Kim Steegen

Susceptibility of Human Immunodeficiency Virus Type 1 to the Maturation Inhibitor Bevirimat Is Modulated by Baseline Polymorphisms in Gag Spacer Peptide 1

ABSTRACT In this study, we evaluated baseline susceptibility to bevirimat (BVM), the first in a new class of antiretroviral agents, maturation inhibitors. We evaluated susceptibility to BVM by complete gag genotypic and phenotypic testing of 20 patient-derived human immunodeficiency virus type 1 isolates and 20 site-directed mutants. We found that reduced BVM susceptibility was associated with naturally occurring polymorphisms at positions 6, 7, and 8 in Gag spacer peptide 1.

Accumulation of HIV-1 drug resistance after continued virological failure on first-line ART in adults and children in sub-Saharan Africa

OBJECTIVES Limited availability of viral load (VL) monitoring in HIV treatment programmes in sub-Saharan Africa can delay switching to second-line ART, leading to the accumulation of drug resistance mutations (DRMs). The objective of this study was to evaluate the accumulation of resistance to reverse transcriptase inhibitors after continued virological failure on first-line ART, among adults and children in sub-Saharan Africa. METHODS HIV-1-positive adults and children on an NNRTI-based first-line ART were included. Retrospective VL and, if VL ≥1000 copies/mL, pol genotypic testing was performed. Among participants with continued virological failure (≥2 VL ≥1000 copies/mL), drug resistance was evaluated. RESULTS At first virological failure, DRM(s) were detected in 87% of participants: K103N (38.7%), G190A (21.8%), Y181C (20.2%), V106M (8.4%), K101E (8.4%), any E138 (7.6%) and V108I (7.6%) associated with NNRTIs, and M184V (69.7%), any thymidine analogue mutation (9.2%), K65R (5.9%) and K70R (5.0%) associated with NRTIs. New DRMs accumulated with an average rate of 1.45 (SD 2.07) DRM per year; 0.62 (SD 1.11) NNRTI DRMs and 0.84 (SD 1.38) NRTI DRMs per year, respectively. The predicted susceptibility declined significantly after continued virological failure for all reverse transcriptase inhibitors (all P < 0.001). Acquired drug resistance patterns were similar in adults and children. CONCLUSIONS Patterns of drug resistance after virological failure on first-line ART are similar in adults and children in sub-Saharan Africa. Improved VL monitoring to prevent accumulation of mutations, and new drug classes to construct fully active regimens, are required.

High prevalence of the K65R mutation in HIV-1 subtype C infected patients failing tenofovir-based first-line regimens in South Africa.

Background Tenofovir (TDF) has replaced stavudine (d4T) as the preferred nucleoside reverse transcriptase inhibitor (NRTI) in first-line regimens in South Africa, but limited information is available on the resistance patterns that develop after the introduction of TDF. This study investigated the antiretroviral drug resistance patterns in South African HIV-1 subtype C-infected patients failing stavudine- (d4T) and tenofovir- (TDF) based first-line regimens and assess the suitability of TDF as the preferred first-line nucleotide reverse transcriptase inhibitor (NRTI). Methods Resistance patterns of HIV-1 from 160 adult patients virologically failing TDF- (n = 80) and d4T- (n = 80) based first-line regimens were retrospectively analyzed. The pol gene was sequenced using an in-house protocol and mutations were analysed using the IAS-USA 2014 Drug Resistance Mutation list. Results Compared to d4T-exposed patients (n = 7), patients failing on a TDF-containing regimen (n = 43) were almost 5 times more likely to present with a K65R mutation (aRR 4.86 95% CI 2.29 – 10.34). Y115F was absent in the d4T group, and detected in 13.8% (n = 11) of TDF-exposed patients, p = 0.0007. Virus from 9 of the 11 patients (82.0%) who developed the Y115F mutation also developed K65R. Intermediate or high-level resistance to most NRTIs was common in the TDF-treatment group, but these patients twice more likely to remain susceptible to AZT as compared to those exposed to d4T (aRR 2.09 95% CI 1.13 – 3.90). Conclusion The frequency of the TDF induced K65R mutation was higher in our setting compared to non-subtype C dominated countries. However, despite the higher frequency of cross-resistance to NRTIs, most patients remained susceptible to AZT, which is reflected in the South African treatment guidelines that recommend AZT as an essential component of second-line regimens.

A Pragmatic Approach to HIV-1 Drug Resistance Determination in Resource-Limited Settings by Use of a Novel Genotyping Assay Targeting the Reverse Transcriptase-Encoding Region Only

ABSTRACT In resource-limited settings (RLS), reverse transcriptase (RT) inhibitors form the backbone of first-line treatment regimens. We have developed a simplified HIV-1 drug resistance genotyping assay targeting the region of RT harboring all major RT inhibitor resistance mutation positions, thus providing all relevant susceptibility data for first-line failures, coupled with minimal cost and labor. The assay comprises a one-step RT-PCR amplification reaction, followed by sequencing using one forward and one reverse primer, generating double-stranded coverage of RT amino acids (aa) 41 to 238. The assay was optimized for all major HIV-1 group M subtypes in plasma and dried blood spot (DBS) samples using a panel of reference viruses for HIV-1 subtypes A to D, F to H, and circulating recombinant form 01_AE (CRF01_AE) and applied to 212 clinical plasma samples and 25 DBS samples from HIV-1-infected individuals from Africa and Europe. The assay was subsequently transferred to Uganda and applied locally on clinical plasma samples. All major HIV-1 subtypes could be detected with an analytical sensitivity of 5.00E+3 RNA copies/ml for plasma and DBS. Application of the assay on 212 clinical samples from African subjects comprising subtypes A to D, F to H (rare), CRF01_AE, and CRF02_AG at a viral load (VL) range of 6.71E+2 to 1.00E+7 (median, 1.48E+5) RNA copies/ml was 94.8% (n = 201) successful. Application on clinical samples in Uganda demonstrated a comparable success rate. Genotyping of clinical DBS samples, all subtype C with a VL range of 1.02E+3 to 4.49E+5 (median, 1.42E+4) RNA copies/ml, was 84.0% successful. The described assay greatly reduces hands-on time and the costs required for genotyping and is ideal for use in RLS, as demonstrated in a reference laboratory in Uganda and its successful application on DBS samples.

Mutational Correlates of Virological Failure in Individuals Receiving a WHO-Recommended Tenofovir-Containing First-Line Regimen: An International Collaboration

Tenofovir disoproxil fumarate (TDF) genotypic resistance defined by K65R/N and/or K70E/Q/G occurs in 20% to 60% of individuals with virological failure (VF) on a WHO-recommended TDF-containing first-line regimen. However, the full spectrum of reverse transcriptase (RT) mutations selected in individuals with VF on such a regimen is not known. To identify TDF regimen-associated mutations (TRAMs), we compared the proportion of each RT mutation in 2873 individuals with VF on a WHO-recommended first-line TDF-containing regimen to its proportion in a cohort of 50,803 antiretroviral-naïve individuals. To identify TRAMs specifically associated with TDF-selection pressure, we compared the proportion of each TRAM to its proportion in a cohort of 5805 individuals with VF on a first-line thymidine analog-containing regimen. We identified 83 TRAMs including 33 NRTI-associated, 40 NNRTI-associated, and 10 uncommon mutations of uncertain provenance. Of the 33 NRTI-associated TRAMs, 12 – A62V, K65R/N, S68G/N/D, K70E/Q/T, L74I, V75L, and Y115F – were more common among individuals receiving a first-line TDF-containing compared to a first-line thymidine analog-containing regimen. These 12 TDF-selected TRAMs will be important for monitoring TDF-associated transmitted drug-resistance and for determining the extent of reduced TDF susceptibility in individuals with VF on a TDF-containing regimen.

Intensive adherence counselling for HIV‐infected individuals failing second‐line antiretroviral therapy in Johannesburg, South Africa

In resource‐limited settings, where genotypic drug resistance testing is rarely performed and poor adherence is the most common reason for treatment failure, programmatic approaches to handling treatment failure are essential. This study was performed to describe one such approach to adherence optimisation.

Moderate levels of pre-treatment HIV-1 antiretroviral drug resistance detected in the first South African national survey

Background In order to assess the level of transmitted and/or pre-treatment antiretroviral drug resistance to HIV-1, the World Health Organization (WHO) recommends that regular surveys are conducted. This study’s objective was to assess the frequency of HIV-1 antiretroviral drug resistance in patients initiating antiretroviral treatment (ART) in the public sector throughout South Africa. Methods A prospective cross-sectional survey was conducted using probability proportional to size sampling. This method ensured that samples from each province were proportionally collected, based on the number of patients receiving ART in each region. Samples were collected between March 2013 and October 2014. Pol sequences were obtained using RT-PCR and Sanger sequencing and submitted to the Stanford Calibrated Population Resistance tool v6.0. Results A total of 277 sequences were available for analysis. Most participants were female (58.8%) and the median age was 34 years (IQR: 29–42). The median baseline CD4-count was 149 cells/mm3 (IQR: 62–249) and, based on self-reporting, participants had been diagnosed as HIV-positive approximately 44 days prior to sample collection (IQR: 23–179). Subtyping revealed that 98.2% were infected with HIV-1 subtype C. Overall, 25 out of 277 patients presented with ≥1 surveillance drug resistance mutation (SDRM, 9.0%, 95% CI: 6.1–13.0%). Non-nucleoside reverse transcriptase inhibitor (NNRTI) mutations were the most numerous mutations detected (n = 23). Only two patients presented with a protease inhibitor (PI) mutation. In four patients ≥4 SDRMs were detected, which might indicate that these patients were not truly ART-naïve or were infected with a multi-resistant virus. Conclusions These results show that the level of antiretroviral drug resistance in ART-naïve South Africans has reached moderate levels, as per the WHO classification. Therefore, regular surveys of pre-treatment drug resistance levels in all regions of South Africa is highly recommended to monitor the changing levels of pre-treatment antiretroviral drug resistance.

HIV-1 Phenotypic Reverse Transcriptase Inhibitor Drug Resistance Test Interpretation Is Not Dependent on the Subtype of the Virus Backbone

To date, the majority of HIV-1 phenotypic resistance testing has been performed with subtype B virus backbones (e.g. HXB2). However, the relevance of using this backbone to determine resistance in non-subtype B HIV-1 viruses still needs to be assessed. From 114 HIV-1 subtype C clinical samples (36 ARV-naïve, 78 ARV-exposed), pol amplicons were produced and analyzed for phenotypic resistance using both a subtype B- and C-backbone in which the pol fragment was deleted. Phenotypic resistance was assessed in resulting recombinant virus stocks (RVS) for a series of antiretroviral drugs (ARVs) and expressed as fold change (FC), yielding 1660 FC comparisons. These Antivirogram® derived FC values were categorized as having resistant or sensitive susceptibility based on biological cut-off values (BCOs). The concordance between resistance calls obtained for the same clinical sample but derived from two different backbones (i.e. B and C) accounted for 86.1% (1429/1660) of the FC comparisons. However, when taking the assay variability into account, 95.8% (1590/1660) of the phenotypic data could be considered as being concordant with respect to their resistance call. No difference in the capacity to detect resistance associated with M184V, K103N and V106M mutations was noted between the two backbones. The following was concluded: (i) A high level of concordance was shown between the two backbone phenotypic resistance profiles; (ii) Assay variability is largely responsible for discordant results (i.e. for FC values close to BCO); (iii) Confidence intervals should be given around the BCOs, when assessing resistance in HIV-1 subtype C; (iv) No systematic resistance under- or overcalling of subtype C amplicons in the B-backbone was observed; (v) Virus backbone subtype sequence variability outside the pol region does not contribute to phenotypic FC values. In conclusion the HXB2 virus backbone remains an acceptable vector for phenotyping HIV-1 subtype C pol amplicons.

A synthetic HIV-1 subtype C backbone generates comparable PR and RT resistance profiles to a subtype B backbone in a recombinant virus assay.

In order to determine phenotypic protease and reverse transcriptase inhibitor-associated resistance in HIV subtype C virus, we have synthetically constructed an HIV-1 subtype C (HIV-1-C) viral backbone for use in a recombinant virus assay. The in silico designed viral genome was divided into 4 fragments, which were chemically synthesized and joined together by conventional subcloning. Subsequently, gag-protease-reverse-transcriptase (GPRT) fragments from 8 HIV-1 subtype C-infected patient samples were RT-PCR-amplified and cloned into the HIV-1-C backbone (deleted for GPRT) using In-Fusion reagents. Recombinant viruses (1 to 5 per patient sample) were produced in MT4-eGFP cells where cyto-pathogenic effect (CPE), p24 and Viral Load (VL) were monitored. The resulting HIV-1-C recombinant virus stocks (RVS) were added to MT4-eGFP cells in the presence of serial dilutions of antiretroviral drugs (PI, NNRTI, NRTI) to determine the fold-change in IC50 compared to the IC50 of wild-type HIV-1 virus. Additionally, viral RNA was extracted from the HIV-1-C RVS and the amplified GPRT products were used to generate recombinant virus in a subtype B backbone. Phenotypic resistance profiles in a subtype B and subtype C backbone were compared. The following observations were made: i) functional, infectious HIV-1 subtype C viruses were generated, confirmed by VL and p24 measurements; ii) their rate of infection was slower than viruses generated in the subtype B backbone; iii) they did not produce clear CPE in MT4 cells; and iv) drug resistance profiles generated in both backbones were very similar, including re-sensitizing effects like M184V on AZT.

HIV-1 antiretroviral drug resistance patterns in patients failing NNRTI-based treatment: results from a national survey in South Africa

Background Routine HIV-1 antiretroviral drug resistance testing for patients failing NNRTI-based regimens is not recommended in resource-limited settings. Therefore, surveys are required to monitor resistance profiles in patients failing ART. Methods A cross-sectional survey was conducted amongst patients failing NNRTI-based regimens in the public sector throughout South Africa. Virological failure was defined as two consecutive HIV-1 viral load results >1000 RNA copies/mL. Pol sequences were obtained using RT–PCR and Sanger sequencing and submitted to Stanford HIVdb v7.0.1. Results A total of 788 sequences were available for analysis. Most patients failed a tenofovir-based NRTI backbone (74.4%) in combination with efavirenz (82.1%) after median treatment duration of 36 months. K103N (48.9%) and V106M (34.9%) were the most common NNRTI mutations. Only one-third of patients retained full susceptibility to second-generation NNRTIs such as etravirine (36.5%) and rilpivirine (27.3%). After M184V/I (82.7%), K65R was the most common NRTI mutation (45.8%). The prevalence of K65R increased to 57.5% in patients failing a tenofovir regimen without prior stavudine exposure. Cross-resistance to NRTIs was often observed, but did not seem to affect the predicted activity of zidovudine as 82.9% of patients remained fully susceptible to this drug. Conclusions The introduction of tenofovir-based first-line regimens has dramatically increased the prevalence of K65R mutations in the HIV-1-infected South African population. However, most patients failing tenofovir-based regimens remained fully susceptible to zidovudine. Based on these data, there is currently no need to change either the recommended first- or second-line ART regimens in South Africa.