Presence Of Drug Resistance Mutations Research Articles

High Rates of Drug Resistance Among Newly Diagnosed HIV-infected Children in the National Prevention of Mother-to-child Transmission Program in Togo

Prevention of mother-to-child transmission (PMTCT) programs have been largely scaled-up, but data on infant HIV drug resistance from PMTCT programs implemented in resource-limited countries are lacking. Remnant dried blood spots from HIV-infected children (aged <18 months) tested through the Togo national early infant diagnosis program during 2012 and 2013 were collected and assessed for HIV drug resistance. Pol-RT (reverse transcriptase) region was amplified, sequenced and analyzed for the presence of drug resistance mutations (DRMs). Overall, 121 of 201 (60.2%) newly diagnosed children had detectable DRMs. Among the 131 of 201 (65.2%) children with reported exposure to maternal and/or infant antiretrovirals (ARVs), DRMs were detected in 99 children (75.6%). Importantly, in 41 of 201 children for whom no exposure to ARVs was reported, DRMs were detected in 11 children (26.8%). For 29 children, no data on ARV exposure were available. For the 121 of 201 children with DRMs, 99 of 121 (81.8%) had only nonnucleoside reverse transcriptase inhibitor DRMs detected but 21 of 121 (17.3%) had both nonnucleoside reverse transcriptase inhibitor and nucleoside reverse transcriptase inhibitor (NRTI) DRMs. Among breast-fed children, drug resistance was more frequent when mothers were on antiretroviral therapy (ART), 61 of 75 (81.3%) versus 14 of 39 (35.9%) when mothers were not on ART (P < 0.001). Nucleoside reverse transcriptase inhibitor resistance was more common when mothers were on ART. Scale-up and improvement of PMTCT strategies resulted in a global decrease of pediatric HIV infections, but our study shows high rates of drug resistance in infants for whom prevention failed.

Should viral load thresholds be lowered?: Revisiting the WHO definition for virologic failure in patients on antiretroviral therapy in resource-limited settings.

The World Health Organization (WHO) guidelines on antiretroviral therapy (ART) define treatment failure as 2 consecutive viral loads (VLs) ≥1000 copies/mL. There is, however, little evidence supporting 1000 copies as an optimal threshold to define treatment failure. Objective of this study was to assess the correlation of the WHO definition with the presence of drug-resistance mutations in patients who present with 2 consecutive unsuppressed VL in a resource-limited setting.In 10 nurse-led clinics in rural Lesotho children and adults on first-line ART for ≥6 months received a first routine VL. Those with plasma VL ≥80 copies/mL were enrolled in a prospective study, receiving enhanced adherence counseling (EAC) and a follow-up VL after 3 months. After a second unsuppressed VL genotypic resistance testing was performed. Viruses with major mutations against ≥2 drugs of the current regimen were classified as "resistant".A total of 1563 adults and 191 children received a first routine VL. Of the 138 adults and 53 children with unsuppressed VL (≥80 copies/mL), 165 (116 adults; 49 children) had a follow-up VL after EAC; 108 (74 adults; 34 children) remained unsuppressed and resistance testing was successful. Ninety of them fulfilled the WHO definition of treatment failure (both VL ≥1000 copies/mL); for another 18 both VL were unsuppressed but with <1000 copies/mL. The positive predictive value (PPV) for the WHO failure definition was 81.1% (73/90) for the presence of resistant virus. Among the 18 with VL levels between 80 and 1000 copies/mL, thereby classified as "non-failures", 17 (94.4%) harbored resistant viruses. Lowering the VL threshold from 1000 copies/mL to 80 copies/mL at both determinations had no negative influence on the PPV (83.3%; 90/108).The current WHO-definition misclassifies patients who harbor resistant virus at VL below 1000 c/mL as "nonfailing." Lowering the threshold to VL ≥80 copies/mL identifies a significantly higher number of patients with treatment-resistant virus and should be considered.

Identification of Immunogenic Cytotoxic T Lymphocyte Epitopes Containing Drug Resistance Mutations in Antiretroviral Treatment-Naïve HIV-Infected Individuals.

BackgroundTherapeutic HIV vaccines may prove helpful to intensify antiretroviral treatment (ART) efficacy and may be an integral part of future cure strategies.MethodsWe examined IFN-gamma ELISpot responses to a panel of 218 HIV clade B consensus-based HIV protease-reverse transcriptase peptides, designed to mimic previously described and predicted cytotoxic T lymphocyte epitopes overlapping drug resistance (DR) positions, that either included the consensus sequence or the DR variant sequence, in 49 ART-naïve HIV-infected individuals. Next generation sequencing was used to assess the presence of minority DR variants in circulating viral populations.ResultsAlthough a wide spectrum of differential magnitudes of response to DR vs. WT peptide pairs was observed, responses to DR peptides were frequent and strong in the study cohort. No difference between the median magnitudes of response to DR vs. WT peptides was observed. Interestingly, of the 22 peptides that were recognized by >15% of the participants, two-thirds (64%) corresponded to DR peptides. When analysing responses per peptide pair per individual, responses to only WT (median 4 pairs/individual) or DR (median 6 pairs/individual) were more common than responses to both WT and DR (median 2 pairs/individual; p<0.001). While the presence of ELISpot responses to WT peptides was frequently associated with the presence of the corresponding peptide sequence in the patient’s virus (mean 68% of cases), responses to DR peptides were generally not associated with the presence of DR mutations in the viral population, even at low frequencies (mean 1.4% of cases; p = 0.0002).ConclusionsOur data suggests that DR peptides are frequently immunogenic and raises the potential benefit of broadening the antigens included in a therapeutic vaccine approach to immunogenic epitopes containing common DR sequences. Further studies are needed to assess the quality of responses elicited by DR peptides.

Ribonuclease H/DNA Polymerase HIV-1 Reverse Transcriptase Dual Inhibitor: Mechanistic Studies on the Allosteric Mode of Action of Isatin-Based Compound RMNC6.

The DNA polymerase and ribonuclease H (RNase H) activities of human immunodeficiency virus type 1 (HIV-1) are needed for the replication of the viral genome and are validated drug targets. However, there are no approved drugs inhibiting RNase H and the efficiency of DNA polymerase inhibitors can be diminished by the presence of drug resistance mutations. In this context, drugs inhibiting both activities could represent a significant advance towards better anti-HIV therapies. We report on the mechanisms of allosteric inhibition of a newly synthesized isatin-based compound designated as RMNC6 that showed IC50 values of 1.4 and 9.8 μM on HIV-1 RT-associated RNase H and polymerase activities, respectively. Blind docking studies predict that RMNC6 could bind two different pockets in the RT: one in the DNA polymerase domain (partially overlapping the non-nucleoside RT inhibitor [NNRTI] binding pocket), and a second one close to the RNase H active site. Enzymatic studies showed that RMNC6 interferes with efavirenz (an approved NNRTI) in its binding to the RT polymerase domain, although NNRTI resistance-associated mutations such as K103N, Y181C and Y188L had a minor impact on RT susceptibility to RMNC6. In addition, despite being naturally resistant to NNRTIs, the polymerase activity of HIV-1 group O RT was efficiently inhibited by RMNC6. The compound was also an inhibitor of the RNase H activity of wild-type HIV-1 group O RT, although we observed a 6.5-fold increase in the IC50 in comparison with the prototypic HIV-1 group M subtype B enzyme. Mutagenesis studies showed that RT RNase H domain residues Asn474 and Tyr501, and in a lesser extent Ala502 and Ala508, are critical for RMNC6 inhibition of the endonuclease activity of the RT, without affecting its DNA polymerization activity. Our results show that RMNC6 acts as a dual inhibitor with allosteric sites in the DNA polymerase and the RNase H domains of HIV-1 RT.

Prevalencia de la resistencia genotípica a los fármacos antirretrovirales en pacientes VIH positivos de Tegucigalpa

El objetivo de la investigación fue determinar la prevalencia de resistencia del VIH a las drogas antirretrovirales en pacientes tratados y no tratados. Para tal efecto, se analizaron 100 muestras de pacientes VIH positivos, 50 previos a iniciar tratamiento y 50 bajo tratamiento con evidencia de falla terapéutica, de Tegucigalpa.Secuencias del gen pol del VIH-1 fueron generadas para determinar la presencia de mutaciones asociadas a resistencia, utilizando la herramienta calibrada para la vigilancia de la resistencia propuesta por la OMS para pacientes pretratados y la lista de mutaciones de la base de datos de Stanford para pacientes bajo tratamiento.Los resultados muestran que la prevalencia de farmacorresistencia en pacientes previos a iniciar tratamiento fue del 19 % [IC 95 %: 9-33 %], el 8.3 % presentaron mutaciones contra los NRTI, el 12.5 % contra los NNRTI y el 6.25 % contra los PI. Las mutaciones con mayor frecuencia fueron: M184V, K103N, M46I. La prevalencia de mutaciones asociadas a resistencia en los pacientes en falla terapéutica fue del 82 % [IC 95 %:60-90 %], el 58 % contra los NRTI, 74 % contra los NNRTI y el 22 % contra los PI.En conclusión, se observó un marcado aumento en la prevalencia de mutaciones asociadas a resistencia en pacientes no tratados, comparada con estudios anteriores, lo cual es de mucha preocupación, ya que limita la eficacia del tratamiento de primera línea. Aunque la prevalencia de resistencia adquirida en pacientes con falla terapéutica se mantiene ligeramente constante, sigue siendo alta, requiriendo de cambios de tratamiento a segunda o tercera línea en estos pacientes, ocasionando un impacto económico en la salud pública.Revista Ciencia y Tecnología, N° 15, diciembre 2014: 147-160

Lamivudine Resistance and Precore Variants in Iranian Patients With Chronic Hepatitis B: Correlation With Virological and Clinical Features.

Background:Long-term lamivudine therapy, despite its initial effectiveness against hepatitis B virus (HBV), is associated with the emergence of drug resistance mutations in polymerase protein.Objectives:The aim of the present study was to determine the prevalence of precore and lamivudine drug resistance mutations in lamivudine treated patients with chronic B hepatitis.Patients and Methods:Sequential sera were obtained from 88 chronic HBV carriers who received lamivudine for more than 24 months. Polymerase and precore regions were directly sequenced for these groups: I (before treatment), II, and III (12 and 24 months after treatment, respectively).Results:All patients (100%) were contained genotype D, subtype ayw2. One (1.1%), 12 (13.6%), and 22 (25%) members of groups I, II, and III had the replacement of either isoleucine or valine instead of methionine in tyrosine-methionine-aspartate-aspartate (YMDD) motif, respectively. The frequency of mutations from 0 time point to 12 and 24 months showed that there was an increasing trend between sequential samples (P < 0.001). In group I, 31 (35.2%); II, 36 (41.0%) and III, 41 (46.6%) members had the precore stop codon mutations. The frequency of mutations from 0 time point to 12 and 24 months showed that there was an ascending trend between sequential samples. Indeed, frequency of precore stop codon was significantly increased with the passage of time (P < 0.001).Conclusions:Presence of drug resistance mutations among the patients was significant. Precore mutations were common amongst Iranian HBV chronic carriers under lamivudine therapy and these mutations were accompanied by clinical relapse.

Use of Dried Plasma Spots for HIV-1 Viral Load Determination and Drug Resistance Genotyping in Mexican Patients.

Monitoring antiretroviral therapy using measurements of viral load (VL) and the genotyping of resistance mutations is not routinely performed in low- to middle-income countries because of the high costs of the commercial assays that are used. The analysis of dried plasma spot (DPS) samples on filter paper may represent an alternative for resource-limited settings. Therefore, we evaluated the usefulness of analyzing DPS samples to determine VL and identify drug resistance mutations (DRM) in a group of HIV-1 patients. The VL was measured from 22 paired plasma and DPS samples. In these samples, the average VL was 4.7 log10 copies/mL in liquid plasma and 4.1 log10 copies/mL in DPS, with a correlation coefficient of R = 0.83. A 1.1 kb fragment of HIV pol could be amplified in 14/22 (63.6%) of the DPS samples and the same value was amplified in plasma samples. A collection of ten paired DPS and liquid plasma samples was evaluated for the presence of DRM; an excellent correlation was found in the identification of DRM between the paired samples. All HIV-1 pol sequences that were obtained corresponded to HIV subtype B. The analysis of DPS samples offers an attractive alternative for monitoring ARV therapy in resource-limited settings.

Drug resistance is rarely the cause or consequence of long-term persistent low-level viraemia in HIV-1-infected patients on ART.

The introduction of highly sensitive HIV-1 viral load assays with a lower quantification limit of 20 copies/ml uncovered that in a number of patients on ART, the viral load systematically fluctuates around or slightly above the detection limit of the assays. This study aimed to analyse the presence or occurrence of drug resistance mutations in HIV-1-infected patients during long-term persistent low-level viraemia (PLLV) under ART. A retrospective study was carried out in which baseline and on-therapy presence of drug resistance mutations in the HIV-1 protease and reverse transcriptase genes were analysed in patients with PLLV between 20 and 250 copies/ml. For all available plasma samples collected during PLLV, resistance analysis was attempted with an ultrasensitive amplification and sequencing protocol. Resistance analysis was successful for 154 samples collected longitudinally from 23 patients over a median period of 4.7 years (IQR 3.3-5.7). Twenty of these patients were on a boosted protease inhibitor (PI)-based regimen (87%). Single drug resistance mutations were detected in isolated samples of 4 patients, 2 of the 3 patients who initiated a non-nucleoside reverse transcriptase inhibitor-based regimen and 2 of the 20 on a PI-based regimen. Only one of the detected mutations decreased susceptibility to the therapy regimen taken at the time of sample collection. Drug resistance mutations were not found in the three patients who developed virological failure (viral load >250 copies/ml) during the study. Long episodes of PLLV in patients on boosted PI-based regimens rarely result in the selection of new drug-resistant variants.

Increased replicative fitness can lead to decreased drug sensitivity of hepatitis C virus.

Passage of hepatitis C virus (HCV) in human hepatoma cells resulted in populations that displayed partial resistance to alpha interferon (IFN-α), telaprevir, daclatasvir, cyclosporine, and ribavirin, despite no prior exposure to these drugs. Mutant spectrum analyses and kinetics of virus production in the absence and presence of drugs indicate that resistance is not due to the presence of drug resistance mutations in the mutant spectrum of the initial or passaged populations but to increased replicative fitness acquired during passage. Fitness increases did not alter host factors that lead to shutoff of general host cell protein synthesis and preferential translation of HCV RNA. The results imply that viral replicative fitness is a mechanism of multidrug resistance in HCV. Importance: Viral drug resistance is usually attributed to the presence of amino acid substitutions in the protein targeted by the drug. In the present study with HCV, we show that high viral replicative fitness can confer a general drug resistance phenotype to the virus. The results exclude the possibility that genomes with drug resistance mutations are responsible for the observed phenotype. The fact that replicative fitness can be a determinant of multidrug resistance may explain why the virus is less sensitive to drug treatments in prolonged chronic HCV infections that favor increases in replicative fitness.

Prevalence of drug resistance in human immunodeficiency virus type 1-infected treatment-naive children in Pune, India.

Abstract We report the presence of drug resistance mutations in 7.4% (2/27) of the treatment-naive, HIV-1-infected children in Pune, India, who had HIV-1 RNA levels >1,000 copies/ml. Nonnucleoside reverse transcriptase inhibitor (NNRTI) mutations, namely A98G and K103N, were observed in two separate sequences. In addition, three study sequences displayed three separate HIV-1 protease minor resistance mutations-L10I, A71T, and T74S. These preliminary data from Pune, India, reporting the presence of HIVDR in treatment-naive, HIV-1-infected children, reinforces the need to conduct large-scale studies to assess the prevalence of primary HIVDR in the pediatric population, which in turn will aid in planning protocols and policies related to antiretroviral treatment for the pediatric population.

Virological efficacy and immunological recovery among Ethiopian HIV-1 infected adults and children

BackgroundIntroduction of antiretroviral therapy (ART) in sub-Saharan Africa was a hot debate due to many concerns about adherence, logistics and resistance. Currently, it has been significantly scaled up. However as the WHO clinico-immunological approaches for initiation and monitoring of ART in the region lacks viral load determination and drug resistance monitoring, HIV infected adults and children may be at risk for “unrecognized” virologic failure and the subsequent development of antiretroviral drug resistance. This study evaluates the virological efficacy and immunological recovery of HIV/AIDS patients under ART.MethodsConsecutive 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 were enrolled following the WHO protocol for assessment of acquired drug resistance. Magnitude of viral suppression, genotypic drug resistance mutations and patterns of CD4+ T cell recovery were determined using standard virological and immunological methods.ResultsVirological suppression (HIV RNA < 40 copies/ml) was observed in 82 and 87% of adults and children on a median time of 24 months on ART, respectively. Mutation K103N conferring resistance to non nucleoside reverse transcriptase inhibitors and thymidine analogue mutations (M41L, L210W) were found only in one adult and child patient, respectively. Median CD4+ T cell count has increased from baseline 124 to 266 (IQR: 203–306) and 345 (IQR: 17–1435) to 998 (IQR: 678–2205) cells/mm3 in adults and children respectively after 12 months of ART. Nevertheless, small but significant number of clinically asymptomatic adults (16%) and children (13%) had low level viraemia (HIV-1 RNA 41–1000 copies/ml).ConclusionsMajority 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.

Sequence and structure based models of HIV-1 protease and reverse transcriptase drug resistance.

BackgroundSuccessful management of chronic human immunodeficiency virus type 1 (HIV-1) infection with a cocktail of antiretroviral medications can be negatively affected by the presence of drug resistant mutations in the viral targets. These targets include the HIV-1 protease (PR) and reverse transcriptase (RT) proteins, for which a number of inhibitors are available on the market and routinely prescribed. Protein mutational patterns are associated with varying degrees of resistance to their respective inhibitors, with extremes that can range from continued susceptibility to cross-resistance across all drugs.ResultsHere we implement statistical learning algorithms to develop structure- and sequence-based models for systematically predicting the effects of mutations in the PR and RT proteins on resistance to each of eight and eleven inhibitors, respectively. Employing a four-body statistical potential, mutant proteins are represented as feature vectors whose components quantify relative environmental perturbations at amino acid residue positions in the respective target structures upon mutation. Two approaches are implemented in developing sequence-based models, based on use of either relative frequencies or counts of n-grams, to generate vectors for representing mutant proteins. To the best of our knowledge, this is the first reported study on structure- and sequence-based predictive models of HIV-1 PR and RT drug resistance developed by implementing a four-body statistical potential and n-grams, respectively, to generate mutant attribute vectors. Performance of the learning methods is evaluated on the basis of tenfold cross-validation, using previously assayed and publicly available in vitro data relating mutational patterns in the targets to quantified inhibitor susceptibility changes.ConclusionOverall performance results are competitive with those of a previously published study utilizing a sequence-based strategy, while our structure- and sequence-based models provide orthogonal and complementary prediction methodologies, respectively. In a novel application, we describe a technique for identifying every possible pair of RT inhibitors as either potentially effective together as part of a cocktail, or a combination that is to be avoided.

Drug‐resistance development differs between HIV‐1‐infected patients failing first‐line antiretroviral therapy containing nonnucleoside reverse transcriptase inhibitors with and without thymidine analogues

We evaluated the emergence of drug resistance in patients failing first-line regimens containing one nonnucleoside reverse transcriptase inhibitor (NNRTI) administered with zidovudine (ZDV) + lamivudine (the ZDV group) or non-thymidine analogues (non-TAs) (tenofovir or abacavir, + lamivudine or emtricitabine; the non-TA group). Three hundred HIV-1-infected patients failing a first-line NNRTI-containing regimen (nevirapine, n = 148; efavirenz, n = 152) were included in the analysis. Virological failure was defined as viraemia ≥ 400 HIV-1 RNA copies/mL for the first time at least 6 months after starting the NNRTI-based regimen. For each patient, a genotypic resistance test at failure was available. The presence of drug-resistance mutations in HIV-1 reverse transcriptase was evaluated by comparing patients treated with NNRTI + zidovudine + lamivudine vs. those treated with NNRTI + non-TA. A total of 208 patients were failing with NNRTI + zidovudine + lamivudine and 92 with NNRTI + non-TA. No significant differences were observed between the non-TA group and the ZDV group regarding the time of virological failure [median (interquartile range): 12 (8-25) vs. 13 (9-32) months, respectively; P = 0.119] and viraemia [median (interquartile range): 4.0 (3.2-4.9) vs. 4.0 (3.3-4.7) log₁₀ copies/mL, respectively; P = 0.894]. Resistance to reverse transcriptase inhibitors (RTIs) occurred at a significant lower frequency in the non-TA group than in the ZDV group (54.3 vs. 75.5%, respectively; P = 0.001). This difference was mainly attributable to a significantly lower prevalence of NNRTI resistance (54.3 vs. 74.0%, respectively; P = 0.002) and of the nucleoside reverse transcriptase inhibitor (NRTI) mutation M184V (23.9 vs. 63.5%, respectively; P < 0.001) in the non-TA group compared with the ZDV group. As expected, the mutation K65R was found only in the non-TA group (18.5%; P < 0.001). At first-line regimen failure, a lower prevalence of RTI resistance was found in patients treated with NNRTI + non-TA compared with those treated with NNRTI + zidovudine + lamivudine. These results confirm that the choice of backbone may influence the prevalence of drug resistance at virological failure.

428 VERTICAL HEPATITIS B VIRUS TRANSMISSION DESPITE SEROVACCINATION OF THE NEWBORN IN HIGHLY VIRAEMIC MONO-INFECTED MOTHERS FROM VARIOUS ETHNIC ORIGINS: A RETROSPECTIVE STUDY IN PARIS, FRANCE

4/8 (50%) rapidly developed lamivudine-resistance mutations. Patients with HBV-reactivation were characterized by a degree of HBsAg-variability higher than that in stably-HBV-infected patients, particularly in a-determinant critical for modulating HBVinfectivity and evasion from neutralizing-antibodies (mean geneticdivergence: 0.058 versus 0.017, P < 0.001). Furthermore, both the percentage of patients with at least 1 immune-escape mutation, and the median number of immune-escape mutations in a-determinant, were significantly higher in patients with HBV-reactivation than in controls (64.3%[9/14] versus 27%[27/100], P = 0.008; and 1[IQR: 0–2] versus 0[IQR: 0–2], P = 0.002, respectively). Moreover, ≥1 specific HBsAg-mutation (M103I-T123N-S143L-D144E-S154L-V190A) was present in 8/14 (57.1%) patients with HBV-reactivation status (independently from the presence of drug-resistance mutations), while in controls they were absent (0/100 for M103I-T123ND144E-S154L), or nearly absent (1/100 for S143L and 2/100 for V190A). All the identified mutations (except for V190A, situated in the membrane-embedded C-terminal domain) are localized in a-determinant, and are known to be associated with decreased HBsAg-antigenicity. T123N introduces an N-linked glycosylationsite, thus potentially reducing HBsAg immunogenic-surface. Conclusions: HBV-reactivation correlates with an increased HBV ability to evade the pressure imposed by humoral immuneresponse. This highlights the need of a careful monitoring, and of establishing an adequate, high-genetic-barrier therapy (if required) in order to prevent HBV-reactivation and consequent liver damage in the setting of immune-suppression.

Characterization of antiviral resistance mutations among the Eastern Indian Hepatitis B virus infected population

BackgroundAntiviral therapy using nucleos(t)ide analogues (NAs) is an effective control measure of chronic hepatitis B virus (HBV) infection; however they need long term treatment. Presence of drug-resistance mutations may get in the way of the efficacy of antiviral therapy. Our study was aimed at defining the prevalence of HBV drug-resistance in HBVrt region in a population of 147 HBsAg positive patients.FindingsHBV/D has shown multiple types of HBVrt mutations both among treatment naïve (65.0%, 13 of 20 HBV/D) and treated patients (56.2%, 9 of 16 HBV/D). In additional, several mutations, with a suggested role in drug resistance, were detected among the treatment naïve as well as the treated patients. The mutations reported to be involved in reduction of drug effectiveness, was common among non-responders to therapy as well as among the naïve patients. Notably, classical antiviral resistance mutations (rtL80I/V-rtI169T-rtV173L-rtL180M-rtA181T/V/S-rtT184A/S/G/C-rtA194T-rtS202C /G/I -rtM204V/I-rtN236T-rtM250V) were not detected.ConclusionThe prevalence of putative NAr mutations among non responders to therapy suggests that they might have role in reduced efficacy of currently available antivirals and requires further investigations.

Characterizing the emergence and persistence of drug resistant mutations in HIV-1 subtype C infections using 454 ultra deep pyrosequencing.

BackgroundThe role of HIV-1 RNA in the emergence of resistance to antiretroviral therapies (ARTs) is well documented while less is known about the role of historical viruses stored in the proviral DNA. The primary focus of this work was to characterize the genetic diversity and evolution of HIV drug resistant variants in an individual’s provirus during antiretroviral therapy using next generation sequencing.MethodsBlood samples were collected prior to antiretroviral therapy exposure and during the course of treatment from five patients in whom drug resistance mutations had previously been identified using consensus sequencing. The spectrum of viral variants present in the provirus at each sampling time-point were characterized using 454 pyrosequencing from multiple combined PCR products. The prevalence of viral variants containing drug resistant mutations (DRMs) was characterized at each time-point.ResultsLow abundance drug resistant viruses were identified in 14 of 15 sampling time-points from the five patients. In all individuals DRMs against current therapy were identified at one or more of the sampling time-points. In two of the five individuals studied these DRMs were present prior to treatment exposure and were present at high prevalence within the amplified and sequenced viral population. DRMs to drugs other than those being currently used were identified in four of the five individuals.ConclusionThe presence of DRMs in the provirus, regardless of their observed prevalence did not appear to have an effect on clinical outcomes in the short term suggesting that the drug resistant viral variants present in the proviral DNA do not appear to play a role in the short term in facilitating the emergence of drug resistance.

Pattern of HIV-1 Drug Resistance among Adults on ART in Nigeria

Background: The development of anitiretroviral drug resistance may limit the benefit of antiretroviral therapy. Therefore the need to closely monitor these mutations, especially the use of ART is increasing. This study was therefore designed to determine the ARV drug resistance pattern among ART na?ve and expose individuals attending a PEPFAR supported by antiretroviral clinic in Nigeria. Methodology: The study participants included patients attending the PEPFAR supported by University College Hospital (UCH), Ibadan ART clinic who have been on HIV treatment for at least one year with consecutive viral load of over 2000 copies/ml as well some ART Na?ve individuals with high (>50,000 copies/ml) baseline viral level attending the hospital for pre-ART assessment. Blood sample was collected from each individual for CD4 enumeration, viral load level determination and DNA sequencing for genotypic typing. Antiretroviral drug resistance mutations (DRM) were determined by using the Viroseq software and drug mutations generated by using a combination of Viroseq and Stanford algorithm. DRM were classified as major or minor mutations based on the June 2013 Stanford DR database. Results: The most common major NRTI, NNRTI and PI mutation were D67N (33.3%), Y181C (16.7%) and M46L/I (55.6%) respectively. Lamivudine (3TC) and emtricitabine (FTC); nevirapine (NVP) and nelfinavir (NFV) were the most common NRTI, NNRTI, and PI drugs to which the virus in the infected individuals developed resistance. Isolates from 4 patients were resistant to triple drug class, including at least one NRTI, NNRTI and a PI. Only one (4.8%) of the isolates from drug Na?ve individuals had major DRM that conferred resistance to any drug. Conclusion: Demonstration of high rates of antiretroviral DRM among patients on 1st and 2nd line ART and the presence of DRM in drug Na?ve individuals in this study show the importance of surveillance for resistance to ARV in line with the magnitude of scaling up of treatment program in the country.

HIV-1 Protease and Substrate Coevolution Validates the Substrate Envelope As the Substrate Recognition Pattern

Drug resistance of HIV-1 protease alters the balance in the molecular recognition events in favor of substrate processing versus inhibitor binding. To develop robust inhibitors targeting ensembles of drug-resistant variants, the code of this balance needs to be cracked. For this purpose, the principles governing the substrate recognition are required to be revealed. Previous crystallographic studies on the WT protease-substrate complexes showed that the substrates have a conserved consensus volume in the protease active site despite their low sequence homology. This consensus volume is termed as the substrate envelope. The substrate envelope was recently reevaluated by taking the substrate dynamics into account, and the dynamic substrate envelope was reported to better define the substrate specificity for HIV-1 protease. Drug resistance occurs mostly through mutations in the protease, occasionally accompanied by cleavage site mutations. In this study, three coevolved protease-substrate complexes (AP2VNC-p1V82A, LP1'Fp1-p6D30N/N88D, and SP3'Np1-p6D30N/N88D) were investigated for structural and dynamic properties by molecular modeling and dynamics simulations. The results show the substrate envelope is preserved by these cleavage site mutations in the presence of drug-resistance mutations in the protease, if not enhanced. This study on the conformational and mutational ensembles of protease-substrate complexes validates the substrate envelope as the substrate recognition motif for HIV-1 protease. The substrate envelope hypothesis allows for the elucidation of possible drug resistance mutation patterns in the polyprotein cleavage sites.

Polymorphisms of HIV RT gene among the ART naïve native drug exposed rural PLHA

Background:The number of people living with human immunodeficiency virus (HIV) is increasing day by day in India. The disease has now spread from urban areas to rural areas. The proof reading of the reverse transcriptase enzyme is poor, which may lead to genetic diversity within the HIV strains, which in turn leads to problems like failure or resistance in antiretroviral treatment. This study is designed to find out the polymorphisms of the reverse transcriptase gene of HIV, after the native drug pressure among antiretroviral therapy (ART) naïve rural people living with HIV/AIDS (RPLHA).Materials and Methods:A total of 207 HIV-Reactive patients were allowed to take native drugs from the local area and were advised to attend the center for HIV after six months for a follow-up. At the time of the follow-up visit, a second blood sample was taken from 20 reactive native-drug exposed ART-naïve patients. The plasma was separated and transported at 20°C to the YRG Care Center for genotyping.Results:Among the 20 HIV-reactive samples processed for gene sequencing analysis to detect the genotypic variations, only one sample (5%) showed high-level mutational resistance variations and the predominant polymorphisms detected were V35T (100%), K122E (94.44%), and V60I (88.88%).Conclusions:The presence of drug-resistance mutations, although minimal, was important, as the drug-resistant strains could spread among the RPLHA and to their sexual partners. There was a definite need to generate a drug resistance database and the polymorphic pattern of Indian strains concern to the future clinical management of the disease, and a vaccine design to contain the disease.

Reverse transcriptase drug resistance mutations in HIV-1 subtype C infected patients on ART in Karonga District, Malawi

BackgroundDrug resistance testing before initiation of, or during, antiretroviral therapy (ART) is not routinely performed in resource-limited settings. High levels of viral resistance circulating within the population will have impact on treatment programs by increasing the chances of transmission of resistant strains and treatment failure. Here, we investigate Drug Resistance Mutations (DRMs) from blood samples obtained at regular intervals from patients on ART (Baseline-22 months) in Karonga District, Malawi. One hundred and forty nine reverse transcriptase (RT) consensus sequences were obtained via nested PCR and automated sequencing from blood samples collected at three-month intervals from 75 HIV-1 subtype C infected individuals in the ART programme.ResultsFifteen individuals showed DRMs, and in ten individuals DRMs were seen from baseline samples (reported to be ART naïve). Three individuals in whom no DRMs were observed at baseline showed the emergence of DRMs during ART exposure. Four individuals who did show DRMs at baseline showed additional DRMs at subsequent time points, while two individuals showed evidence of DRMs at baseline and either no DRMs, or different DRMs, at later timepoints. Three individuals had immune failure but none appeared to be failing clinically.ConclusionDespite the presence of DRMs to drugs included in the current regimen in some individuals, and immune failure in three, no signs of clinical failure were seen during this study. This cohort will continue to be monitored as part of the Karonga Prevention Study so that the long-term impact of these mutations can be assessed. Documenting proviral population is also important in monitoring the emergence of drug resistance as selective pressure provided by ART compromises the current plasma population, archived viruses can re-emerge