The development of Resistance to Antiretroviral Therapy for HIV Infected Individuals:a Systematic Review

Background : Antiretroviral treatment (ART) has reduced morbidity and mortality due to AIDS. However, treatment options can be impaired by the development of antiretroviral drug resistance. Resistant virus strains can be transmitted to new hosts and, subsequently, can lead to antiretroviral treatment failure. Methods : Th A systematic review of articles on resistance to antiretroviral therapy for HIV-infected individuals, published from January 1, 2014 to June 16, 2014, on SCOPUS and PUBMED databases wascarried out. Search terms were "AIDS" (Medical Subject Headings [MeSH]), "HIV" (MeSH), “treatment” (keyword), “resistance” (keyword) and " antiretroviral therapy" (MeSH). Of the 118 retrieved studies, 24 met the eligibility criteria. Results : The main classes of antiretroviral drugs are Non Nucleoside Reverse Transcriptase Inhibitors (NNRTIs), Nucleoside Reverse Transcriptase Inhibitors (NRTIs), and Protease Inhibitors (PIs). Antiretroviral treatment selects for drug resistance. Resistant strains become prevalent in the population and are transmitted to new patients. Discussion : A higher prevalence of certain mutations are present and characterize resistant strains that are prevalent in the population making it more diffiult to treat these infected individuals. Poor compliance with antiretroviral therapy increases the potential for developing these and other resistant strains. Conclusions : Knowledge of drug resistance generated by mutations is useful for optimizing treatment regimens and to get better responses. Promoting compliance is important to limiting the selection of new resistant strains.


Introduction
The wide use of combination antiretroviral therapy (cART), for example, succeeded in sustained inhibition of viral replication and reduced significantly the morbidity and mortality of HIV related disease. [1][2][3][4][5] However, treatment options have been impaired by the development of antiretroviral drug resistance. Resistant virus strains can develop in a patient but are likely to be acquired from others leading to antiretroviral treatment failure. [6,7] Antiretroviral therapy resistance is a concern not just for individual patients, but also for society due to the high potential transmission of drug-resistant HIV. This compromises the effectiveness of available first-line regimens for a substantial portion of the population and increases the need for costly second-line drugs. [8] This study is based on the following research questions: what are the main mutations conferring resistance to antiretroviral treatments and how do individuals acquire resistant strains? This issue has gained great impact in recent years with the constant and intense research on the many HIV antiretroviral therapies. Thus, this systematic review aims to present the main classes of drugs for ART and the resistance mechanisms that could lead to antiretroviral treatment failure.

Methods
We performed a qualitative systematic review of articles about the resistance to antiretroviral drugs used to treat HIV/AIDS in previously chosen electronic databases.
A search of the literature was conducted via Pub-Med and SCOPUS online databases in June 2014 and was limited to articles published from January 1, 2014 to June 16, 2014. The reason for limiting the search to 2014 was that several studies on the subject of HIV and resistance to antiretroviral therapies are performed every year, being necessary to recovery most recent data on resistance to antiretroviral drugs. Initially, the search terms browsed in SCOPUS database were: 1. "AIDS" (Medical Subject Headings [MeSH] term); 2. "HIV" ([MeSH]) 3. "treatment" (keyword). 4. "resistance" (keyword); and 5. "antiretroviral therapy" ([MeSH]) The following searches were performed: 1 OR 2 AND 3 AND 4 AND 5. In addition to MeSH terms, we opted to add the keywords "treatment" and "resistance" to the search strategy, because, despite not being included in the MeSH thesaurus, they are frequently used to describe studies that deal with the theme object of the present review. The search strategy and the retrieved articles were reviewed on two separate occasions to ensure adequate sampling. A similar search strategy was performed in the PubMed database, using the aforementioned terms and their correspondent terms.
The article analysis followed previously determined eligibility criteria. We adopted the following inclusion criteria: (1) references written in English; (2) studies pertaining resistance to antiretroviral therapy for HIVinfected individuals; (3) original articles with online accessible full text available in database SCOPUS,

Results
Initially, the aforementioned search strategies resulted in 118 references. After browsing the title and abstract of the retrieved citations for eligibility based on study inclusion criteria, 91 articles were excluded and 27 articles were further retrieved and included in the final sample ( Figure 1). Articles from SCOPUS and PubMed database matched the inclusion criteria of the present study. Table 1 provides an overview of all studies included in the final sample and of all data elements  Prevalence of resistance is projected to increase substantially. However, introduction of policies to increase ART coverage is not expected to lead to appreciably higher prevalence of HIV positive people with resistance and viral load more than 500 copies/ml. Concern over resistance should not stop expansion of treatment availability.

PRISMA 2009 Flow Diagram
Fall-Malick, F-Z et al. [10] Journal of Medical Virology Eighty-six subjects were included and 65 samples were amplified successfully and sequenced.
Phylogenetic analysis revealed 17 HIV-1 variants with the predominance of CRF02_AG (n=42; 64.6%). A high rate of DRM was found in this study and shows the potential need for a structured virological surveillance including viral load quantification and genotyping. Further studies may also be needed in regards to the great variability of HIV-1 strains in Mauritania.
Mulu, A. et al. [15] BMC Infectious Diseases Consecutive HIV-1 infected adults (N = 100) and children (N = 100) who have been receiving ART for up to 6 years at Gondar University Hospital, Ethiopia.
Majority of both adults (82%) and children (87%) who received ART showed high viral suppression and immunological recovery. This indicates that despite limited resources in the setting virological efficacy can be sustained for a substantial length of time and also enhance immunological recovery irrespective of age. However, the presence of drug resistance mutations and low level viraemia among clinically asymptomatic patients highlights the need for virological monitoring. Baesi Two hundred sixty five patients had VL data available at baseline and at 6 months.
Our data confirm that the currently recommended first-line ART regimen is efficient in the vast majority of individuals initiating therapy in Jimma, Ethiopia eight years after the introduction of ART. However, the documented occurrence of transmitted resistance and accumulation of acquired HIVDR mutations among failing patients justify increased vigilance by improving the availability and systematic use of VL testing to monitor ART response, and underlines the need for rapid, inexpensive tests to identify the most common drug resistance mutations. Strong evidence for consistent HIV-1C clade homogeneity and low influx of other variant into the country was found. The level of drug resistance observed in chronically infected treatment naïve patients which exceeds the WHO estimates suggests the need for incorporation of HIV-1 drug resistance testing prior to ART initiation. The occurrence of monophyletic transmission clusters affecting (24/160) individuals indicates their potential risk related practice. Thus, an intensified public health intervention program and monitoring of HIV drug resistance testing appears indispensable.

Journal of Infection in Developing Countries
Fifty-four HIV-1 strains isolated from treatmentnaive patients (n = 54) were included in this study.
This study confirms that HIV-1 subtype C predominates in northern India. Protease secondary mutations associated with drug resistance to protease inhibitors (PIs) were present with high frequency in the HIV-1 C subtype strains isolated from north Indian ARV treatment-naive patients, but no primary resistance mutations were found in this region.

Ross, LL. et al. [41]
PLoS ONE Viral transmission clusters (VTCs) were initially predicted from a phylogenetic analysis of population level HIV-1 pol sequences obtained from 690 antiretroviral-naïve subjects in 2007.
Our analysis shows specific geographical and drug resistance trends that correlate well with transmission clusters defined by HIV sequences of similarity. Furthermore, our study demonstrates the utility of molecular and epidemiological analysis of VTCs for identifying population-specific risks associated with HIV-1 transmission and developing effective local healthcare strategies.
Low frequency DRMs detected before ART initiation and at VF in patients experiencing VF on first-line ART can increase the overall burden of resistance to PI, NRTI and NNRTI.
Antunes DA. et al. [44] PLoS One. Bioinformatics tools were used to evaluate the impact of the unusual mutations D30V and V32E over the dynamics of the PR-Nelfinavir complex.
The D30V mutation triggered a subtle change in the PR structure, which was also observed for the wellknown. Nelfinavir resistance mutation D30N, while the V32E exchange presented a much more dramatic impact over the PR flap dynamics. Journal of Infectious Diseases Using prospectively collected data from patients in South Africa(417).
No adherence, suggested by subtherapeutic ART with/without major resistance mutations, significantly contributed to failure when switching regimen. Unresolved no adherence, not NRTI resistance, drives early second-line failure.
The structural correlation of natural polymorphisms and unusual mutations with drug resistance is useful for the identification of HIV-1 variants with potential resistance to PIs. The D29V mutation likely confers a selection advantage in viruses; however, in silico, presence of this mutation results in unstable enzyme/PI complexes, that possibly induce resistance to PIs.

Megens, S. et al. [48]
Virology Origin and the effect of insertion D67D-THGERDLGPA within HIV-1 RT from a patient failing antiviral therapy.
These results suggest that a particular sequence within human chromosome 17 is prone to horizontal gene transfer into the HIV-1 RT finger subdomain. This insertion confers selective advantage to HIV-1 by its contribution to multi-drug resistance and restoration of impaired replication capacity. Educational level and social support against HIV-related stigma and discrimination were not significantly associated with adherence. Adherence to antiretroviral therapy among the respondents is very poor. Urgent interventions based on modifiable factors and mainly targeting females and younger age group are needed to improve adherence to antiretroviral therapy among people living with HIV.
Bulteel, N. et al. [66] Journal of Infection 5455 patients received either (or both) 3TC, TDF and EFV or FTC, TDF and EFV contributing 6465 treatment episodes over 9962 person-years follow up.
We have not found evidence of an increased risk of development of M184V and K65R in patients exposed to 3TC.

Discussion
The increased use of antiretroviral therapy has reduced significantly the number of deaths related to HIV/AIDS and improved the life expectancy of people living with HIV. [10] It has been suggested that scaling up of ART results in a level of virological suppression at the population level that will reduce HIV transmission. [11][12][13][14][15] With the increased numbers of patients being treated, there is increased potential for development and spread of resistant virus. Individuals infected with HIV acquire antiretroviral resistance in one of two ways: (1) transmitted drug resistance (TDR) occurs when antiretroviralnaive individuals are infected with viruses already harboring resistance mutations, and (2) acquired drug resistance (ADR) emerges from the selective pressure of antiretrovirals in individuals without fully suppressed viral loads either because of poor adherence or the use of a non-suppressive regimen. [16,17] As access to antiretroviral therapy (ART) expands in resource-limited countries, concerns will increase regarding the numbers of patients failing treatment and the subsequent emergence of drugresistant viruses. Resistant virus selected during treatment may subsequently be transmitted to newly infected individuals, undermining the effectiveness of currently recommended or available first-line ART. [18,19] Also, factors associated with the development of drug resistance include poor adherence, suboptimal ART doses, and initiation of therapy late in the course of HIV infection. [20,21] Highly active antiretroviral therapy (HAART) has been shown to reduce significantly the mortality and morbidity of patients infected with HIV at various disease stages. [2, 22-25] Although HAART may limit the extent of selection during treatment, the success of HAART could still be compromised by the emergence of HIV drug resistance. [10,26] Resistance can be acquired as the pool of variants within the population increases. [19] Resistance to an antiretroviral drug class is defined by the presence of at least one primary resistance mutation (PRM), as included in the mutation list paneled by the International Antiviral Society in 2013 [27], within the genes for the nucleos(t)ide reverse transcriptase inhibitors (NRTIs), non nucleos(t)ide reverse transcriptase inhibitors (NNRTIs), and protease inhibitors (PIs). [28] Viral replication under suboptimal antiretroviral pressure leads to accumulation of resistance mutations. As the number of mutants increases, future therapeutic choices become more limited [29] especially with regard to monotherapy. In addition, mutations conferring resistance to one drug frequently confer cross resistance to other antiretroviral drugs within the same class. [30, 31] The significance of drug-specific or drug class-specific transmitted drug resistance to treatment outcome is not fully understood, especially when one or more drugs or drug classes in the regimen are still effective. [32] Non nucleoside reverse transcriptase inhibitors (NNRTIs) are important and common components of highly active antiretroviral therapy (HAART). Rilpivirine, a second-generation NNRTI, is active against wild-type viruses and retains activity against some NNRTI-resistant HIV-1 strains. [33-35] This drug is currently indicated in the USA and Europe in combination with other antiretroviral drugs for the treatment of antiretroviral-naive HIV-1-infected patients with (HIV-1 RNA≤100000 copies/mL).
[48], a virus strain without the insertion already displayed some NRTI resistance due to the TAM1 mutations and the level of resistance was strongly enhanced after the addition of the insertion. Surprisingly, NNRTI resistance was observed in the absence of major NNRTIassociated mutations and the level of resistance was further elevated after the selection of strains with K103N and Y181C mutations. [54] Ibrahim et al. [55] also showed, in a health facilitybased cross sectional study, that poor adherence to antiretroviral therapies could have serious consequences for individuals who are infected with HIV including failure of prevention of viral replication, an increased possibility of developing viral resistance, the progression of the disease to stages of clinical complications, and reduction of survival. [55][56][57][58] Factors associated with poor adherence based on the findings of this study include gender, age, employment, marital status, and health status. [55] The results reveal that females, younger people and unemployed individuals were more likely to be associated with poor adherence to antiretroviral therapy. Lack of support from either the family or community is also an important risk factor for poor adherence to antiretroviral therapy. [55] Moreover, low adherence to ART is associated with an increased risk of resistance to HIV-1 reverse transcriptase inhibitors (RTIs). The emergence of drug resistant virus limits antiretroviral choice due to cross-resistance to other antiretroviral agents [59,60] and is strongly associated with progression of HIV-1 infection and increased mortality. [61][62][63][64][65][66] Choi et al. [67] assumed that the earlier that patients were diagnosed and treated, the higher the level of drug resistance that would develop in them. Based on the treatment history of these patients, mutations giving resistance to the extensively used NRTIs were more prevalent than to PIs. [67] As the efficacy of the combination antiretroviral therapy (cART) decreased, the number of patients at risk to resistant virus increased with a possible concomitant increase in risk of TDR. [7] The consequences of prolonged continuation of non suppressive cART were observed in a cross-sectional study conducted by Mutwa et al. [68] Their genotypic results, with 90% of children with virologic failure and available genotyping determinations, showed the development of major NRTIs and NNRTI-associated resistance mutations. [68][69][70][71][72] A major obstacle to the long-term efficacy of ART is the emergence of drug resistance mutations in the polymerase gene of HIV-1, the primary target for ART. This reduces anti-retroviral (ARV) drug susceptibility [73] and limits the therapeutic options of newly infected patients. [74] The transmission of resistant variants to uninfected individuals has become a serious clinical and public-health concern.
[73] Knowledge of prevalent drug resistant variants is useful for optimizing therapeutic schemes and obtaining a better patient response. [21,75] Despite stable ART, many patients experience episodes of low-level viremia (LLV), defined as plasma viral load (pVL) measurements between 50 and 1000 HIV-1 RNA copies/mL. This low level of virus production in the presence of ART increases the potential for mutants to arise and their selection. An increased risk of virologic failure has been associated with episodes of LLV in several studies, [74][75][76][77][78][79] but not in others. [80][81][82] Finally, it is important to expand the surveillance for antiretroviral resistance amongst these patients as well as treatment naive patients in order to facilitate the early detection of transmitted drug resistant strains to individual patients as well as the population. [25].

Conclusion
Recent findings reveal that the development of resistance to antiretroviral therapy by HIV-infected patients is associated with increased access to drugs used in the treatment, and with low adherence. The appearance of these resistant strains is associated with specific mutations that not only reduce the effect of the drug class in use on the patient but also provide the emergence of cross-resistance with other classes. Poor adherence to treatment, the use of sub-optimal doses and treatments initiated too late for efficacy provide conditions that allow the emergence of resistant mutations classes of drugs currently used in treatment of HIV/AIDS as NNRTIs, NRTIs and PI's and contribute to the reduction of effectiveness of treatment.
Knowledge of the mutations that confer drug resistance is useful for optimizing treatment regimens. Selection of resistant viruses by poor adherence to antiretroviral treatment and the acquisition of resistant viruses are important factors for the failure of antiretroviral treatment. With knowledge of the prevalent mutations conferring resistance to the antiHIV drugs, alternative drugs can be used. As