Low Drug Resistance Levels Among Drug-Naive Individuals with Recent HIV Type 1 Infection in a Rural Clinical Cohort in Southwestern Uganda

Abstract

To investigate the prevalence of transmitted drug resistance (TDR) among individuals with recent HIV-1 infection between February 2004 and January 2010 in a rural clinical cohort, samples from 72 participants were analyzed. Results from the 72 participants showed no protease inhibitor and nucleoside reverse transcriptase inhibitor-associated mutations. One participant (1.4%, 95% CI: 0.04–7.5%) had two nonnucleoside reverse transcriptase inhibitor mutations (G190E and P225H). HIV-1 subtype frequencies were A 22 (30.6%), D 38 (52.8%), and C 1 (1.4%); 11 (15.3%) were A/D unique recombinant forms. Seven years after the scale up of antiretroviral therapy (ART) in a rural clinical cohort in Uganda, the prevalence of TDR among recently HIV-1-infected individuals was low at 1.4%. Since our findings from an HIV study cohort may not be generalizable to the general population, routine TDR surveys in specific populations may be necessary to inform policy on the magnitude and prevention strategies of TDR.

Introduction

I n spite of the global decline in new HIV-1 infections, sub-Saharan Africa has contributed more than 80% of new infections according to the 2010 UNAIDS report.¹ Antiretroviral therapy (ART) has tremendously reduced HIV/AIDS-related deaths and morbidity as well as improved the livelihoods of patients with access to treatment. It is estimated that worldwide only 5.2 million of the 15 million HIV-1-infected patients requiring life-saving ART have access to treatment¹; however, the availability of ART continues to increase in the developing world allowing more patients access each year. The Ugandan Government has been making ART available to eligible patients since 2004, and the number of people on ART has increased from 10,000 in 2004² to 218,359 according to the March 2010 Ministry of Health report.³

The major challenges associated with the scaling up of ART in resource-limited settings such as sub-Saharan Africa are an intermittent drug supply leading to stock outs, limited patient monitoring, poor adherence, and incorrect prescribing practices.⁴ The development of HIV-1 drug resistance (HIV-DR) is the ultimate outcome of poor adherence and this will further complicate the availability of ART.⁵ The development of HIV-DR in patients on therapy and the subsequent transmission of resistance mutations mean that the standard first line therapy may be compromised before treatment is started.^6,7 The lack of affordable baseline resistance genotyping in resource-limited settings means that some patients (with HIV-DR) may be started on ineffective ART regimens.

Several studies have been undertaken to monitor the emergence of and to determine the prevalence of transmitted drug resistance (TDR) in those likely to be recently HIV infected as well as drug resistance in drug-naïve chronic patients among different risk groups with variable results.^8

–13 Among 104 drug-naive recently HIV-1-infected adults in the rural Rakai district between 1998 and 2003 a TDR prevalence of 5.8% was found.⁸ In Entebbe between 2006 and 2007, a study among 46 newly diagnosed HIV-1-infected pregnant women aged less than 25 years found no evidence of resistance mutations for any of the three drug classes.¹²

A study among 70 newly HIV-1 diagnosed young individuals attending Voluntary Counseling and Testing (VCT) clinics between 2009 and 2010 in Kampala found a prevalence of 8.6%.¹¹ A multicenter study (2006–2009) among recently infected African volunteers found an overall TDR prevalence of 6.7% in the two Ugandan sites.¹⁴ Among 38 recently HIV-1-infected female sex workers in Kampala between 2008 and 2010, 2.6% had resistance mutations.¹³ The prevalence of TDR among 47 recently infected fisher-folks between 2009 and 2010 around the Lake Victoria basin in Uganda was 6.4% (Nazziwa et al., unpublished data). In a cross-sectional survey (2007–2009) in six African countries among drug-naive HIV-1-infected adults, the overall prevalence in all the three Ugandan sites was 11.6%.¹⁰

We have previously found no HIV drug resistance among drug-naive chronic patients between 1998 and 2004 in a rural clinical cohort (RCC) in southwestern Uganda.⁹ In this study, we present data on the current prevalence of TDR among recently HIV-1-infected individuals in RCCs 7 years (2004–2010) after the scale up of ART in this cohort.

Materials and Methods

Study population, sample collection, and processing

To study the natural history of HIV disease progression, the Natural History Cohort (NHC) was established in 1990 in rural southwestern Uganda by the Medical Research Council/Uganda Virus Research Institute (MRC/UVRI) Uganda Research Unit on AIDS. The study participants were a random selection of one-third of the HIV-seropositive adults identified from the initial survey round of a general population cohort (GPC) as prevalent cases, seroconvertors identified during subsequent GPC survey rounds (incident cases), and age- and sex-matched HIV-negative controls.¹⁵ In January 2004, the NHC was renamed the Rural Clinical Cohort (RCC) and ART has since been provided to all eligible HIV-infected individuals according to the National ART guidelines.^16,17 Study participants are reviewed at the clinic after every 3 months when clinical data are collected and a blood sample is drawn for CD4 cell count measurements, full blood cell counts, serum biochemistry, and virology studies. Participant demographic data such as age, sex, marital status, education level, and occupation among others are captured.

A total of 72 participants who seroconverted and enrolled in the RCC between February 2004 and January 2010, with no prior exposure to ART and with available CD4 cell count results (Beckton-Dickinson, Franklin Lakes, NJ), were included in the survey. The estimated date of seroconversion was the mid-point between the last seronegative date and the first seropositive date. Blood specimens were collected in EDTA tubes and transported to the MRC/UVRI laboratory within 12 h of collection. Plasma and packed cells were prepared by centrifugation at a relative centrifugal force of 400×g for 10 min and stored at −80°C.

Ethical considerations

The study was approved by the Uganda Virus Research Institute, Institutional Review Board and the Uganda National Council for Science and Technology. All participants provided written informed consent for collection, storage, and analysis of specimens.

Viral genetic subtyping and determination of HIV-1 drug resistance

The polymerase chain reaction (PCR) and sequencing reactions were conducted using previously described protocols.^11
–13 The sequences were submitted to the Stanford University HIV Drug Resistance database (http://cpr.stanford.edu/cpr.cgi, accessed October 2011). The surveillance drug resistance mutations (SDRMs) were identified using the 2009 WHO list for surveillance of TDR,¹⁸ using the Stanford calibrated population resistance (CPR) analysis tool version 5.0 beta.¹⁹ Sequences with genetic mixtures of wild-type and mutant sequences at amino acid sites that code for SDRMs were considered to be drug resistant. The REGA²⁰ and SCUEAL²¹ subtyping tools were used to infer HIV-1 subtype and recombination patterns from the pol sequences. Sequences from this study were deposited in GenBank under accession numbers JQ266020–JQ266091.

Statistical methods

We extracted the sociodemographic and laboratory data of the 72 individuals from the Rural Clinical Cohort database in MS Access (Microsoft, Redmond, CA). Data were exported to STATA release 10.1 (StataCorp LP, College Station, TX) for descriptive analyses.

Results

Social demographics

Between 2004 and 2010, 72 recently infected individuals were enrolled in the study, with a mean age (standard deviation, SD) of 33.4 years (SD 11.5) and median age (interquartile range, IQR) of 33 years (IQR 16–71) with 79.2% aged 25 years and above; of these 66.7% were females. The overall mean (SD) CD4 cell count was 578.4 (248.6) and the median (IQR) CD4 cell count (cells/mm³) was 505 (170–1309). Of the participants 47.2% were either separated or divorced, 36.1% did not complete primary education, and 55.6% were farmers by occupation (Table 1). The mean time between the last seronegative and first seropositive test was 17.7 months (range 8.7–48.2 months).

Table 1.

Sociodemographic Characteristics of the 72 Participants

Characteristic	n (%)
Age (years)
<25	15 (20.8%)
25+	57 (79.2%)
Mean (SD)	33.4 (11.5)
Median (IQR)	33 (16–71)
Sex
Female	48 (66.7%)
Male	24 (33.3%)
Marital status
Married	25 (34.7%)
Separated/divorced	34 (47.2%)
Single (never married)	7 (9.7%)
Unknown	4 (5.6%)
Widowed	2 (2.8%)
Education level
Did not complete primary	26 (36.1%)
Completed primary	10 (13.9%)
Did not complete secondary	6 (8.3%)
Completed secondary	1 (1.4%)
Completed higher education	3 (4.2%)
Never	8 (11.1%)
Unknown	18 (25.0%)
Occupation
Builder	1 (1.4%)
Farmer	40 (55.6%)
Teacher	2 (2.8%)
Trader/seller	4 (5.6%)
Unknown	18 (25.0%)
Other	7 (9.7%)
Subtype distribution
A	22 (30.6%)
C	1 (1.4%)
D	38 (52.8%)
Recombinants	11 (15.3%)
CD4 counts^a
Mean (SD)	578.4 (248.6)
Median (IQR)	505 (170–1309)

Data are missing for 15 participants (CD4).

HIV-1 subtype frequencies and drug resistance patterns

The HIV-1 subtype distribution among samples from the 72 recently infected individuals was subtype A 22 (30.6%), C 1 (1.4%), and D 38 (52.8%); 11 (15.3%) were A/D unique recombinant forms (Table 1). In all the 72 sequences, there were no protease inhibitor (PI)-associated mutations and no nucleoside reverse transcriptase inhibitor (NRTI) mutations. However, one participant (1.4%, 95% CI: 0.04–7.5%) who was infected with an A/D recombinant form had two nonnucleoside reverse transcriptase inhibitor (NNRTI) mutations (G190E and P225H). These G190E and P225H NNRTI mutations confer high-level resistance to nevirapine (NVP) and efavirenz (EFV), respectively.

Discussion

In this first survey to determine the prevalence of TDR among study participants in a clinical cohort in rural Uganda after the introduction of ART (2004–2010), we found a low prevalence of TDR (1.4%) among individuals with recent HIV-1 infection. A previous survey done among drug-naive chronically infected patients in this cohort before the start of ART in 2004 (1998–2004) found no drug resistance.⁹

In Uganda, different rates of TDR prevalence have been reported from studies among different populations but with no clear-cut pattern between study populations (urban versus rural) and HIV risk groups (sex workers versus the general population). Whereas we previously observed no resistance before ART became available,⁹ findings from a cohort in the rural Rakai district, in the same geographic region as the RCC, showed a TDR prevalence of 5.8% among recently infected drug-naive individuals.⁸ Even after the initiation of ART, a different TDR prevalence has been reported, regardless of the year of sampling following the start of ART in a population. We found a low TDR prevalence (1.4%) 7 years after the initiation of ART, which was comparable to the 2.6% that was reported among female sex workers 4–6 years after ART became available in Uganda,¹³ yet no TDR was reported among recently infected women attending antenatal clinics in an urban area 2–3 years after ART initiation.¹² A TDR prevalence of 6.7% was reported from two Ugandan sites 4–5 years after ART initiation¹⁴ and 6.4% (between 2009 and 2010) in the fisher-folk community 5–6 years after ART initiation (Nazziwa et al., unpublished data). A TDR prevalence of 8.6% was reported among young individuals attending voluntary counseling and testing clinics 5–6 years after ART initiation.¹¹

The observed differences in the prevalence of TDR in the different populations and geographic regions may be due to several factors. The year of sampling following the initiation of ART in a population will affect the prevalence of TDR; as expected, a low prevalence prior to ART initiation in drug-naive chronic patients was shown in Uganda⁹ and this was shown to increase over the years after ART inititation¹¹ despite the fact that some populations have maintained low TDR prevalence after years of ART use.^12,13

The prevalence of TDR has been shown to vary between different populations and this may explain our observations.^11
–13 The RCC is a cohort within a research program; participants are routinely followed up with provision of ART to those who become eligible. There is also a timely and a continuous supply of ART with home visits for those who may not have access to the clinic. In the RCC, the standard of care is high with good follow-up; therefore the emergence and transmission of drug resistance may be less likely compared to the general Ugandan population, which probably explains the low prevalence of TDR in our report. For this reason, it therefore remains important to carry out routine surveys of TDR as well as sentinel surveillance of drug resistance at treatment centers.

There are high-throughput assays such as deep sequencing that have been developed to effectively detect resistant viruses present as minority populations as low as 1%; the use of such assays may therefore lead to a more precise estimation of TDR.²²

In conclusion, 7 years after the scale up of ART in a rural clinical cohort in Uganda, there is evidence of the emergence of TDR among recently HIV-1-infected individuals, but this has remained low. Therefore, though our findings from a rural clinical cohort may not be generalizable to the general population, they indicate that routine TDR surveys in specific populations may be necessary to inform policy concerning the magnitude and prevention strategies of TDR.

Footnotes

Acknowledgments

Funding for this study was provided by the Medical Research Council of the United Kingdom. We thank the staff and the study participants of the Rural Clinical Cohort and Drs. Nicaise Ndembi and Jonathan Kayondo for revising this manuscript.

D.S. and P.K. designed the study. B.N.M. coordinated the field and clinic work. A.K., F.L., B.M., and M.N. performed laboratory testing. D.S. analyzed the data and wrote the first draft of the manuscript. D.S., C.M.P., B.N.M., and P.K. contributed to subsequent drafts and reviewed and approved the final manuscript.

Author Disclosure Statement

No competing financial interests exist.

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