also established an effective HIV drug resistance surveillance program that monitors the most common DRMs worldwide and recommends resistance testing for all individuals who initiate or restart ART [9,10]. Additionally, international databases and collaborations also consolidate and analyze the described DRMs, assessing resistance levels and estimating the likelihood of treatment failure for ART-naïve and experienced patients [11].

The prevalence of pre-treatment drug resistance (PDR) mutations in ART-naïve patients to commonly used antiretroviral therapy drugs varies significantly depending on patient type, country, and other factors. It ranges from 5% to 30% in adults starting any ART, with 5.4% corresponding to Nucleoside Reverse Transcriptase Inhibitors (NRTIs), 12.9% to Non-Nucleoside Reverse Transcriptase Inhibitors (NNRTIs), 0.4% to Protease Inhibitors (PIs), and 0.6% to Integrase Strand Transfer Inhibitors (INSTIs). These values increase in patients with prior ART experience. In Latin America, the prevalence of NNRTI resistance is notably higher, at 16.7% among treatment-naïve patients and 26.7% among those previously exposed to ART. This underscores the challenges of continuing NNRTI-based regimens in the region and highlights the need for regimens with drugs that have a high genetic barrier, such as dolutegravir (DTG). It also emphasizes the importance of ensuring that all people living with HIV (PLHIV) are included in national treatment programs [8,12].

In 2020, Ecuador reported 47,000 people living with HIV (PLHIV), including 2,823 new cases. From these, 85% knew their status, 74% received ART, and 65% achieved viral suppression. The HIV/AIDS mortality rate was 4.8 per 100,000 inhabitants, with 44 deaths. Most cases occurred in key and vulnerable populations, such as young adults (ages 19-49), sex workers, and men who have sex with men (MSM), primarily in the provinces of Guayas, Manabí, and El Oro [13]. National efforts have reduced mortality and morbidity and increased timely diagnosis through rapid testing. Care and follow-up are provided in national public hospitals offering ART regimens [14]. Since 2021, most healthcare centers have transitioned from NNRTI- to DTG-based regimens. ART resistance in naïve and experienced patients in Ecuador is not well characterized, potentially leading to poor clinical outcomes. This study aimed to determine the prevalence and profile of HIV-1 drug resistance mutations (DRMs) at an HIV healthcare center in Quito between 2019 and 2021.

In this study, we described the presence of HIV-1 drug resistance mutations (DRMs) at a healthcare center in Quito, Ecuador, along with the circulating subtypes and their potential impact on various ART regimens over two different years. To our knowledge, this is the most recent study in Ecuador to report on the prevalence of HIV DRMs across different drug classes (NNRTIs, NRTIs, PIs, and INSTIs) before and during the transition to DTG-based regimens. We identified an overall DRM prevalence of 9.52% associated with low-level, intermediate, and high resistance prediction to the different available ART regimens. Most were primarily associated with NNRTIs, while none were detected for INSTI drugs. According to a recent meta-analysis, the worldwide prevalence of resistance to NNRTIs is 26.31% (95% CI, 20.76–32.25) [21].

Subtype B is the most prevalent HIV-1 subtype in Western and Central Europe, North America, Latin America, and the Caribbean, accounting for 60.5% of cases in these regions [8]. In the Andean region, the prevalence can reach up to 99% [22]. In our study, subtype B was detected in 78.57% of the samples, aligning with previous reports from Ecuador [23]. Circulating Recombinant Forms (CRFs) are considered HIV-1 strains that result from the recombination of different HIV subtypes and represent approximately 29% of HIV infections worldwide [24]. Among these, CRF02_AG was identified in 11.9% of our samples, confirmed by the presence of the K20I signature mutation in the PR gene [20]. This form has a global prevalence of 7%, while Latin America is less common, with a prevalence of 0.9% [8](World Health Organization, 2021). A previous Ecuadorian study reported a 4% prevalence of CRF02_AG in antiretroviral therapy (ART) treated patients during 2018 [23].

CRF19_cxp and CRF24_BG, recombinant forms of subtypes D-A and B-G, respectively, were previously reported in countries like Cuba and Spain but had not been documented in Ecuador [25,26]. These CRFs are particularly significant due to their association with rapid disease progression to AIDS. In our study, both CRFs were identified, with one case found in an ART-experienced patient from Cuba who exhibited persistent low-level viremia. Migration is known to play a critical role in the introduction and spread of HIV subtypes [24]; Ecuador has experienced significant migration from countries such as Cuba, Spain, and Venezuela in recent decades, likely explaining the detection of these CRFs in our population. However, the specific impact of these recombinant forms on HIV-1 transmission dynamics and ART resistance in Ecuador requires further investigation.

Despite this, our study has several limitations. First, the small sample size was due to resource constraints and did not include other national healthcare centers; this may lead to significant variations in our findings compared to national estimations. Geographic variability and selection bias are also concerns; although there is a significant proportion of HIV cases in Quito-Pichincha (19.56%), other provinces such as Guayas (75%), Manabí (8.46%), and Esmeraldas (4.86%) have reported higher prevalence rates in Ecuador [27]. These findings indicate that the prevalence of circulating mutations warrants further attention. In recent years, next-generation sequencing (NGS) technologies have significantly enhanced the accurate identification of drug resistance mutations (DRMs) compared to traditional Sanger sequencing, which only detects variants with DRMs at levels exceeding 20% of the viral population across various samples. This limitation of the Sanger method may help to explain the low prevalence of DRMs observed in our study. Despite this, Sanger sequencing is still recommended as the standard method for HIV DRM surveillance by the World Health Organization (WHO) [16]. Additionally, patient adherence to antiretroviral therapy (ART) and the transition to dolutegravir (DTG)-based regimens may not fully reflect DRM trends from previous years, potentially introducing further limitations to our analysis.

In 2023, the Pan-American Health Organization (PAHO) reported that 1.7 million people who live with HIV (PLHIV) were receiving ART with a treatment coverage of 73% for the Americas and the Caribbean [28]. While a definitive cure has not yet been found, ART has significantly improved the quality of life and life expectancy of HIV patients through long-term viral suppression and reducing clinical symptoms and complications. During the same year, in Ecuador, at least 85% of PLHIVs knew their status and were under an ART regimen [27], remarking the continuous efforts to improve and reach the established goals for 2030. However, at least 36% of patients under ART could not reach viral suppression (less than 1,000 copies/mL), increasing the risk of treatment failure and drug resistance.

The emergence of antiretroviral therapy (ART) resistance in recent decades has significantly compromised the benefits of treatment, particularly for the most widely used regimens. Determining and characterizing patterns of HIV-1 drug resistance mutations (DRMs) and polymorphisms is crucial for understanding viral transmission dynamics, adaptation, and resistance to current antiretroviral therapy (ART) regimens. This knowledge aids in developing accurate interventions. It is well-established that resistance to NNRTIs, such as nevirapine (NVP) or efavirenz (EFV), is the most common and can develop in at least 10% of ART-naive patients receiving these drugs as first-line therapy (ranging from 9-14% depending on the country), with this rate tripling in ART-experienced patients [12]. According to the WHO 2021 [8], the overall prevalence of pre-treatment drug resistance (PDR) for NNRTIs in countries such as Colombia, Argentina, Brazil, Mexico, and Cuba ranges from 6 to 22% [9,28,29] and in neighboring countries like Peru, the prevalence of pre-treatment drug resistance in 2021 was reported at 9.8% [30]. This is consistent with our study, which identified a prevalence of 9.52% for NNRTs, and half of them were considered as transmitted, reflecting the widespread use of EFV-based regimens as first-line regimens before 2021. Furthermore, a previous study conducted in Latin America reported a prevalence of drug resistance in a limited number of samples from Ecuador during 2012, which was 4.3%, suggesting the progressive increase of DRMs over the years [23].

In our study, half of the NNRTI resistance mutations (DRMs) were classified as transmitted, primarily consisting of minor, accessory, or polymorphic mutations with minimal impact on ART susceptibility. One such mutation is V108I. It was detected in a treatment-naive patient on a darunavir/ritonavir (DRV/r) regimen. This mutation, selected by most NNRTIs, contributes to reduced susceptibility only when combined with other DRMs, leading to low-level resistance (LLR) to nevirapine (NVP), potential low-level resistance (PLLR) to efavirenz (EFV), and etravirine (DOR) [31]. This mutation has been reported in 50% of drug resistance mutations (DRMs) in Argentina but shows a lower prevalence in Mexico and Colombia (<5%) [8,20,32]. In our study, it was observed in 2.38% of cases. Also, the E138A mutation was identified in one treatment-naive patient under an INSTI-based regimen. It could be found in 2-5% of treatment-naive patients under rilpivirine (RPV) and etravirine (ETR) regimens, with prevalence varying by subtype and region. This mutation confers potential low-level resistance (PLLR) to ETR and low-level resistance (LLR) to RPV but does not significantly impact ART efficacy [8,20,33,34].

In contrast, protease inhibitors (PIs) and integrase strand transfer inhibitors (INSTIs) exhibit lower resistance rates due to their higher genetic barrier. Nonetheless, their frequent use and viral characteristics still impact their effectiveness. Resistance rates for PIs ranged from 0.1-1.4%, and for INSTIs, available data showed rates of 7.6% for Argentina and 0.5% for Mexico [8]. For PIs, one important DRM was found in an ART-naïve patient under an NRTI-based regimen. The M46L mutation is classified as a surveillance DRM (SDRM). It can occur in various HIV-1 subtypes among both ART-experienced or naive patients, with a reported prevalence of 0.37% in subtype B samples. M46L impacts the structural integrity of the enzyme binding site, conferring resistance to indinavir, nelfinavir, and saquinavir [35,36,37]. It has been documented in multiple countries, including the United States, Argentina, Brazil, Peru, Spain, and Cuba [12,38]. For our study, the prevalence of PI drug resistance was 2.38%.

No major DRMs were identified for INSTIs; however, minor accessory mutations like E157Q were found in two ART-naïve patients receiving RAL and efavirenz EFV regimens, as well as in one ART-experienced patient on an LP/r regimen. The E157Q mutation occurs in 1-5% of INSTI-naïve patients and has a minimal impact on susceptibility to RAL, EVG, or DTG when combined with other mutations [39]. It has been reported in African studies with a prevalence of 1.56% [40]. Additionally, L74M is a common polymorphic mutation found in 1-5% of naive patients, especially in the CRF02_AG subtype. It was found in two naive patients on RAL and DTG-based regimens, though it did not affect susceptibility.

The low prevalence of DRMs for PIs and INSTI regimens in the present study would be explained by their less frequent use as first-line regimens during 2019-2021 in Ecuador. It could also be attributed to several factors, including high viral suppression rates, good treatment adherence, limited ART exposure, and the geographical and epidemiological characteristics of ART-naive patients in Quito. Other potential explanations include the small sample size or the recent introduction of highly effective dolutegravir (DTG)- based regimens. Similar findings have been reported in previous studies from Sweden, Iceland, China, and the U.S., where low levels of transmitted DRMs have been associated with strong ART adherence and near-complete viral suppression, particularly concerning NRTIs, PIs, and INSTIs [41,42,43]. This suggests that the widespread use of potent regimens and comprehensive treatment strategies may continue to play a key role in reducing transmitted drug resistance across various populations [41,43,44,45].

In 2018, the WHO recommended the inclusion of DTG in first-line regimens, highlighting the need for strategies to prevent HIV drug resistance and the transmission of viral populations carrying associated mutations [46]. It reported high levels of HIV viral load suppression (>90%) in populations receiving dolutegravir (DTG)-based antiretroviral therapy as a first-line regimen. According to PAHO data, Ecuador is one of the countries where more than 80% of PLWH currently receive a DTG-based regimen [46]. Still, from 2020, the DTG switch was performed according to international guidelines as a first-line treatment [47]. During our study, this transition was under implementation, finding that 48% of patients received an EFV and 24.1% DTG regimens. However, no national surveillance studies evaluate the direct impact of DRMs and their clinical response when EFV and DTG regimens are used [27].

On the other hand, acquired drug resistance mutations (ADRs) are typically observed in ART-experienced patients, where certain viral populations develop resistance under drug-selective pressure, often leading to a virological failure (VF) [47]. These mutations are frequently associated with NNRTI regimens, with a prevalence of approximately 27%, and in some African countries, this can rise to as high as 50% [48,49]. A 2018 study conducted in Ecuador involving 101 HIV-positive adults and children with VF found a prevalence of ADRs of 34.9% [23]. In contrast, our study observed a lower ADR prevalence of 4.74%, which may reflect a lower proportion of patients experiencing VF in the healthcare center. However, this could be different on a national scale.

The most common acquired drug resistance (ADR) mutation to NNRTIs is K103N. It was identified in an HIV-1 subtype B sample from a low-adherent, ART-experienced patient. K103N is considered a surveillance drug resistance mutation (SDRM), occurring in approximately 1.15% of treatment-naive patients and up to 37.11% of ART-experienced patients. This mutation alters the binding site of NNRTIs to the reverse transcriptase (RT) enzyme, significantly reducing the efficacy of NNRTIs like efavirenz (EFV) and nevirapine (NVP), with a 20- to 50-fold decrease in susceptibility. However, the presence of K103N does not impact other NNRTIs [20]. The importance of this mutation was noted in one meta-analysis performed in Latin America [28]. Another study suggested that this mutation may cause reduced viral suppression in patients under dolutegravir (DTG)-based regimens, particularly in South African populations, raising concerns about the potential for multi-class drug resistance [50]. This underscores the importance of continuous surveillance and tailored ART regimens to prevent treatment failure in the presence of specific mutations.

F277C is a non-polymorphic mutation selected in persons receiving DOR and rarely in persons receiving ETR and RPV. It usually occurs in combination with other DRMs and, in this setting, has been related to the high level of resistance for DOR. The mutation is traditionally associated with moderate to high-level reductions in susceptibility to NNRTIs such as nevirapine (NVP), efavirenz (EFV), etravirine (ETR), and rilpivirine (RPV). In the earlier study from Ecuador by [23], this ADR mutation was not reported. In our findings, the F277C mutation was identified in a single subtype B sample from an ART-experienced patient on an EFV regimen, but it did not result in any significant reduction in susceptibility to NNRTIs.

Although INSTI drugs have a high genetic barrier, a concerning increase in acquired drug resistance has been reported in some countries. One study performed in Mexico in 2020 identified a cross-resistance between different drugs of this group and VF to raltegravir and a lower proportion to dolutegravir [51]. In Sudan, DTG drug resistance mutations have been observed at a prevalence of less than 0.2% (95 % CI: 0.0-1.2 %); however, acquired drug resistance (ADR) surveys suggest that resistance rates to DTG may be below 3 % globally, though in low- and middle-income countries, prevalence can reach 4.8 %, and in certain African countries, it can rise as high as 19.6% [12]. Given these concerns, the WHO recommends routine drug resistance surveillance as the global use of DTG-based regimens expands [12].

In conclusion, our findings indicate that the prevalence of drug resistance mutations (DRMs) among the studied population is low, and most of these mutations do not significantly compromise susceptibility to key antiretroviral drug classes, including INSTIs, NRTIs, and PIs. However, some non-nucleoside reverse transcriptase inhibitor (NNRTIs) mutations could produce PLLR to LLR. The clinical impact of these results should be evaluated based on individualized responses to ART. Finally, it is important to highlight the potential role of viral mutations classified as polymorphisms. Although they are often not associated with resistance, it has been observed that they could contribute to resistance in the context of cumulative mutations or when they occur in certain HIV subtypes [52].

Our results also suggest that, given the low prevalence of DRMs, maintaining current ART regimens is a viable option. However, further studies are required to assess this approach nationally. Since HIV-1 DRM testing is not yet mandatory for treatment-naïve or experienced patients in many settings, implementing routine surveillance and follow-up of drug resistance is crucial. Countries like Brazil, Mexico, Argentina, Peru, and Honduras form part of the WHO HIVResNET for drug resistance, enhancing their responsiveness in the control and prevention of HIV transmission; although the economic resources required vary, Ecuador should consider its implementation as a public policy. Additionally, understanding the impact of switching to dolutegravir (DTG)-based regimens when necessary will ensure long-term treatment success.

To Doctor Santiago Ávila and Claudia García from the Laboratory of Virological Diagnosis at the Center for Research in Infectious Diseases in the Instituto Nacional de Enfermedades Respiratorias "Ismael Cosio Villegas" in Mexico City for their kind support in performing some of the analyses.

The authors confirm the contribution to the paper as follows: study conception and design: E. Teran, P. Quirola; data collection: P. Quirola, N. Cevallos; analysis and interpretation of results: P. Quirola, P. Espinosa; draft manuscript preparation: P. Quirola, E. Teran. All authors reviewed the results and approved the final version of the manuscript. Some sentences were improved using ChatGPT.

The authors declare that they have no known competing financial interests or personal relationships that could have influenced the work reported in this paper.