Prevalence of drug-resistant mutations in newly diagnosed drug-naive HIV-1-infected individuals in a treatment site in the Waterberg district, Limpopo province.
Aim. We studied the prevalence of resistance mutations in
drug-naive HIV-infected individuals at the Bela-Bela treatment site to
gather information on the presence of antiretroviral (ARV)
drug-resistant viruses in drug-naive populations, so as to improve
Subjects and methods. Drug-naive HIV-1-infected individuals were sequentially recruited between February 2008 and December 2008 from individuals visiting the voluntary counselling and testing (VCT) services of the Bela-Bela HIV/AIDS Wellness Clinic. Viral sub-typing was done by phylogenetic analysis; drug-resistant mutations were determined according to the Stanford HIV Drug Resistance Interpretation and the International AIDS Society-USA Guidelines.
Results. A drug-resistant mutation prevalence of 3.5% (95% confidence interval 0.019796-0.119077) comprising Y181C and L33F was observed; 98% of the viruses were HIV-1 subtype C on the protease (PR) and reverse transcriptase (RT) gene regions.
Conclusion. The prevalence of drug-resistant mutations in drug-naive persons may be low in Bela-Bela after 8 years of access to antiretroviral treatment (ART), and resistance testing before initiating treatment may not be needed.
S Afr Med J 2011;101:335-337.
Anti-HIV agents (Research)
Antiviral agents (Health aspects)
Antiviral agents (Research)
Microbial mutation (Research)
HIV infection (Drug therapy)
HIV infection (Research)
HIV infection (Genetic aspects)
Viral drug resistance (Genetic aspects)
Viral drug resistance (Research)
|Publication:||Name: South African Medical Journal Publisher: South African Medical Association Audience: Academic Format: Magazine/Journal Subject: Health Copyright: COPYRIGHT 2011 South African Medical Association ISSN: 0256-9574|
|Issue:||Date: May, 2011 Source Volume: 101 Source Issue: 5|
|Topic:||Event Code: 310 Science & research|
|Geographic:||Geographic Scope: South Africa Geographic Code: 6SOUT South Africa|
Highly active antiretroviral therapy (HAART) has dramatically
reduced morbidity and mortality among HIV/AIDS patients. However, a
major drawback of HAART is the development of drug resistance. (1,2)
South Africa has a high HIV prevalence, and access to treatment is
expanding. A regimen based on the genetic resistance profiles of
patients' viruses before initiating therapy generally correlates
with a better outcome, compared with the absence of such resistance
data. (2) The emergence of drug resistance in drug-naive populations
should be monitored, particularly in regions such as Limpopo Province,
where such data are scarce. We aimed to determine the prevalence of ARV
drug-resistance among drug-naive HIV-infected individuals in a community
where treatment has been provided for a considerable time.
Study subjects were recruited from the HIV/AIDS Wellness Clinic in Bela-Bela, Waterberg District, Limpopo Province. Treatment at the clinic began in 2001. HIV-positive individuals without prior exposure to ARV were recruited sequentially between February 2008 and December 2008. Five ml of venous blood was collected into EDTA vacutainer tubes from each consenting subject. Demographic data were obtained by questionnaire administration. Viral RNA was isolated using the viral mini RNA kit (Qiagen). Viral DNA was synthesised by a one-tube reverse transcription-polymerase chain reaction (RT-PCR), followed by a nested PCR. (3) The PCR products were verified for expected size by electrophoresis of 1% agarose gel. PCR amplicons were purified with a QIA quick PCR purification kit (Qiagen), and direct population-based sequencing was performed on both strands. HIV subtyping was done by phylogenetic analysis. Predicted amino acids for the PR and RT genes were aligned using the BioEdit programme (http://www.mbio.ncsu.edu/BioEdit/bioedit. html). Owing to the broad scope of viral mutations, drug-resistant mutations were identified using the Stanford HIV Drug Resistance Interpretation Algorithm (http://hivdb.stanford.edu/) and the International AIDS Society (IAS)-USA Guidelines. (4) Two algorithms were used to describe as broadly as possible the significance of observed amino acid substitutions. The Bayesian analysis was used to calculate the 95% confidence interval (CI), to estimate the confidence limits around the prevalence estimate. The PR and RT gene sequences reported here have been submitted to GenBank with the following respective accession numbers: PR [GU139577-GU139633] and RT [GU139634-GU139688]. Approval of the study protocol was obtained from the Health, Safety, and Research Ethics Committee of the University of Venda, Thohoyandou.
A total of 79 HIV-infected individuals were sequentially recruited. The mean age was 43.5 (18-69) years. The most important risk factor for HIV transmission was sexual intercourse (88.6%), but the questionnaire was not designed to determine whether this was heterosexual or homosexual. In 90% of the cases the most probable place of HIV infection was South Africa. Viral DNA was obtained for 62 out of 79 (78%) individuals. Reliable nucleotide sequences were obtained for 57 PR genes and 55 RT genes. Using the Stanford and IAS guidelines, major drug-resistant mutations were detected in 2 out of 57 subjects (3.5%) (95% CI 0.019796-0.119077). One primary NNRTI mutation (Y181C) and one major PI mutation (L33F) were detected in two different patients. No primary NRTI mutation was recorded. The detected mutations, nucleotide substitution, and their potential significance are shown in Table I.
Phylogenetic analysis of the PR and RT genes showed that 56/57 (98.2%) of the isolates were HIV-1 subtype C on both gene regions. One isolate (08BBVCT31ZA) was HIV-1 subtype B on the PR and RT genes (phylogenetic trees not shown). Mean genetic distance for the PR sequences ranged from 0.0101 to 0.2035 and 0.0331 to 0.1377 for the RT sequences. Considering the first 300 amino acids in the RT gene, sequence alignment showed that the consensus sequences of the test viruses were identical to the global subtype C consensus, except at 2 positions (V60I and Q174K). The consensus sequence differed from the global subtype B consensus at 17 positions (V35T, E36A, T39E, S48T, V60I, K122E, D123G, K173A, Q174K, D177E, T200A, Q207E, R211K, V245Q, A272P, K277R, and T286A). Amino acid alignment of the PR gene showed that the test consensus was identical to the global subtype C consensus, except at position I13T. It differed from the global subtype B consensus at 8 positions (T12S, I15V, L19I, M36I, R41K, H69K, L89M, and I93L).
The development of drug resistance is an important attribute of HIV biology. An inevitable drawback of HAART is the emergence of drug-resistant variants in patients under treatment, which complicates treatment options and hampers good prognosis. Antiretroviral therapy was started at the Bela-Bela Wellness Clinic about 8 years ago, within which time resistance could have possibly emerged. The low rate (3.5%) of drug-resistant mutations detected in this study is similar to reports in other parts of South Africa. Drug-resistance studies among naive patients reported a prevalence in Gauteng Province in 2002 and 2004 of 4.2%; (5) in Cape Town of 2.5%;6 and 3.6% in Free State Province. (7) Low levels (<5%), or the absence of drug-resistant mutations, have been reported in Zambia (8) and Malawi. (9) Higher rates have been reported in several developed countries with more than a decade of ART history. (10,11)
Low rates of ARV drug resistance are expected in developing countries because most patients start therapy on highly potent regimens, unlike in the developed world where ART scale-up began with resistance-associated monotherapy and one-class dual therapy. In this study, one primary NNRTI mutation (Y181C) was observed (Y181C mutation causes high-level resistance to nevirapine and delavirdine and low-level resistance to efavirenz), while no primary NRTI mutation was noted. This mutation occurred in a 40-year-old married woman. At the time of the investigation, the South African national drug regimen guideline stipulated the use of stavudine, lamivudine and efavirenz as first-line therapy for those who had never been exposed to ARVs, with nevirapine replacing efavirenz for women of child-bearing age. (12) It is not clear whether the patient who harboured the Y181C mutation had been enrolled in a mother-to-child transmission prevention programme in which nevirapine was used.
One major PI mutation (L33F) was detected. This mutation is classified as a major mutation by the Stanford Drug Resistance Interpretation Algorithm, and as a minor mutation by the IAS-USA guidelines (L33F mutation is selected by fosamprenavir/ritonavir, duranavir/ritonavir, lopinavir/ritonavir, atazanavir/ritonavir, and tipranavir/ritonavir, and contributes to resistance to these drugs).
This study indicates that the prevalence of drug-resistant HIV among the drug-naive population in Bela-Bela is low after 8 years of free ART. We used population-based sequencing in our study, which might have contributed to an underestimation of drug-resistance prevalence. Patients may harbour populations of minor-resistance viruses that are usually not detected by population sequencing. This prevalence study also used chronically infected patients, which could mean that resistance acquired might have disappeared over time, which might have also led to an underestimation of the results.
Apparently, the testing of patients for drug-resistant viruses before initiation of therapy may not be required. However, subsequent periodic studies are required in Bela-Bela and other treatment sites to monitor the emergence and spread of drug-resistant viruses. Such data are important for guiding policy on sentinel surveillance and treatment algorithms. A high prevalence could indicate the need for regular sentinel surveillance or for baseline genotypic drug-resistance testing before treatment.
This study was supported by a research grant awarded to PB by the South African National Department of Health, under the Comprehensive HIV and AIDS Care, Management and Treatment Plan for South Africa. Financial support from the National Research Foundation is also acknowledged. PB, NN and CM are board members of the HIV/AIDS Prevention Group, an NGO running the Bela-Bela Wellness Clinic. The views expressed here are those of the authors.
Accepted 18 October 2010.
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(12.) Department of Health. National Antiretroviral Treatment Guidelines. Pretoria: Department of Health, 2004.
Julius Nwobegahay, Pascal Bessong, Tracy Masebe, Lufuno Mavhandu, Cecile Manhaeve, Norbert Ndjeka, Gloria Selabe
AIDS Virus Research Laboratory, Department of Microbiology, University of Venda, Viohoyandou, Limpopo Province
J Nwobegahay, MSc
P Bessong, PhD
T Masebe, MSc
L Mavhandu, MSc
The HIV/AIDS Prevention Group, Bela-Bela, Limpopo Province
C Manhaeve, MA
N Ndjeka, MD
HIV/AIDS and Hepatitis Unit, Department of Medical Virology, University of Limpopo, MEDUNSA Campus, Garankuwa G Selabe, PhD
Corresponding author: P Bessong (email@example.com)
Table I. Drug resistance-associated mutations, frequency, coding nucleotides and HIV-1 subtypes from HIV-infected patients in the Waterberg District Protease Coding nucleotides gene (N=57) Frequency Wild type Mutant L10I/F 2 (3.5) CTC ATT/CTT K20R 19 (33.3) AAG AGG L23F 2 (3.5) CTA TTT D30G 1 (1.7) GAT CGT L33F 1 (1.7) TTA AGA M36I 46 (80.7) ATG ATA L63S 7 (12.3) CTC TCT/AGT A71L/T 2 (3.5) GCT TGT/AAA T74S 4 (7.0) ACA TCA L89M 46 (80.7) CTG ATG I93L 54 (94.7) ATT CTT Protease gene HIV-1 (N=57) subtype Comment L10I/F C Minor mutation to atazanavir/ritonavir according to Stanford and IAS K20R C Polymorphism (Stanford); minor mutation (IAS) L23F C Highly unusual minor mutation with unknown significance (Stanford); undocumented by IAS D30G C Minor mutation (Stanford); undocumented (IAS) L33F C Major mutation to protease inhibitors (Stanford); but a minor mutation by IAS M36I C Minor mutation to atazanavir, ritonavir, indinavir, nelfinavir (Stanford and IAS) L63S C Minor mutation (Stanford); undocumented (IAS) A71L/T C Minor mutation to atazanavir/ritonavir (Stanford and IAS) T74S C Minor mutation (Stanford); undocumented by IAS L89M C Polymorphism (Stanford); undocumented (IAS) I93L C Common polymorphism (Stanford); undocumented (IAS) Reverse Coding nucleotides transcriptase (N=55) Frequency Wild type Mutant T69N 1 (1.8) ACT AAT (NRTI mutation) K100D 1 (1.8) TTA GTA (NNRTI mutation) K101Q 1 (1.8) AAA CAG (NNRTI mutation) V118I 3 (5.5) GTT ATT (NRTI mutation) E138A 1 (1.8) GAG GCA (NNRTI mutation) V179D 1 (1.8) GTT GAT (NNRTI mutation) Y181C 1 (1.8) TAT GTC (NNRTI mutation) L210V 1 (1.8) TTG AAA (NRTI mutation) Reverse transcriptase HIV-1 (N=55) subtype Comment T69N C Secondary mutation (NRTI (Stanford); primary mutation) mutation (IAS) K100D C Secondary mutation (NNRTI (Stanford); mutation) undocumented(IAS) K101Q C Secondary mutation (NNRTI (Stanford); mutation) undocumented (IAS) V118I B & C Secondary mutation (NRTI (Stanford); mutation) undocumented (IAS) E138A C Polymorphism (NNRTI (Stanford); but a mutation) secondary mutation to etravirine (IAS) V179D C Minor mutation (NNRTI according to Stanford mutation) and IAS Y181C C Primary mutation to (NNRTI efavirenz/etravirine/ mutation) nevirapine (Stanford and IAS) L210V C Secondary mutation (NRTI (Stanford); mutation) undocumented (IAS)
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