Michelle Gordon

Prevalence of HIV-1 Drug Resistance after Failure of a First Highly Active Antiretroviral Therapy Regimen in KwaZulu Natal, South Africa

BACKGROUND Emergence of human immunodeficiency virus type 1 (HIV-1) drug resistance may limit the benefits of antiretroviral therapy in resource-limited settings. The prevalence of resistance was assessed among patients from KwaZulu Natal, South Africa, following failure of their first highly active antiretroviral therapy (HAART) regimen. METHODS Genotypic resistance testing was performed on plasma virus samples from patients who experienced virologic failure of their first HAART regimen at 2 clinics in KwaZulu Natal. Clinical and demographic data were obtained from medical records. Regression analysis was performed to determine factors associated with > or =1 significant drug resistance mutation. RESULTS From January 2005 through August 2006, a total of 124 antiretroviral-treated adults who experienced virologic failure were enrolled. The predominant subtype was HIV-1C. Virus samples from 83.5% of participants carried > or =1 significant drug resistance mutation. Dual-class drug-resistant virus was present in 64.3% of participants, and 2.6% had virus with triple-class drug resistance. The most common mutation was M184V/I (64.3% of patients); K103N was present in virus from 51.3%, and V106M was present in virus from 19.1%. Thymidine analog resistance mutations were found in virus from 32.2% of patients, and protease resistance mutations were found in virus from 4.4%. CONCLUSIONS Antiretroviral drug-resistant virus was detected in >80% of South African patients who experienced failure of a first HAART regimen. Patterns of drug resistance reflected drugs used in first-line regimens and viral subtype. Continued surveillance of resistance patterns is warranted to guide selection of second-line regimens.

Molecular Characteristics of Human Immunodeficiency Virus Type 1 Subtype C Viruses from KwaZulu-Natal, South Africa: Implications for Vaccine and Antiretroviral Control Strategies

ABSTRACT The KwaZulu-Natal region of South Africa is experiencing an explosive outbreak of human immunodeficiency virus type 1 (HIV-1) subtype C infections. Understanding the genetic diversity of C viruses and the biological consequences of this diversity is important for the design of effective control strategies. We analyzed the protease gene, the first 935 nucleotides of reverse transcriptase, and the C2V5 envelope region of a representative set of 72 treatment-naïve patients from KwaZulu-Natal and correlated the results with amino acid signature and resistance patterns. Phylogenetic analysis revealed multiple clusters or “lineages” of HIV-1 subtype C that segregated with other C viruses from southern Africa. The same pattern was observed for both black and Indian subgroups and for retrospective specimens collected prior to 1990, indicating that multiple sublineages of HIV-1 C have been present in KwaZulu-Natal since the early stages of the epidemic. With the exception of three nonnucleoside reverse transcriptase inhibitor mutations, no primary resistance mutations were identified. Numerous accessory polymorphisms were present in the protease, but none were located at drug-binding or active sites of the enzyme. One frequent polymorphism, I93L, was located near the protease/reverse transcriptase cleavage site. In the envelope, disruption of the glycosylation motif at the beginning of V3 was associated with the presence of an extra protein kinase C phosphorylation site at codon 11. Many polymorphisms were embedded within cytotoxic T lymphocyte or overlapping cytotoxic T-lymphocyte/T-helper epitopes, as defined for subtype B. This work forms a baseline for future studies aimed at understanding the impact of genetic diversity on vaccine efficacy and on natural susceptibility to antiretroviral drugs.

High rate of K65R for antiretroviral therapy-naive patients with subtype C HIV infection failing a tenofovir-containing first-line regimen.

Objective:We sought to determine the rate of the K65R mutation in patients receiving tenofovir (TDF)-based antiretroviral therapy (ART) with subtype C HIV infection. Design:Retrospective cohort study. Methods:All patients initiated on stavudine (d4T) with lamivudine (3TC) or TDF with 3TC and a nonnucleoside reverse transcriptase inhibitor at McCord Hospital in Durban, South Africa had their charts reviewed. All patients with virologic failure, defined as a viral load more than 1000 copies/ml after 5 months of a first ART regimen, had genotypic resistance testing performed prospectively using a validated in-house assay. Important resistance mutations were selected based upon published mutations in subtype B virus in the Stanford HIV Drug Resistance database. Results:A total of 585 patients were initiated on TDF-containing first-line ART from 3 August 2010 to 17 March 2011. Thirty-five (6.0%) of these patients had virologic failure and 23 of 33 (69.7%) of the virologic failure patients had the K65R mutation. The median (interquartile range) for the baseline CD4 cell count was 105 cells/&mgr;l (49–209) and viral load at virologic failure was 47 571 copies/ml (20 708–202 000). During the same period, 53 patients were initiated on d4T-containing regimens. Two (3.8%) of these patients had virologic failure and one of the virologic failure patients had the K65R mutation. Conclusion:Preliminary data show very high rates (>65%) of K65R for patients failing TDF-based first-line regimens at McCord Hospital with few additional nucleoside reverse transcriptase inhibitor mutations compared with subtype B. These rates may reflect faster in-vivo selection, longer time on a failing regimen or transmitted drug resistance.

Variability at Human Immunodeficiency Virus Type 1 Subtype C Protease Cleavage Sites : an indication of viral fitness

ABSTRACT Naturally occurring polymorphisms in the protease of human immunodeficiency virus type 1 (HIV-1) subtype C would be expected to lead to adaptive (compensatory) changes in protease cleavage sites. To test this hypothesis, we examined the prevalences and patterns of cleavage site polymorphisms in the Gag, Gag-Pol, and Nef cleavage sites of C compared to those in non-C subtypes. Codon-based maximum-likelihood methods were used to assess the natural selection and evolutionary history of individual cleavage sites. Seven cleavage sites (p17/p24, p24/p2, NC/p1, NC/TFP, PR/RT, RT/p66, and p66/IN) were well conserved over time and in all HIV-1 subtypes. One site (p1/p6gag) exhibited moderate variation, and four sites (p2/NC, TFP/p6pol, p6pol/PR, and Nef) were highly variable, both within and between subtypes. Three of the variable sites are known to be major determinants of polyprotein processing and virion production. P2/NC controls the rate and order of cleavage, p6gag is an important phosphoprotein required for virion release, and TFP/p6pol, a novel cleavage site in the transframe domain, influences the specificity of Gag-Pol processing and the activation of protease. Overall, 58.3% of the 12 HIV-1 cleavage sites were significantly more diverse in C than in B viruses. When analyzed as a single concatenated fragment of 360 bp, 96.0% of group M cleavage site sequences fell into subtype-specific phylogenetic clusters, suggesting that they coevolved with the virus. Natural variation at C cleavage sites may play an important role, not only in regulation of the viral cycle but also in disease progression and response to therapy.

Outcomes after virologic failure of first-line ART in South Africa.

Objective:To determine initial 24-week outcomes among prospectively enrolled patients with failure of initial antiretroviral therapy (ART). Methods:Baseline virologic failure was defined as HIV-1 viral load greater than 1000 copies/ml. Second-line ART was informed by results of genotype testing and selected from agents in the South-African public sector. Twenty-four week endpoints included virologic suppression and mortality. Results:The cohort consisted of 141 patients (median CD4 cell count and viral load at failure of 173 cells/μl and 17 500 copies/ml). The median prior duration of initial ART was 12.0 months. At least one major resistance mutation was found in 87% of patients.After 24 weeks of follow-up, intent-to-treat virologic suppression (<50 copies/ml) was 65%, as-treated virologic suppression was 78%, the median CD4 cell count improvement was 88 cells/μl and the mortality was 6%. The median CD4 cell count at initial virologic failure among those who died was 70 cells/μl, compared to 182 cells/μl among patients who survived (P = 0.01). Patients with wild-type virus at initial failure (N = 19) had inferior outcomes after switch. The presence of nucleoside analogue resistance mutations at failure did not affect early efficacy of boosted-protease inhibitor regimens. Conclusions:Virologic monitoring linked to resistance testing helped demonstrate the efficacy of lopinavir/ritonavir-containing second-line regimens in South Africa. A switch to second-line regimens in patients with virologic failure and drug resistance has substantial and rapid immunological and clinical benefits. Resistance testing identified a high-risk group without resistance who might benefit from increased medication access and/or adherence support.

Tracing the origin of Brazilian HTLV-1 as determined by analysis of host and viral genes.

We compared the genetic diversity of the Brazilian human T-cell lymphotropic virus type 1 isolates with those found in KwaZulu–Natal (KZN), South Africa, and with the genetic background of the hosts. The seroprevalence rate in KZN was 1.7%. All sequences belonged to the A subgroup. The presence of South African sequences in two different clusters from Brazil, and the finding of the βA-globin haplotype in infected hosts are consistent with the transmission of this virus from southern Africa to Brazil.

MDR1 and CYP3A4 polymorphisms among African, Indian, and white populations in KwaZulu-Natal, South Africa

To the Editor: Dr Quick, in his commentary on essential medicines, noted the extent of “unsafe and wasteful drug use,” especially citing the extent of inappropriate prescribing of antibiotics. Comments have been made for decades about inappropriate prescribing of antibiotics, apparently with little effect. Perhaps what appears to be irrational prescribing of antibiotics from the perspective of an auditor is very rational from the perspective of the prescribing physician, even if inappropriate medically. If I were to have to see 6 patients per hour, I would barely have time to take a history from a patient with a cough and fever, examine his or her nose, throat, and chest, and write a prescription for an antibiotic. The patient would leave happy, and I would get emotional satisfaction from the expression of appreciation by the patient, irrespective of any financial aspects of the office visit. If, instead of writing a prescription, I were to explain to the patient about the difference between viral and bacterial infections, as well as the ineffectiveness of antibiotics for viral respiratory diseases (when drugs for other viral diseases such as human immunodeficiency virus are known), and send the patient home with instructions to take over-the-counter antipyretics, the patient would likely leave disgruntled. I would need more than 10 minutes to do all of this, plus a more careful history and physical examination to be reasonably certain that the infection was only viral. Thus, from the point of view of some physicians in busy practices, prescribing the antibiotic appears to be the rational act. One can always justify to oneself the need for an antibiotic for a febrile coughing patient. When this way of ending an office visit appears rational to the doctor but is not beneficial to the patient and is not rational for the public health, one needs better ways to end office visits than by prescribing unnecessary medicines. I know of no way to end an office visit as satisfactorily and as efficiently as by writing a prescription. The patient knows that the visit is over and is expected to leave. He has something in his hand that he thinks will help him and obtaining it required an office visit. The World Health Organization, professional societies, and other groups interested in medical treatment, such as health maintenance organizations and governments, should work on finding other efficient ways to end office visits for common complaints that are congruent with expert opinion about how these complaints should be treated. This would be helpful in improving prescribing and decreasing “unsafe and wasteful drug use.”

Drug resistance and coreceptor usage in HIV type 1 subtype C-infected children initiating or failing highly active antiretroviral therapy in South Africa.

HIV-1 drug resistance monitoring in resource-poor settings is crucial due to limited drug alternatives. Recent reports of the increased prevalence of CXCR4 usage in subtype C infections may have implications for CCR5 antagonists in therapy. We investigated the prevalence of drug resistance mutations and CXCR4 coreceptor utilization of viruses from HIV-1 subtype C-infected children. Fifty-one children with virological failure during highly active antiretroviral therapy (HAART) and 43 HAART-naive children were recruited. Drug resistance genotyping and coreceptor utilization assessment by phenotypic and genotypic methods were performed. At least one significant drug resistance mutation was present in 85.4% of HAART-failing children. Thymidine analogue mutations (TAMs) were detected in 58.5% of HAART-failing children and 39.0% had ≥3 TAMs. CXCR4 (X4) or dual (R5X4)/mixed (R5, X4) (D/M)-tropic viruses were found in 54.3% of HAART-failing and 9.4% of HAART-naive children (p<0.0001); however, the HAART-failing children were significantly older (p<0.0001). In multivariate logistic regression, significant predictors of CXCR4 usage included antiretroviral treatment, older age, and lower percent CD4(+) T cell counts. The majority of genotypic prediction tools had low sensitivity (≤65.0%) and high specificity (≥87.5%) for predicting CXCR4 usage. Extensive drug resistance, including the high percentage of TAMs found, may compromise future drug choices for children, highlighting the need for improved treatment monitoring and adherence counseling. Additionally, the increased prevalence of X4/D/M viruses in HAART-failing children suggests limited use of CCR5 antagonists in salvage therapy. Enhanced genotypic prediction tools are needed as current tools are not sensitive enough for predicting CXCR4 usage.

Resistance to antiretroviral drugs in newly diagnosed, young treatment‐naïve HIV‐positive pregnant women in the province of KwaZulu‐Natal, South Africa

In 2004, KwaZulu‐Natal initiated one of the worlds largest HIV/AIDS treatment programs. Studies in South Africa have shown that patients on antiretroviral therapy (ART) develop rapidly and transmit drug resistant mutations. Since resistance testing is not widely available in Kwazulu‐Natal, the Department of Health conducted the first HIV drug resistance (HIVDR) threshold survey in 2005, which did not identify any mutations associated with HIVDR. The objective of this study was to conduct a follow‐up threshold survey to update the information on HIVDR. This study was conducted in 2009 in five antenatal care sites in Kwazulu‐Natal using the HIVDR threshold survey method developed by WHO. Two hundred and thirteen newly‐diagnosed HIV positive, drug‐naïve primigravidae, less than 22 years of age were included in the survey. Of the 82 HIV positive specimens, 17 had insufficient volume for genotyping and, of the remaining 65, 47 were genotyped sequentially. Drug resistance was identified by sequencing the HIV‐1 pol gene, using the ViroSeq® HIV‐1 genotyping system v2.0. Of the 47 samples that were genotyped, only one presented with a K103N mutation, which equates to a prevalence of transmitted HIVDR of <5%. The low prevalence of transmitted HIVDR is in keeping with statistical models of the early stages of ART rollout. As ART coverage is increasing continuously, there is a need to ensure that vigilance of HIVDR continues so that the emergence and spread of HIVDR is minimized. This survey should be repeated in 2011, in accordance with WHO guidelines. J. Med. Virol. 83:1508–1513, 2011.

BioAfrica's HIV-1 Proteomics Resource: Combining protein data with bioinformatics tools

Most Internet online resources for investigating HIV biology contain either bioinformatics tools, protein information or sequence data. The objective of this study was to develop a comprehensive online proteomics resource that integrates bioinformatics with the latest information on HIV-1 protein structure, gene expression, post-transcriptional/post-translational modification, functional activity, and protein-macromolecule interactions. The BioAfrica HIV-1 Proteomics Resource http://bioafrica.mrc.ac.za/proteomics/index.html is a website that contains detailed information about the HIV-1 proteome and protease cleavage sites, as well as data-mining tools that can be used to manipulate and query protein sequence data, a BLAST tool for initiating structural analyses of HIV-1 proteins, and a proteomics tools directory. The Proteome section contains extensive data on each of 19 HIV-1 proteins, including their functional properties, a sample analysis of HIV-1HXB2, structural models and links to other online resources. The HIV-1 Protease Cleavage Sites section provides information on the position, subtype variation and genetic evolution of Gag, Gag-Pol and Nef cleavage sites. The HIV-1 Protein Data-mining Tool includes a set of 27 group M (subtypes A through K) reference sequences that can be used to assess the influence of genetic variation on immunological and functional domains of the protein. The BLAST Structure Tool identifies proteins with similar, experimentally determined topologies, and the Tools Directory provides a categorized list of websites and relevant software programs. This combined database and software repository is designed to facilitate the capture, retrieval and analysis of HIV-1 protein data, and to convert it into clinically useful information relating to the pathogenesis, transmission and therapeutic response of different HIV-1 variants. The HIV-1 Proteomics Resource is readily accessible through the BioAfrica website at: http://bioafrica.mrc.ac.za/proteomics/index.html