The proportion of individuals with a history of exposure (‘pre-exposure’) to antiretrovirals (ARVs) prior to formal initiation into antiretroviral treatment (ART) is unknown.
This study describes the detection of ARVs in plasma and/or hair, of persons who self-reported no pre-exposure to ART at their first-time initiation onto ART in three clinics in the province of Limpopo, South Africa (SA).
Concentrations of tenofovir (TDF), emtricitabine (FTC) and efavirenz (EFV) in the plasma and hair of individuals initiating ART were analysed using a validated liquid chromatography tandem mass spectrometry (LC-MS/MS) method. Next generation sequences of HIV polymerase gene were analysed with Geneious software 11.15, and drug resistance (DR) mutations were determined according to the Stanford HIV Drug-Resistance database. Participants’ demographic data were collected on a structured questionnaire. Data that describe prior exposure to ARV were also collected by this self-reporting method.
Paired blood and hair samples were collected from 77 individuals newly initiated onto ART from 2017 to 2019. We detected at least one of the drugs in the plasma or hair of 41/77 (53.2%) patients who responded with a ‘no’ to the question ‘have you received ARVs before initiation onto ART?’ Thirty-one participants (
Antiretroviral levels in the hair of individuals initiating treatment imply prolonged prior-exposure to that ARV. The presence of ARV in plasma and hair of persons living with HIV (PLWH) who deny ARV-use, requires an explanation. A larger study at multiple sites and regular DR surveillance of ART-naïve PLWH will be necessary to confirm the generalisability of these findings to the wider South African population.
Considering evidence that treatment significantly reduces HIV transmission at the population level,
Several key assumptions are included in UTT. These include the fact that the tested person ‘newly’ diagnosed with HIV is naïve to antiretroviral therapy (ART) and is infected with a strain of HIV susceptible to the recommended first-line ART regimen. Further, it is assumed that the prevalence of circulating drug-resistant virus in the pre-treated population is negligible and will not impact treatment outcomes. The World Health Organization (WHO) categorises the pre-treatment -drug-resistance at the population level as low if the incidence of drug resistance < 5%; moderate if 5% – 15% and high if > 15%. In South Africa (SA), the level of drug resistance in the pre-treated population has increased over time but is heterogeneous across and within provinces. At least one pre-treated population with a moderate level of resistance has been reported in each province, except the Northern Cape.
Another important UTT assumption is that recipients of ART will be adherent to the treatment. Poor adherence leads to inadequate viral suppression, the development of drug-resistance, treatment-failure and an increased risk of death.
In this study we explore the relationship of prior ARV exposure to subsequent resistance on reinitiating treatment by analysing the plasma and/or hair samples of individuals at baseline, and at 6 months post-initiation for the presence of drug-resistant virus and to confirm non-adherence.
This was an exploratory study, involving the use of available plasma samples collected prior to initiation of treatment (baseline) and at 6 months post-initiation amongst persons living with HIV (PLWH) who were participants in an unpublished parent HIV drug resistance and treatment outcome study. Both at ART initiation and at the 6-month post-initiation visit, strands of hair were cut approximately 3 mm away from the scalp at the back of each participant’s head, and stored in a sterile container for subsequent analysis. Matched plasma samples were taken at the same time. Study participants were recruited from three HIV-screening and ART-initiating sites in the province of Limpopo, SA namely, the University of Venda Campus Health Clinic in Thohoyandou, the Rethabile Community Health Centre in Polokwane and the Seshego Community Clinic in Seshego. Participants provided informed consent before recruitment into the study. All participants were adults, aged ≥ 18 years and were reportedly taking ART for the first time. No additional inclusion or exclusion criteria were applied. All participants were started on a fixed-dose combination (FDC) of tenofovir (TDF) + emtricitabine (FTC) + efavirenz (EFV): TEE. All demographic data including sex and age were collected by means of a structured questionnaire. Data on prior ARV drug exposure relied on self-reporting by participants.
CD4+ cell count and VL measurement of study participants is shown in
Nucleoside/tide variant frequencies coding for drug resistance mutations were evaluated at both majority (> 20%) and minority (> 5% and > 1%) thresholds, using the find variation/single nucleotide polymorphisms feature from the annotation and prediction menu in Geneious,
Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to quantify the individual components of TEE in the samples of hair and plasma, and was conducted at FARMOVS Bioanalytical Services Division, the University of the Free State, SA, a South African National Accreditation System accredited laboratory.
For the plasma preparation, the run comprised eight standard (STD) levels over the range of 20.00 ng/mL – 2560 ng/mL, with six levels of quality control (QC) samples extending over this range. Two replicates were included per level for each STD and each QC sample. One blank sample and one zero sample were included in the sample run.
The hair preparation run comprised eight STD levels over the ranges of 0.158 ng/mL – 20 ng/mL for EFV, 0.118 ng/mL – 15 ng/mL for FTC and 0.063 ng/mL – 8 ng/mL for TDF, with three levels of QC samples extending over this range. Two replicates were included per level for each STD and each QC sample. The STDs and QCs were interspersed amongst the study samples in a predetermined manner, with one blank sample and one zero sample included in the study sample run. See the detailed methodology for both matrices in
Data including the participants’ age, CD4 and HIV-RNA VL were calculated and reported as medians and interquartile ranges (IQR). The Spearman’s correlation coefficient (
The study protocol was approved by the University’s Human and Clinical Trial Research Ethics Committee (SMN/17/MBY/05/2106), and permission to access public sector health facilities was granted by the National Health Research Ethics Committee (NHREC) and obtained from the Provincial Department of Health of South Africa, and the relevant district managers. Signed informed consent was obtained from all participants prior to collection of specimens and demographic data. Confidentiality and anonymity were maintained.
Paired blood and hair samples were collected from 77 PLWH newly started on ART between 2017 and 2019.
Characteristics of the study cohort.
Parameters | Values |
---|---|
Number of patients | 77 |
Median age, IQR | 35 (27.25–42) |
Females (%) | 69 (89.60%) |
Males (%) | 8 (10.40%) |
Median viral load copies/mL (IQR) at baseline | 25 150 (6427.5–84 514) |
Median viral load copies/mL (IQR) post 6 months | 38 (30–54.50) |
Median CD4 counts cells/μL (IQR) at baseline | 259 (137–382) |
Median CD4 counts cells/μL (IQR) post 6 months | 572 (347–781) |
IQR, interquartile range; CD4, cluster of differentiation 4.
Considering the presence of TDF, FTC or EFV in the plasma or hair of participants, 41/77 (53.2%) were found to have been exposed to ARVs prior to treatment initiation. Thirty-four (
Characteristics of participants with at least one antiretroviral drug detected in hair or plasma prior to treatment initiation.
No | Sample code | Sex | Age | Drug concentrations (ng/mg) in hair at baseline |
Drug concentrations (ng/mL) in plasma at baseline |
||||
---|---|---|---|---|---|---|---|---|---|
TDF | FTC | EFV | TDF | FTC | EFV | ||||
1 | AHDR-R280 | F | 33 | 0.028 | 00.000 | 00.000 | 00.000 | 00.000 | 00.000 |
2 | AHDR-R283 | M | 32 | 0.330 | 00.000 | 00.000 | 00.000 | 00.000 | 00.000 |
3 | AHDR-R287 | M | 31 | 0.027 | 0.300 | 1.070 | 00.000 | 00.000 | 00.000 |
4 | AHDR-R289 | F | 40 | 00.000 | 00.000 | 0.068 | 00.000 | 00.000 | 00.000 |
5 | AHDR-R292 | F | 25 | 00.000 | 00.000 | 0.096 | 00.000 | 00.000 | 00.000 |
6 | AHDR-R295 | F | 48 | 00.000 | 0.863 | 17.432 | 00.000 | 00.000 | 1183.000 |
7 | AHDR-R296 | F | 36 | 0.025 | 00.000 | 00.000 | 19.570 | 00.000 | 00.000 |
8 | AHDR-R297 | F | 55 | 0.058 | 0.497 | 9.000 | 59.450 | 26.570 | 1051.000 |
9 | AHDR-R298 | F | 23 | 00.000 | 00.000 | 00.000 | 4.356 | 00.000 | 00.000 |
10 | AHDR-R299 | F | 37 | 00.000 | 00.000 | 0.079 | 00.000 | 00.000 | 00.000 |
11 | AHDR-R302 | F | 65 | 0.490 | 1.300 | 13.230 | 51.110 | 114.500 | 895.800 |
12 | AHDR-R306 | F | 32 | 0.025 | 00.000 | 00.000 | 00.000 | 00.000 | 00.000 |
13 | AHDR-R307 | M | 40 | 0.043 | 00.000 | 0.880 | 00.000 | 00.000 | 00.000 |
14 | AHDR-R311 | F | 42 | 00.000 | 00.000 | 0.138 | 00.000 | 00.000 | 00.000 |
15 | AHDR-R315 | F | 55 | 00.000 | 00.000 | 0.257 | 00.000 | 00.000 | 00.000 |
16 | AHDR-R319 | F | 37 | 00.000 | 00.000 | 0.075 | 15.700 | 00.000 | 00.000 |
17 | AHDR-R320 | F | 52 | 00.000 | 00.000 | 00.000 | 61.880 | 00.000 | 00.000 |
18 | AHDR-R322 | F | 57 | 0.027 | 0.201 | 16.700 | 43.890 | 139.100 | 1651.000 |
19 | AHDR-R326 | F | 23 | 0.208 | 0.930 | 5.900 | 11.670 | 00.000 | 238.300 |
20 | AHDR-R329 | F | 31 | 0.090 | 0.770 | 0.290 | 00.000 | 00.000 | 00.000 |
21 | AHDR-R332 | M | 43 | 0.032 | 00.000 | 00.000 | 00.000 | 00.000 | 00.000 |
22 | AHDR-R337 | F | 42 | 0.088 | 00.000 | 2.400 | 00.000 | 00.000 | 00.000 |
23 | AHDR-R338 | F | 28 | 0.030 | 00.000 | 0.190 | 00.000 | 00.000 | 00.000 |
24 | AHDR-R339 | F | 31 | 00.000 | 00.000 | 0.330 | 00.000 | 00.000 | 00.000 |
25 | AHDR-R346 | F | 32 | 00.000 | 00.000 | 1.200 | Not done | Not done | Not done |
26 | AHDR-R347 | F | 48 | 00.000 | 00.000 | 00.000 | 14.160 | 00.000 | 00.000 |
27 | AHDR-R348 | F | 25 | 0.038 | 00.000 | 00.000 | 18.030 | 00.000 | 00.000 |
28 | THDR-R26 | F | 22 | 00.000 | 00.000 | 0.170 | Not done | Not done | Not done |
29 | AHDR-S083 | M | 28 | 00.000 | 0.230 | 00.000 | 25.530 | 00.000 | 00.000 |
30 | AHDR-S087 | F | 30 | 00.000 | 00.000 | 00.000 | 35.250 | 00.000 | 00.000 |
31 | AHDR-S090 | F | 39 | 00.000 | 00.000 | 00.000 | 36.430 | 00.000 | 00.000 |
32 | AHDR-S091 | F | 35 | 00.000 | 00.000 | 00.000 | 23.060 | 00.000 | 00.000 |
33 | AHDR-S094 | F | 53 | 00.000 | 00.000 | 0.267 | 35.330 | 290.600 | 769.900 |
34 | AHDR-S095 | F | 23 | 00.000 | 00.000 | 0.071 | 28.820 | 00.000 | 00.000 |
35 | AHDR-S096 | M | 39 | 00.000 | 00.000 | 00.000 | 52.860 | 00.000 | 00.000 |
36 | AHDR-S099 | F | 26 | 00.000 | 00.000 | 0.110 | 25.030 | 00.000 | 00.000 |
37 | AHDR-S103 | F | 22 | 00.000 | 00.000 | 0.180 | 223.300 | 13.100 | 00.000 |
38 | AHDR-S105 | F | 21 | 00.000 | 00.000 | 0.104 | 00.000 | 00.000 | 00.000 |
39 | AHDR-S112 | F | 27 | 00.000 | 0.210 | 2.670 | 13.030 | 00.000 | 9.500 |
40 | AHDR-U01 | F | 22 | 00.000 | 00.000 | 00.000 | 27.310 | 00.000 | 00.000 |
41 | AHDR-U02 | M | 20 | 00.000 | 0.270 | 8.000 | 31.310 | 00.000 | 00.000 |
TDF, tenofovir; FTC, emtricitabine; EFV, efavirenz.
Emtricitabine and EFV were observed in the plasma or hair of 12/77 (15.6%) and 25/77 (32.4%) of participants, respectively. Six (
Baseline drug resistance/mutation data were available for 13 of the 41 participants (
Participants having at least one drug at baseline in either the hair or plasma matrix with their drug resistance profiles.
No | Sample code | Sex | Drug resistance profiles |
Drugs detected in hair at baseline |
Drugs detected in plasma at baseline |
||||||
---|---|---|---|---|---|---|---|---|---|---|---|
DRM at > 20% threshold | DRM at > 5% threshold | DRM at > 1% threshold | TDF | FTC | EFV | TDF | FTC | EFV | |||
1 | AHDR-R 289 | F | NO DRM | NO DRM | K65R (NRTI) | 0.028 | 00.000 | 00.000 | 00.000 | 00.000 | 00.000 |
2 | AHDR-R 297 | F | NO DRM | K103N (NNRTI) | K103N (NNRTI) | 0.058 | 0.497 | 9.000 | 59.450 | 26.570 | 1051.000 |
3 | AHDR-R 299 | F | NO DRM | K103N (NNRTI) | K103N (NNRTI) | 00.000 | 00.000 | 0.079 | 00.000 | 00.000 | 00.000 |
4 | AHDR-R 302 | F | NO DRM | NO DRM | NO DRM | 0.490 | 1.300 | 13.230 | 51.110 | 114.500 | 895.800 |
5 | AHDR-R 307 | M | NO DRM | NO DRM | NO DRM | 0.043 | 00.000 | 0.880 | 00.000 | 00.000 | 00.000 |
6 | AHDR-R 319 | F | NO DRM | NO DRM | N88D (PI) | 00.000 | 00.000 | 0.075 | 15.700 | 00.000 | 00.000 |
7 | AHDR-R 339 | F | NO DRM | NO DRM | K65R (NRTI) | 00.000 | 00.000 | 0.330 | 00.000 | 00.000 | 00.000 |
8 | AHDR-R 347 | F | NO DRM | NO DRM | NO DRM | 00.000 | 00.000 | 00.000 | 14.160 | 00.000 | 00.000 |
9 | AHDR-R 348 | F | K103N (NNRTI) | K103N, V106A (NNRTI) | K65R, K219E (NRTI), K103N, V106A (NNRTI) | 0.038 | 00.000 | 00.000 | 18.030 | 00.000 | 00.000 |
10 | AHDR-S 94 | F | NO DRM | NO DRM | NO DRM | 00.000 | 00.000 | 0.267 | 35.330 | 290.600 | 769.900 |
11 | AHDR-S 95 | F | NO DRM | NO DRM | NO DRM | 00.000 | 00.000 | 0.071 | 28.820 | 00.000 | 00.000 |
12 | AHDR-S 103 | F | NO DRM | NO DRM | K65R (NRTI) | 00.000 | 00.000 | 0.180 | 223.300 | 13.100 | 00.000 |
13 | AHDR-S 112 | F | NO DRM | NO DRM | I50V (PI) | 00.000 | 0.210 | 2.670 | 13.030 | 00.000 | 9.500 |
DRM, drug-resistant mutation; NRTI, nucleoside reverse transcriptase inhibitors; NNRTI, non-nucleoside reverse transcriptase inhibitor; PI, protease inhibitor; TDF, tenofovir; FTC, emtricitabine; EFV, efavirenz.
We then looked at the resistance profiles of those, 36 of 77 (46.8%), who did not have ARVs in their plasma or hair. Data for this assessment were available for 18/36 (50%) participants. Seven participants 7/18 (38.9 %) had no DRMs. Of the 11 participants (61%) who harboured resistance mutations, the distribution was as follows: K65R (5.5%); D67N (5.6%); K65R, D67G, Y181C, G190E, V82A, I84V were observed in 11/18 (61%) participants at the > 20%, > 5% and > 1% threshold levels, respectively. Details of the distribution of these mutations are shown in
Participants with no drug at baseline in either the hair or plasma matrix with their drug resistance profiles.
No | Sample code | Sex | Drug resistance profiles |
Drugs detected in hair at baseline |
Drugs detected in plasma at baseline |
||||||
---|---|---|---|---|---|---|---|---|---|---|---|
DRM at > 20% threshold | DRM at > 5% threshold | DRM at > 1% threshold | TDF | FTC | EFV | TDF | FTC | EFV | |||
1 | AHDR-R294 | F | NO DRM | NO DRM | NO DRM | 00.000 | 00.000 | 00.000 | 00.000 | 00.000 | 00.000 |
2 | AHDR-R300 | F | NO DRM | NO DRM | K65R (NRTI), V82A (PI) | 00.000 | 00.000 | 00.000 | 00.000 | 00.000 | 00.000 |
3 | AHDR-R303 | F | K65R (NRTI) | NO DRM | Y181C (NNRTI) | 00.000 | 00.000 | 00.000 | 00.000 | 00.000 | 00.000 |
4 | AHDR-R305 | F | NO DRM | NO DRM | NO DRM | 00.000 | 00.000 | 00.000 | 00.000 | 00.000 | 00.000 |
5 | AHDR-R308 | F | NO DRM | D67N (NRTI) | NO DRM | 00.000 | 00.000 | 00.000 | 00.000 | 00.000 | 00.000 |
6 | AHDR-R313 | F | NO DRM | NO DRM | K65R (NRTI) | 00.000 | 00.000 | 00.000 | 00.000 | 00.000 | 00.000 |
7 | AHDR-R324 | F | NO DRM | NO DRM | NO DRM | 00.000 | 00.000 | 00.000 | 00.000 | 00.000 | 00.000 |
8 | AHDR-R330 | F | NO DRM | NO DRM | NO DRM | 00.000 | 00.000 | 00.000 | 00.000 | 00.000 | 00.000 |
9 | AHDR-R331 | F | NO DRM | NO DRM | NO DRM | 00.000 | 00.000 | 00.000 | 00.000 | 00.000 | 00.000 |
10 | AHDR-R335 | F | NO DRM | NO DRM | I84V (PI) | 00.000 | 00.000 | 00.000 | 00.000 | 00.000 | 00.000 |
11 | AHDR-R340 | F | NO DRM | NO DRM | D67G (NRTI) | 00.000 | 00.000 | 00.000 | 00.000 | 00.000 | 00.000 |
12 | AHDR-R342 | F | NO DRM | NO DRM | K65R(NRTI),G190E (NNRTI) | 00.000 | 00.000 | 00.000 | 00.000 | 00.000 | 00.000 |
13 | AHDR-R343 | F | NO DRM | NO DRM | K65R (NRTI) | 00.000 | 00.000 | 00.000 | 00.000 | 00.000 | 00.000 |
14 | AHDR-R344 | F | NO DRM | NO DRM | K65R (NRTI), V82A (PI) | 00.000 | 00.000 | 00.000 | 00.000 | 00.000 | 00.000 |
15 | AHDR-S088 | F | NO DRM | NO DRM | NO DRM | 00.000 | 00.000 | 00.000 | 00.000 | 00.000 | 00.000 |
16 | AHDR-S102 | F | NO DRM | NO DRM | NO DRM | 00.000 | 00.000 | 00.000 | 00.000 | 00.000 | 00.000 |
17 | AHDR-S110 | F | NO DRM | NO DRM | K65R (NRTI) | 00.000 | 00.000 | 00.000 | 00.000 | 00.000 | 00.000 |
18 | AHDR-S111 | F | NO DRM | NO DRM | K65R (NRTI) | 00.000 | 00.000 | 00.000 | 00.000 | 00.000 | 00.000 |
DRM, drug-resistant mutation; NRTI, nucleoside reverse transcriptase inhibitors; NNRTI, non-nucleoside reverse transcriptase inhibitor; PI, protease inhibitor; TDF, tenofovir; FTC, emtricitabine; EFV, efavirenz.
More DRMs were observed in participants who had prior exposure to ARV drugs compared to those who did not, although this difference was not significant (χ2 = 0.798;
We looked at the adherence profile at 6 months post-treatment initiation. Of the 77 participants who were recruited at initiation, paired plasma and hair samples were available for only 21, at the 6 month assessment. The adherence-analysis using plasma and hair levels of TDF/FTC/EFV, was based on the 21 participants. The concentrations of these ARVs administered as a FDC tablet, were within an acceptable adherence-range level for all 21 at their 6 month assessment. This observation is supported by a significant increase in the median CD4 = 572 (IQR 347–781) cells/µL at 6 months after starting treatment, compared to the median ART-initiating CD4 count of 259 (137–382) cells/µL (
Study participants’ adherence profiles after 6 months of antiretroviral therapy.
No | Sample code | Age | Sex | CD4 at treatment initiation (cells/µL) | CD4 6 months post-treatment (cells/µL) | Viral load at treatment initiation (copies/mL) | Viral load 6 months post-treatment (copies/mL) | Drugs detected at baseline |
Drugs detected 6 months post-treatment |
||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Hair |
Plasma |
Hair |
Plasma |
||||||||||||||||
TDF | FTC | EFV | TDF | FTC | EFV | TDF | FTC | EFV | TDF | FTC | EFV | ||||||||
1 | AHDR-R 295 | 48 | F | 547 | 672 | 207 | 20 | 00.000 | 0.863 | 17.432 | 00.000 | 00.000 | 1183.000 | 0000.000 | 9.958 | 16.298 | 61.490 | 139.700 | 287.300 |
2 | AHDR-R 298 | 23 | F | 497 | Not done | 6950 | 20 | 00.000 | 00.000 | 00.000 | 4.356 | 00.000 | 00.000 | 0.124 | 0.019 | 12.026 | 58.750 | 182.500 | 2071.000 |
3 | AHDR-R 305 | 31 | F | 664 | Not done | 31100 | 20 | 00.000 | 00.000 | 00.000 | 00.000 | 00.000 | 00.000 | 0.053 | 4.205 | 22.200 | 95.970 | 337.800 | 3738.000 |
4 | AHDR-R 311 | 42 | F | Not done | Not done | 137000 | 22 | 00.000 | 00.000 | 0.138 | 00.000 | 00.000 | 00.000 | 0.138 | 2.027 | 27.166 | 108.700 | 352.200 | 3330.000 |
5 | AHDR-R 315 | 55 | F | 416 | Not done | 919000 | Not done | 00.000 | 00.000 | 0.257 | 00.000 | 00.000 | 00.000 | 0.060 | 3.981 | 22.062 | 189.900 | 228.000 | 2419.000 |
6 | AHDR-R 318 | 39 | F | 113 | Not done | 2600000 | 38 | 00.000 | 00.000 | 00.000 | 00.000 | 00.000 | 00.000 | 0.124 | 1.659 | 9.873 | 72.850 | 456.000 | 3562.000 |
7 | AHDR-R 328 | 36 | F | 484 | 786 | Not done | 20 | 00.000 | 00.000 | 00.000 | 00.000 | 00.000 | 00.000 | 0.055 | 0.656 | 6.969 | 108.500 | 287.400 | 3450.000 |
8 | AHDR-R 331 | 35 | F | 345 | 565 | Not done | 20 | 00.000 | 00.000 | 00.000 | 00.000 | 00.000 | 00.000 | 0.088 | 1.165 | 10.843 | 83.000 | 304.600 | 1283.000 |
9 | AHDR-R 338 | 28 | F | 1007 | 1196 | Not done | 39.36 | 0.030 | 00.000 | 0.190 | 00.000 | 00.000 | 00.000 | 0.024 | 0.670 | 10.900 | 35.720 | 104.900 | 1134.000 |
10 | AHDR-R 340 | 36 | F | 278 | 592 | Not done | 48 | 00.000 | 00.000 | 00.000 | 00.000 | 00.000 | 00.000 | 0.065 | 2.243 | 6.574 | 66.180 | 194.200 | 1688.000 |
11 | AHDR-S 88 | 31 | F | 191 | 142 | 16900 | 20 | 00.000 | 00.000 | 00.000 | 00.000 | 00.000 | 00.000 | 0.087 | 4.563 | 29.440 | 73.200 | 418.900 | 6685.000 |
12 | AHDR-S 92 | 46 | F | 164 | Not done | 3390 | 20 | 00.000 | 00.000 | 00.000 | 00.000 | 00.000 | 00.000 | 0.077 | 3.531 | 10.926 | 69.100 | 229.800 | 2671.000 |
13 | AHDR-S 94 | 53 | F | 101 | 347 | 100000 | 20 | 00.000 | 00.000 | 0.267 | 35.330 | 290.600 | 769.900 | 0.039 | 1.201 | 9.953 | 121.400 | 353.100 | 1944.000 |
14 | AHDR-S 101 | 30 | M | 500 | 572 | 9120 | 20 | 00.000 | 00.000 | 00.000 | 00.000 | 00.000 | 00.000 | 0.053 | 0.152 | 13.088 | 0000.000 | 18.480 | 506.800 |
15 | AHDR-S 102 | 43 | F | 567 | 781 | 2746469 | 20 | 00.000 | 00.000 | 00.000 | 00.000 | 00.000 | 00.000 | 0.256 | 3.336 | 30.200 | 95.000 | 290.400 | 3661.000 |
16 | AHDR-S 106 | 33 | F | 334 | Not done | 68941 | 71 | 00.000 | 00.000 | 00.000 | 00.000 | 00.000 | 00.000 | 0.061 | 2.536 | 30.789 | 82.700 | 352.900 | 2999.000 |
17 | AHDR-S 110 | 29 | F | 74 | Not done | 17600 | 20 | 00.000 | 00.000 | 00.000 | 00.000 | 00.000 | 00.000 | 0.031 | 10.359 | 34.847 | 197.300 | 530.300 | 00.000 |
18 | AHDR-S 111 | 35 | F | Not done | Not done | 6400 | 20 | 00.000 | 00.000 | 00.000 | 00.000 | 00.000 | 00.000 | 0.408 | 2.034 | 1.430 | 57.820 | 265.400 | 1180.000 |
19 | AHDR-S 113 | 54 | F | Not done | 1148 | 5490 | 20 | 00.000 | 00.000 | 00.000 | 00.000 | 00.000 | 00.000 | 0.161 | 8.591 | 29.272 | 96.610 | 344.000 | 2781.000 |
20 | AHDR-S 117 | 41 | F | 85 | 261 | 84600 | 20 | 00.000 | 00.000 | 00.000 | 00.000 | 00.000 | 00.000 | 0.051 | 4.557 | 36.880 | 117.800 | 229.600 | 4358.000 |
21 | AHDR-S 120 | 37 | F | 186 | 415 | 3490 | 20 | 00.000 | 00.000 | 00.000 | 00.000 | 00.000 | 00.000 | 0.995 | 1.874 | 55.300 | 101.600 | 483.100 | 15670.000 |
TDF, tenofovir; FTC, emtricitabine; EFV, efavirenz; CD4, cluster of differentiation 4.
Tenofovir has been the anchor component of most standard first-line ART regimens in low and middle-income countries (LMICs). Taking into account the presence of TDF alone, we observed that 31/77 (40.3%) of the participants had TDF in either their plasma or hair. The concentration of TDF detected in plasma or hair falls within the range of values described in the literature for individuals who are adherent to TDF.
The study participants had answered ‘no’ to a question on whether they had received ARV medications in any form or programme prior to their current diagnosis and treatment initiation. The detection then of ARV drugs in their tissues requires explanation: some of the women may have previously been on a prevention of mother-to-child transmission (PMTCT) programme,
Surprisingly, no significant difference in the prevalence of drug resistance mutations was observed between the participants in whom ARV drugs were detected and those in whom ARV drugs were not detected prior to initiation of treatment. However, mutation distribution amongst both groups was different. Possible explanations for this could be the exposure to TDF for a significant duration of time, or high frequency of minor variants detected using high-sensitivity sequencing technologies,
A monotonic relationship was observed in the CD4+ cell count and viral copy number in all 21 study participants post-treatment. Several studies have established that VL below the level of detection at post-treatment initiation (HIV RNA < 20 copies/mL – 75 copies/mL, depending on the assay used) indicates optimal viral suppression, and such observations are normal in successfully treated patients and do not predict virological failure.
The data presented here should be considered in the context of some limitations. Firstly, the small sample size is unlikely to be representative of all who are initiated on ART in the Limpopo province. Secondly, drug resistance data were not available for all 21 participants, who were assessed for adherence based on CD4+ cell count, VL and ARV levels at 6 months post-treatment initiation. So, it was not possible to determine whether or not they harboured resistance mutations going into treatment, and to make the call regarding whether or not the benefits of adherence cancel out the potential impact of these mutations on treatment outcome (i.e. a significant increase in CD4+ cell count, and undetectable VL). Despite these shortcomings, we have objectively shown, by detecting ARV drugs in plasma or hair, that clinicians may be unknowingly recruiting non-drug naïve individuals into HIV treatment programmes on the standard first-line regimen. These results suggest that reporting previous ARV drug exposure accurately is likely to be of benefit in identifying individuals at increased risk of harbouring resistant mutations, and who will require closer follow-up to ensure long-term viral suppression. Disclosure of prior exposure could also assist in the choice of the initial ARV drug regimen, or opting for intensive adherence for these individuals to achieve the desirable treatment outcomes.
Non-disclosure of previous ART exposure is frequent. Measurement of hair and plasma ARV drugs in PLWH who are NOT yet on ART may identify a group at risk of subsequent treatment failure, and would therefore be a priority group with regard to ART management and follow-up. The presence of resistance related viral mutations and of ARVs in the plasma and hair of ‘ART-naive’ PLWH at the time of ART initiation, suggests the need of surveillance programmes to monitor primary drug-resistance and the establishment of an ‘early-warning’ system to monitor primary HIV-resistance in all areas where ART initiation is provided.
The authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article.
L.G.M.-R. and P.O.B. were responsible for conception and supervision. L.A.M.T. and L.G.M.-R. contributed equally to the article and were responsible for data collection and preparation of the first draft. L.G.M.-R., L.A.M.T., N.D.M., D.K. and P.O.B. were responsible for data analysis, manuscript revision and approval for submission.
L.G.M.-R.’s work on adherence to antiretrovirals was supported by the National Research Foundation (NRF), under the Thuthuka Award (Gun 107394). Additional funding was obtained from P.O.B.’s work on sustainability of antiretrovirals supported by the South African Medical Research Council (SAMRC), under the RCDI Programme, through funding received from the South African National Treasury; and from NRF support (GUN 109312, GUN86037). N.D.M. has postdoctoral support from SAMRC RCDI (P/C 57009).
The data that support the findings of this study are available from the corresponding author, L.G.M.-R., upon reasonable request.
The views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors.
CD4+ cell counts were measured on the BD FACSPresto instrument (Beckton Dickinson) at baseline and after 6 months on ART. Viral loads were determined by using the HIV-1 RNA 3.0 assay (Bayer Healthcare, Leverkusen, Germany). Plasma was prepared by centrifugation of 5 mL whole blood at 4000 rpm for 5 min, and stored in sterile cryovials at –80°C for subsequent experiments.
Available genetic drug resistance data were obtained by next-generation sequencing on an Illumina MiniSeq platform. Sequences were checked for quality with the Fast quality control (QC) program and trimmed using Geneious Prime version 2020.1.2.
The analytical standards used in both hair and plasma experiments were
The analytes were isolated from the biological matrix by methanol protein precipitation followed by solid phase extraction. The internal standards (ISTD) were spiked into the extract solution to a concentration of ~1000 ng/mL Efavirenz-d5, ~150 ng/mL Lamivudine and ~500 ng/mL Tenofovir-d7, and the ISTD working solution was added to each sample excluding blank samples.
Calibration standards (STDs) and QC samples were prepared gravimetrically in human K2EDTA plasma. A stock solution of each analyte was prepared and used to spike a pool of plasma for the preparation of the STDs. For the preparation of the QCs, a second stock solution of each analyte was prepared and used to spike a pool of plasma. Each pool was then serially diluted with normal blank plasma to attain the desired concentrations and within 1% of target concentrations according to a standard protocol. The target concentrations are assigned as the nominal concentrations. The STDs and QCs were aliquoted and stored at –70°C for subsequent analysis.
The run-containing study samples comprised eight STD levels over the range of 20.00 ng/mL – 2560 ng/mL, with six levels of QC samples extending over this range. Two replicates were included per level for each STD and each QC sample. One blank sample and one zero sample were included in the study sample run.
Approximately 2 mg of hair was weighed in a glass Kimble-tube and a buffer solution consisting of a mixture of trifluoroacetic acid, water and methanol was added. After shaking for 20 h, 1 mL of the extract was evaporated under nitrogen and reconstituted in a solution mixture of methanol and formic acid in water. An ISTD working solution with a concentration of ~400 ng/mL EFV-d5, ~400 ng/mL 3TC and ~50 ng/mL TFV-d7 was prepared, and the working solution was added to each sample excluding blank samples.
Standards and QCs were prepared gravimetrically in a buffer solution. A stock solution of each analyte was prepared and used to spike a pool of buffer for the preparation of the STDs. Quality controls were obtained by preparing a second stock solution of each analyte and using it to spike a pool of buffer. Each pool was then serially diluted with buffer to attain the desired concentrations, acceptable within 1% of the target concentrations. The target concentrations are assigned as the nominal concentrations. The STDs and QCs were aliquoted into individual polypropylene tubes and stored at –20°C until required for the analysis of samples.
The LC-MS/MS comprised an autosampler, binary pump, column compartment and sample cooler (AGILENT), with the SCIEX – API4000 and SCIEX – API5500 used for mass spectrometry in the plasma and hair analysis, respectively. The analytical column used for liquid chromatographic separation for both matrices was the Phenomenex® Gemini C18, 150 × 2.00 mm, 5 µm and the mobile phases used (A [100% methanol] and B [0.1% formic acid]) were delivered with a gradient. The autosampler, equipped with a 96-well tray, was used to inject 5 µL of each plasma sample onto the column; 10 µL of each hair sample was injected onto the column.
For each matrix run, the mass spectrometer was set to electrospray ionisation (ESI) in positive ionization mode, for all three analytes. The multiple reaction monitoring (MRM) mode for each analyte and IS was set as follows:
The qualification run was validated for system suitability (SYS), accuracy, precision, ISTD interference, dilution integrity and carry-over.