Globally, Antiretroviral Therapy has Helped Reduce the Rates of Mortality and Morbidity Associated with HIV-1 Infection. However, the Emergence of HIV Drug Resistance (HIVDR) Remains a Threat to the Effectiveness of Antiretroviral Therapy


Globally, antiretroviral therapy (ART) has been a cornerstone in the fight against HIV-1, significantly reducing the rates of mortality and morbidity associated with the infection. Since the introduction of ART in the mid-1990s, millions of lives have been saved, and the quality of life for people living with HIV (PLHIV) has dramatically improved. Despite these advancements, the emergence of HIV drug resistance (HIVDR) remains a substantial threat to the continued success of ART programs. HIVDR can lead to treatment failure, necessitating more complex and expensive treatment regimens, and potentially contributing to the further spread of resistant strains of the virus.

Global Efforts and the 95-95-95 Goals

Over the past decade, significant efforts have been made by the Joint United Nations Programme on HIV/AIDS (UNAIDS) to achieve the 95-95-95 goals. These ambitious targets aimed that by the end of 2022, 95% of all people living with HIV would know their HIV status, 95% of all people with diagnosed HIV infection would receive sustained ART, and 95% of all people receiving ART would achieve viral suppression. According to UNAIDS, these targets were intended to ensure that 33.6 million PLHIV knew their HIV status, 29.8 million PLHIV were accessing ART, and 27.7 million PLHIV were virally suppressed.

HIV Drug Resistance: A Growing Concern

According to World Health Organization (WHO) reports, the prevalence of drug resistance mutations (DRM) among ART initiators in developing countries increased from 6.8% to 10% between 2010 and 2017. HIVDR has been reported for all major antiretroviral (ARV) drugs, potentially resulting in delayed viral suppression or treatment failure in individuals initiating ART. Surveillance of HIVDR is conducted in most countries, enabling the monitoring of HIV susceptibility and tracing of virus mobility. Understanding HIVDR at the population level can aid countries in selecting the optimal first-line ARV, particularly when HIVDR testing is not conducted prior to ART initiation.

The HIV Epidemic in Pakistan

In Pakistan, the HIV/AIDS epidemic has increased significantly over the last two decades. From 2010 to 2019, Pakistan experienced a 78.5% increase in new HIV infections, with an annual incidence of 20,000 HIV infections. As of December 2023, an estimated 270,000 people are living with HIV (PLHIV) in Pakistan. The country’s progress toward the 95-95-95 goals has been lagging, with only 24.5% of PLHIV aware of their HIV status and just 16.2% on ART. Data on viral suppression are sparsely available, with UNAIDS country progress reports and recent studies estimating viral suppression rates to be 23% and 40%, respectively.

Key Populations and High-Risk Groups

Recent surveillance reports indicate that the HIV epidemic in Pakistan is concentrated among key population groups with consistently high prevalence rates. For example, the prevalence among people who inject drugs (PWID) is 38.4%, among transgender sex workers is 7.5%, and among male sex workers is 5.6%. In Pakistan, rising new HIV cases pose serious challenges related to access and adherence to ART. In 2004, the National AIDS Control Programme (NACP) initiated the ART program in all four provinces with the support of the Global Fund. Currently, there are 89 ART centers across the country providing antiretrovirals free of cost to all HIV patients.

ART Regimen and Challenges in Pakistan

The primary ART regimen for PLHIV in Pakistan consists of two nucleoside reverse transcriptase inhibitors (NRTIs), specifically lamivudine (3TC) and tenofovir, combined with the integrase inhibitor dolutegravir (DTG). Abacavir (ABC) and zidovudine (AZT) may also be included. ART in Pakistan is typically initiated without conducting baseline or periodic sequencing, increasing the likelihood that individuals may receive ineffective treatment. Limited data on HIV-1 drug resistance mutations among Pakistani PLHIV highlight the presence of DRM among 6.5% of ART-naïve individuals and 36.4% of ART-experienced individuals.

Case Study: Karachi

Karachi, the capital city of Sindh and the largest financial city in Pakistan, reported its first cases of HIV-1 in 1987. According to the Sindh province HIV/AIDS epidemiology database, Karachi had the highest number of HIV-1 cases in 2021, with 6768 cases, where 40% of cases were reported in Karachi’s central district alone. The HIV-1 epidemic in Karachi is primarily associated with PWIDs, with an HIV prevalence of 48.7%. Additionally, Karachi is experiencing an increase in HIV prevalence among women and children.

Genetic and Drug Resistance Analysis in Karachi

A comprehensive genetic and drug resistance analysis was conducted using samples obtained from ART-naïve and ART-experienced individuals living with HIV in Karachi. This analysis of DRMs associated with protease inhibitors (PIs) and reverse transcriptase inhibitors (RTIs) remains crucial for surveillance and monitoring of drug resistance patterns. Optimizing alternate ART regimens can ensure viral suppression in PLHIV who may not be eligible for a switch to or initiation of dolutegravir (DTG) therapy due to factors such as hyperglycemia or diabetes.

Findings and Analysis

In this study, 268 HIV-1 pol sequences from PLHIV in Karachi were analyzed to identify drug resistance mutations, quantify levels of drug resistance, and determine the distribution of HIV-1 genetic diversity in the city. The study participants were divided into three groups based on their ART history: ART-experienced (n = 168), ART-naïve (n = 37), and unknown status (n = 68). Among the study participants, 88% were male, and 61.9% were PWIDs. This finding is consistent with previous studies showing that male PWIDs comprise the predominant key group.

The study revealed that 40% of participants were infected with HIV-1 subtype A1, 33.2% with CRF02_AG, and 7.8% with subtype C. Subtype A1 continues to be the predominant clade in Pakistan, although the proportion of CRF02_AG is gradually increasing. Additionally, sub-subtype A6 was identified for the first time in Pakistan. The overall prevalence of DRMs in the study was 47.76%. The proportion of transmitted NNRTIs and PIs resistance was 93% and 7%, respectively, while NRTIs remained susceptible. The proportion of acquired NNRTIs, NRTIs, and PIs resistance was 70.7%, 20.7%, and 8.5%, respectively.

Transmission Clusters and DRMs

A comparative analysis of DRMs in the three study groups showed multiple DRMs associated with resistance to reverse transcriptase inhibitors. The DRM E138A, which can confer major resistance against rilpivirine (PRV), was particularly high among the three study groups. Previous studies have detected the E138A mutation among subtype A1 sequences of Pakistani PLHIV, reporting varying prevalence in ART-naïve (8–39%) and ART-experienced individuals. The E138A mutation frequently occurs in subtype C and is associated with reduced RPV susceptibility by 2.9-fold. A high prevalence of E138A has also been reported in PLHIV from Mozambique (12%), China (14.3%), and Greece (7.7%).

K103N was the second most prevalent DRM identified among ART-experienced and unknown status group individuals, conferring significant resistance to efavirenz and nevirapine. Additionally, DRM M184I identified among ART-experienced and unknown status groups can confer major resistance against abacavir, emtricitabine, and lamivudine. The presence of DRMs associated with resistance to efavirenz and lamivudine, which are part of the ART regimen administered to PLHIV in Pakistan, may lead to ART failure. DRMs K103N and M184V have been frequently reported in Pakistani PLHIV.

High-Level Resistance in ART-Experienced Groups

High-level resistance against NNRTIs was observed among the ART-experienced group (21.5%) and the unknown status group (11.8%). In both groups, the DRMs E138A and K103N showed high-level resistance against NNRTIs, including EFV, NVP, and RPV. K103N is the most commonly transmitted DRM associated with virological failure and can decrease NVP and EFV susceptibility by 50- and 20-fold, respectively. A considerable number of individuals carrying DRMs (51.8%) were observed in transmission clusters, confirming the transmission of DRMs in the region.

The prevalence of NRTI-associated DRMs in ART-experienced patients in Pakistan (20.7%) is consistent with findings from other countries. Studies from India and China reported NRTI-associated DRMs in 44.5% and 74.4% of ART-experienced PLHIV, respectively. Despite the high prevalence of NRTI mutations in ART-experienced patients, the transmission of these mutations appears to be relatively low. This may be due to the fitness cost associated with certain DRMs against NRTIs, such as DRM M184V, which can significantly reduce viral replication capacity, making these variants less transmissible.

Entropy and Positive Selection Analysis

Shannon entropy analysis revealed that DRM sites 10, 138, 179, and 230 in the three study groups exhibited higher entropy than the mean entropy. High entropy at different codon positions can correlate with an increased probability of mutations. This finding is consistent with previous studies that observed high Shannon entropy in the HIV-1 pol gene of ART-naïve individuals in Pakistan. In our study, DRM sites 138, 188, 103, and 115 were under positive selection pressures. The prevalence of DRM at sites 138 and 103 was frequently observed in all three study groups. Positive selection of pol-encoded amino acids in PLHIV experiencing virological failure can potentially alter the structural and biological functions of the viral proteins. This adaptive process may serve as a mechanism by which the virus evades the pressure exerted by antiretroviral drugs.

ART Challenges and Future Directions in Pakistan

Despite the establishment of ART programs and the availability of free antiretrovirals, Pakistan faces significant challenges in controlling the HIV epidemic. One major challenge is the lack of baseline and periodic sequencing to monitor HIVDR. This gap increases the likelihood of patients receiving ineffective treatment, contributing to the development and spread of drug-resistant HIV strains. The limited data on HIV-1 drug resistance mutations in Pakistan necessitate more comprehensive studies to understand the dynamics of HIVDR and inform treatment strategies.

Regional Variations and Key Populations

The HIV epidemic in Pakistan is marked by significant regional variations and is concentrated among key populations such as PWIDs, transgender sex workers, and male sex workers. Targeted interventions for these high-risk groups are crucial for controlling the epidemic. In Karachi, the high prevalence of HIV among PWIDs highlights the need for harm reduction programs, including needle exchange programs and opioid substitution therapy. Additionally, outreach and education programs tailored to transgender and male sex workers can help reduce transmission rates and improve access to ART.

The Role of Surveillance and Monitoring

Effective surveillance and monitoring systems are essential for tracking the prevalence and spread of HIVDR. Enhanced HIVDR surveillance can help identify emerging resistance patterns and inform the selection of first-line and second-line ART regimens. Implementing routine HIVDR testing before ART initiation and during treatment can improve treatment outcomes and reduce the spread of resistant strains. Strengthening laboratory capacity and training healthcare workers in HIVDR testing and interpretation are critical steps toward achieving this goal.

Community Engagement and Awareness

Community engagement and awareness campaigns play a vital role in addressing the HIV epidemic. Stigma and discrimination associated with HIV can hinder individuals from seeking testing and treatment. Raising awareness about HIV, its transmission, and the benefits of ART can encourage more people to get tested and adhere to treatment. Engaging community leaders, PLHIV networks, and civil society organizations in these efforts can help build trust and promote a supportive environment for PLHIV.

Genetic Diversity and Recombinant Forms

The study highlighted the genetic diversity of HIV-1 in Karachi, with the presence of various subtypes and recombinant forms. Subtype A1 remains predominant, but the increasing proportion of CRF02_AG and the identification of sub-subtype A6 and other unique recombinant forms indicate ongoing viral evolution and migration patterns. This genetic diversity has implications for treatment and vaccine development, as different subtypes and recombinant forms may respond differently to ART and potential vaccines.

Implications for Treatment Strategies

The findings of high-level resistance against NNRTIs and the presence of multiple DRMs underscore the need for personalized treatment strategies. Standardized ART regimens may not be effective for all patients, particularly those with pre-existing DRMs. Incorporating routine HIVDR testing into clinical practice can help tailor treatment to individual needs, improving the likelihood of viral suppression and reducing the risk of resistance development. Exploring the use of newer antiretroviral drugs and drug classes, such as integrase inhibitors, can provide additional options for managing drug-resistant HIV.

In conclusion, while antiretroviral therapy has significantly reduced the mortality and morbidity associated with HIV-1 infection globally, the emergence of HIV drug resistance poses a critical threat to its continued effectiveness. Pakistan’s HIV epidemic, characterized by significant increases in new infections and challenges in achieving the 95-95-95 goals, highlights the need for comprehensive strategies to address HIVDR. Strengthening surveillance and monitoring systems, implementing routine HIVDR testing, and developing targeted interventions for key populations are essential steps toward controlling the epidemic and ensuring the sustainability of ART programs. The genetic diversity and evolving nature of HIV-1 in Pakistan underscore the importance of ongoing research and adaptation of treatment strategies to meet the needs of PLHIV and mitigate the impact of HIV drug resistance.

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