[PMC free article] [PubMed] [CrossRef] [Google Scholar] 72. had low-frequency (or minority) drug-resistant variants in the intrapatient HIV-1 population, which correlated with treatment failure. Moreover, the presence of these minority HIV-1 variants was associated with higher intrapatient HIV-1 diversity, suggesting a dynamic selection or fading of drug-resistant HIV-1 variants from the viral quasispecies in the presence or absence of drug pressure, respectively. This study identified low-frequency HIV drug resistance mutations by deep sequencing in Ugandan patients failing antiretroviral treatment but lacking dominant drug resistance mutations as determined by Sanger sequencing methods. We showed that these low-abundance drug-resistant viruses could have significant consequences for clinical outcomes, especially if treatment is not modified based on a susceptible HIV-1 genotype by Sanger sequencing. Therefore, we propose to make clinical decisions using more sensitive methods to detect minority HIV-1 variants. INTRODUCTION To date, 28 antiretroviral drugs from six drug classes have been approved for treatment of individuals infected with human immunodeficiency virus type 1 (HIV-1). Combinations of specific antiretroviral drugs are the basis for an effective therapy that suppresses viral replication, leading to partial immune reconstitution and considerable reduction in morbidity and mortality (1, 2). Together with prevention and educational efforts, antiretroviral treatment (ART) has been responsible for a worldwide reduction in AIDS-related deaths, as well as a 4-fold reduction in mother-to-child HIV-1 transmissions compared to the pretreatment era in Africa (3). Unfortunately, use of antiretroviral drugs in high-income countries (HICs) has also led to the emergence of HIV-1 drug resistance in many treated individuals (1). With high prevalence of HIV-1 drug resistance in the late 1990s/early 2000s, at least 10% of new infections were established by drug-resistant HIV-1 strains in HICs (4,C6). In low- to middle-income countries (LMICs), poor access to clinical care, intermittent supply of BBT594 antiretroviral drugs, and costs of travel to reach care providers result in suboptimal ART adherence, so that treatment failures and emergence of HIV-1 drug resistance have doubled in the last 10 years (6). With increased treatment access and high frequencies of treatment failures, HIV-1 with primary drug resistance is already found in 2% to 10% of treatment-naive individuals in Uganda, an increase of less than 1% to as high as 6.5% over the past 10 years (7,C11). Similar to other sub-Saharan countries, Uganda still has a high prevalence of people living with HIV-1 (approximately 2.1 million) (3), with BBT594 over 750,000 HIV-infected adults receiving combination antiretroviral therapy (cART) in 2014 (12). Ugandan patients have access to first-line cART, consisting of a combination of two nucleoside/nucleotide reverse transcriptase (RT) inhibitors (NRTIs) and one nonnucleoside reverse transcriptase inhibitor (NNRTI), commonly tenofovir (TDF) or zidovudine (AZT) plus lamivudine (3TC) or emtricitabine (FTC) and efavirenz (EFV) or nevirapine (NVP) (13). Individuals experiencing virologic failure may have limited access to second- and third-line cART regimens due to limited drug availability and high costs (14), highlighting the need to identify the reason(s) for treatment failure. Unfortunately, treatment monitoring using plasma HIV RNA (viral) load measurements is limited in resource-limited settings, and regimen switching is typically guided by CD4+ T-cell counts and various BBT594 clinical criteria. Access to HIV-1 genotyping to detect drug resistance is increasing but is often reserved for clinical studies and is rarely used as the standard of care (11, 15). HIV-1 genotypic (antiretroviral) testing based on population (Sanger) sequencing is currently the most common method to manage patients infected with HIV-1 (1, 16,C18); however, Sanger sequencing can detect only HIV-1 variants present at frequencies above 15% to 20% of the viral quasispecies (19,C23) and thus fails to quantify low levels of HIV-1 drug-resistant variants (18, 24). These variants are usually present as minority members of the virus population, which can be selected to dominate over drug-susceptible variants under drug pressure (25,C27). For this reason, a series of ultrasensitive HIV-1-genotyping assays, based on deep sequencing (next-generation sequencing [NGS]), have been developed to detect drug-resistant HIV-1 variants at levels below 20% of the viral population in an infected individual (24, 28,C31). Several studies have associated early detection of these minority HIV-1 drug-resistant variants with subsequent treatment failure (32,C37); however, with the advent of single-pill once-a-day (QD) cART regimens, treatment failures in HICs are.HIV-1 sequences were interpreted and drug resistance profiles were generated based on the HIVdb Program Genotypic Resistance Interpretation Algorithm from the Stanford University HIV Drug Resistance Database (http://hivdb.stanford.edu). (DeepGen) to quantify low-level drug-resistant HIV-1 variants in 33 patients failing a first-line antiretroviral treatment regimen in the absence of drug-resistant mutations, as screened by standard population-based Sanger sequencing. Using this sensitive assay, we observed that 64% (21/33) of these individuals had low-frequency (or minority) drug-resistant variants in the intrapatient HIV-1 population, which correlated with treatment failure. Moreover, the presence of these minority HIV-1 variants was associated with higher intrapatient HIV-1 diversity, suggesting a dynamic selection or fading of drug-resistant HIV-1 variants from the viral quasispecies in the presence or absence of drug pressure, respectively. This study identified low-frequency HIV drug resistance mutations by deep sequencing in Ugandan patients failing antiretroviral treatment but lacking dominant drug resistance mutations as dependant on Sanger sequencing strategies. We showed these low-abundance drug-resistant infections could possess significant implications for clinical final results, particularly if treatment isn’t modified predicated on a prone HIV-1 genotype by Sanger sequencing. As a result, we propose to create scientific decisions using even more delicate solutions to detect minority HIV-1 variations. INTRODUCTION To time, 28 antiretroviral medications from six medication classes have already been accepted for treatment of people contaminated with individual immunodeficiency trojan type 1 (HIV-1). Combos of particular antiretroviral medications will be the basis for a highly effective therapy that suppresses viral replication, resulting in partial immune system reconstitution and significant decrease in morbidity and mortality (1, 2). As well as avoidance and educational initiatives, antiretroviral treatment (Artwork) continues to be responsible for an international decrease in AIDS-related fatalities, and a 4-fold decrease in mother-to-child HIV-1 transmissions set alongside the pretreatment period in Africa (3). However, usage of antiretroviral medications in high-income countries (HICs) in addition has resulted in the introduction of HIV-1 medication resistance in lots of treated people (1). With high prevalence of HIV-1 medication level of resistance in the past due 1990s/early 2000s, at least 10% of brand-new infections were set up by drug-resistant HIV-1 strains in HICs (4,C6). In low- to middle-income countries (LMICs), poor usage of clinical treatment, intermittent way to obtain antiretroviral medications, and costs of happen to be reach care suppliers bring about suboptimal Artwork adherence, in order that treatment failures and introduction of HIV-1 medication resistance have got doubled within the last a decade (6). With an increase of treatment gain access to and high frequencies of treatment failures, HIV-1 with principal medication resistance has already been within 2% to 10% of treatment-naive people in Uganda, a rise of significantly less than 1% to up to 6.5% within the last a decade (7,C11). Comparable to various other sub-Saharan countries, Uganda still includes a high prevalence of individuals coping with HIV-1 (around 2.1 million) (3), with more than 750,000 HIV-infected adults receiving combination antiretroviral therapy (cART) in 2014 (12). Ugandan sufferers get access to first-line cART, comprising a combined mix of two nucleoside/nucleotide invert transcriptase (RT) inhibitors (NRTIs) and one nonnucleoside invert transcriptase inhibitor (NNRTI), typically tenofovir (TDF) or zidovudine (AZT) plus lamivudine (3TC) or emtricitabine (FTC) and efavirenz (EFV) or nevirapine (NVP) (13). People experiencing virologic failing may possess limited usage of second- and third-line cART regimens because of limited medication availability and high costs (14), highlighting the necessity to identify the reason why(s) for treatment failing. However, treatment monitoring using plasma HIV RNA (viral) insert measurements is bound in resource-limited configurations, and program switching is normally guided by Compact disc4+ T-cell matters and various scientific criteria. Usage of HIV-1 genotyping to detect medication resistance is raising but is frequently reserved for scientific studies and it is seldom used as the typical of treatment SLC3A2 (11, 15). HIV-1 genotypic (antiretroviral) examining based on people (Sanger) sequencing happens to be the most frequent solution to manage sufferers contaminated with HIV-1 (1, 16,C18); nevertheless, Sanger sequencing can detect just HIV-1 variations present at frequencies above 15% to 20% from the viral quasispecies (19,C23) and therefore does not quantify low degrees of HIV-1 drug-resistant variations (18, 24). These variations are often present as minority associates of the trojan people, which may be chosen to dominate over drug-susceptible variations under medication pressure (25,C27). Because of this, some ultrasensitive HIV-1-genotyping assays, predicated on deep sequencing (next-generation sequencing [NGS]), have already been created to detect drug-resistant HIV-1 variations at amounts below 20% from the viral people in an contaminated person (24, 28,C31). Many studies have linked early detection of the.
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