Urine Tenofovir Point-of-care Test to Identify Patients in Need of ART Adherence Support (UTRA Study)
Phase: NA
Conditions: Risk Reduction
Sponsor: University of California, San Francisco

 
 
 
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Urine tenofovir point-of-care test to identify patients in need of ART 
adherence support (UTRA study): Aim 3 
 
CORE STUDY TEAM: 
Gert van Zyl: Co-principal Investigator 
Division of Medical Virology, Faculty of Medicine and Health Sciences, Stellenbosch University 
 
guvz@sun.ac.za  
Catherine Orrell: Co-principal Investigator 
 
Desmond Tutu Health Foundation, University of Cape Town 
 
Catherine.Orrell@hiv-research.org.za 
Monica Gandhi: Co-principal Investigator 
 
Center for AIDS Research (CFAR), University of California San Francisco (UCSF) 
 
Monica.Gandhi@ucsf.edu  
David Glidden: Biostatistician 
 
University of California San Francisco (UCSF) 
 
David.Glidden@ucsf.edu  
Graeme Hoddinott: Socio-behavioural Science Lead 
Desmond Tutu TB Centre, Department of Paediatrics and Child Health, Faculty of Medicine and 
Health Sciences, Stellenbosch University 
 
graemeh@sun.ac.za  
Eric Decloedt: Clinical pharmacologist 
Division of Clinical Pharmacology, Department of Medicine, Faculty of Medicine and Health 
Sciences, Stellenbosch University 
 
ericdecloedt@sun.ac.za 
Tracy Kellermann: Research pharmacologist  
Division of Clinical Pharmacology, Department of Medicine, Faculty of Medicine and Health 
Sciences, Stellenbosch University    
tkellermann@sun.ac.za  
Marije van Schalkwyk: Clinician 
Division of Infectious Diseases, Department of Medicine, Faculty of Medicine and Health 
Sciences    
Stellenbosch University 
 
marije@sun.ac.za 

 
 
 
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SITE RESEARCHERS: 
Yashna Singh:  
 
Desmond Tutu Health Foundation, University of Cape Town 
 
yashna.singh@hiv-research.org.za  
Lauren Jennings:  
 
Desmond Tutu Health Foundation, University of Cape Town 
 
lauren.jennings@hiv-research.org.za   
 
SPONSOR  
 
 
 
 
 
 
FUNDER 
Not applicable: Investigator initiated research 
 
 
NIH/SA MRC R01-AI152119 
 
 

 
 
 
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Table of Contents 
BACKGROUND AND RATIONALE ......................................................................................................................... 5 
AIM: .................................................................................................................................................................. 9 
HYPOTHESES ...................................................................................................................................................... 9 
STUDY DESIGN ................................................................................................................................................... 9 
Study site ................................................................................................................................................................... 9 
Recruitment and enrolment: ..................................................................................................................................... 9 
Participant Inclusion criteria: ............................................................................................................................. 10 
Participant Exclusion criteria: ............................................................................................................................ 10 
Study Procedures ..................................................................................................................................................... 10 
Schedule of events: .................................................................................................................................................. 10 
Visit procedures ....................................................................................................................................................... 11 
Randomisation: ....................................................................................................................................................... 13 
Standard of Care adherence support: ..................................................................................................................... 13 
Intervention Enhanced Adherence Support: ............................................................................................................ 14 
Visit Windows: ......................................................................................................................................................... 14 
Retention ................................................................................................................................................................. 14 
Qualitative procedures: ........................................................................................................................................... 14 
Lab management .................................................................................................................................................... 15 
Data management .................................................................................................................................................. 15 
Study Withdrawal: ................................................................................................................................................... 16 
Adverse event management ................................................................................................................................... 16 
STATISTICAL ANALYSIS: ..................................................................................................................................... 16 
Sample size: ............................................................................................................................................................. 16 
Primary outcome ..................................................................................................................................................... 16 
Secondary outcome ................................................................................................................................................. 17 
Analysis plan ............................................................................................................................................................ 17 
STUDY MONITORING ........................................................................................................................................ 17 
ETHICAL CONSIDERATIONS AND INFORMED CONSENT ...................................................................................... 17 
Ethical Conduct ........................................................................................................................................................ 17 
Compliance with the protocol ................................................................................................................................. 18 
Protocol amendments ............................................................................................................................................. 18 

 
 
 
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Informed consent and procedures ........................................................................................................................... 18 
Potential risks .......................................................................................................................................................... 18 
Potential benefits .................................................................................................................................................... 19 
CONFIDENTIALITY OF DATA ............................................................................................................................... 19 
APPENDIX A: TIMELINE AND GANTT CHART (AIMS 1-3) ...................................................................................... 20 
REFERENCES ...................................................................................................................................................... 21 
 
 

 
 
 
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BACKGROUND AND RATIONALE 
 
Viral load (VL) monitoring is essential for individual management and for treatment as prevention but 
is costly: Achieving a 90% suppression rate in patients receiving antiretroviral therapy (ART) is the third 
UNAIDS 90-90-90 target and is essential for optimizing the individual health benefits of ART. Viremic 
patients are also a source of ongoing transmission and achieving virological suppression in all patients 
with HIV is a cornerstone of treatment as prevention (TasP).1–4 Preventing transmission and improving 
individual benefit requires timely identification of patients with virologic failure (VF) in order to support 
adherence to achieve resuppression of viral load and, in cases where adequate adherence does not 
achieve resuppression, trigger second-line ART (preceded by HIV drug resistance testing in resource rich 
settings). Therefore, the World Health Organization (WHO) recommends routine VL monitoring every 6-
12 months.5 Despite evidence of the cost effectiveness of VL, this testing increases the financial burden 
of HIV monitoring in high-prevalence limited-resource settings.  For instance, South Africa currently has 
more than 7.52 million individuals living with HIV, all requiring life-long treatment and monitoring.6 
Although point-of-care (POC) VL tests may in future provide immediate feedback regarding virologic 
failure, current commercially available assays remain costly, require trained operators and have limited 
throughput.7 Therefore, treatment programs in high-burden settings continue to rely on laboratory-
based VL testing but use them less frequently than is ideal.  An alternative low-cost, low complexity 
assay that would reduce the need or frequency of VL testing without increasing the risk for failure would 
therefore help to ensure sustainability of HIV treatment programs worldwide. 
A low-cost point-of-care test to monitor adherence to antiretroviral therapy (ART) has recently been 
developed: Adherence is critical for maintaining the efficacy of ART. Self-reported adherence is subject 
to both social desirability and recall biases, so pharmacologic adherence measures, where drug 
concentrations are measured in a biological matrix, provide objective and more accurate predictors of 
virologic suppression.8-19 However, no current real-time metric of adherence to antiretrovirals (ARVs) at 
the point of care has existed until now. The current techniques to measure ARVs in plasma,20 urine,21 
dried blood spots (DBS),22 or hair,23 all require spectrometry machines and laboratory-based personnel, 
making them impractical to incorporate into routine clinical care. Antibody-based tests, when converted 
into lateral flow assays, are low-cost, enable drug detection to occur within minutes, and are easy to 
perform by non-trained personnel.24,25 The most well-known lateral flow immunoassay is the urine-
based pregnancy test. 
Tenofovir disoproxil fumarate (TDF)/ lamivudine (3TC) or emtricitabine (FTC) are the backbone of ART 
regimens used worldwide. South Africa is rolling out dolutegravir (DTG)/TDF/3TC (TLD) as first-line 
treatment across the country; and exploring the use of TLD in second-line for those failing a first-line 
non-nucleoside reverse transcriptase-based 
(NNRTI) regimen.26,27 Therefore, a low-cost 
adherence metric that could be deployed in 
resource-limited settings should target tenofovir (TFV, 
the breakdown product of TDF in plasma and urine); as 
this drug is likely to be utilized in both first- and second-
line ART. The UCSF Hair Analytical Laboratory and 
Alere/Abbott™ Rapid Diagnostics recently developed a 
Figure 1: Urine-based lateral flow assay for tenofovir  

 
 
 
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urine-based immunoassay for tenofovir (UTRA: urine tenofovir rapid assay).21,28 Using samples from a 
directly-observed therapy (DOT) study of HIV-noninfected volunteers provided TDF/FTC,29 the specificity 
and sensitivity of the immunoassay to quantitate tenofovir in urine compared to the gold standard 
method of liquid chromatography-tandem mass spectrometry (LC-MS/MS) is 99% and 94%, 
respectively.28 Moreover, the DOT study allowed us to determine the appropriate adherence cut-off for 
the point-of-care (POC) lateral flow assay. An immunoassay cut-off of 1500ng/mL accurately classified 
98% of patients who took a dose within the past 24 hours as adherent.28 Urine collection is noninvasive30 
and preferred over blood sampling.31,32 The lateral flow assay to detect tenofovir in urine has now been 
developed (Figure 1) and further validated against LC-MS/MS (preliminary data section). The test is low 
cost (<$2 per assay), provides results in ~2 minutes, and has been used in the first part of this study.  
Low-cost urine tenofovir rapid assays (UTRA) could be used more frequently than VL testing to predict 
impending failure: Low urine concentrations of tenofovir in a large pre-exposure prophylaxis (PrEP) 
demonstration project heralded future seroconversion events.33 In the setting of routine clinical care, 
this test could be performed at every visit to provide patients with ‘real-time’ feedback on their 
practices.  
There is limited information on the pattern of adherence resulting in failure on new regimens: 
Interruption of treatment for periods longer than 48 hours has been associated with an increased risk of 
failure of non-nucleoside reverse transcriptase inhibitor (NNRTI)-based regimens,34 whereas average 
adherence on protease inhibitor (PI)-based regimens is predictive of failure.35 The patterns of adherence 
associated with failure of modern integrase inhibitor regimens are not yet known. 
Beyond pharmacologic adherence testing, other biomedical assays used to unravel the etiology of 
failure include HIV VL and HIV drug resistance testing (HIVDRT). When combined, these biomedical 
assays can assess which patterns of adherence are predictive of failure (Table 1). The utility and 
feasibility of a particular biomedical assay or combination of assays to unravel the etiology of virologic 
failure is dependent on assay availability, performance, practicality, and cost. Tenofovir diphosphate 
(TFV-DP) concentrations in dried blood spots (DBS) are currently one of the most commonly used 
metrics of long-term adherence in research settings.36–38  However, their cost, complexity, and turn-
around time make them less feasible to employ as routine metrics of monitoring adherence in resource-
limited settings.   
In contrast to the longer-term measures, drug concentrations in both plasma and urine provide 
information on adherence over short time frames (1-7 days). Therefore, adherence monitoring via these 
matrices may be susceptible to “white coat adherence”, where adherence improves transiently before a 
visit (Table 1). Despite this theoretical concern, this phenomenon has not yet been observed; indeed, 
plasma concentrations of TFV/FTC served as the “gold standard” adherence metric in every one of the 
placebo-controlled trials of TDF/FTC-based PrEP.20,39–46 Across these trials, plasma PrEP concentrations 
provided robust and critical information for interpreting trial results. However, further study, including 
Aim 1 of this study, which collected both short-term and long-term adherence metrics, will determine 
the susceptibility of the short-term urine assay to “white coat” patterns in more real-world settings. 

 
 
 
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In our setting, undetectable urine tenofovir was strongly associated with drug resistance in those on 
NNRTI-based ART. This study is the second part of a proposal with three aims. In Aim 1, a pilot cross-
sectional study, we enrolled patients from the Gugulethu Community Health Centre ART service who 
were taking tenofovir-containing ART, and were at risk of virologic failure, as evidenced by a recent 
raised VL and/or gap in treatment (confirmed by pharmacy refill data). In an interim analysis we found 
that only 2 of 48 (4%) participants to date have had undetectable UTRA tests (despite 38% experiencing 
virologic failure). Both cases with negative UTRA tests had either very low or undetectable TFV-DP 
concentrations in DBS (showing high specificity for poor adherence).  The combination of an 
undetectable UTRA test and VF was 100% sensitive in predicting resistance on a low genetic barrier 
efavirenz-based regimen. Therefore, the combination of UTRA and VL testing could be used to target 
follow-up resistance testing or to prioritize transition to second-line regimens.  
The utility of adherence reinforcement may, however, be dependent on the genetic barrier of the ART 
regimen and whether episodes of reduced adherence are detected and managed early. Periods of 
treatment interruption are strongly predictive of treatment failure and drug resistance on NNRTI-based 
regimens.34,47 Indeed, many of these patients may already have drug resistance when identified with 
virologic failure48,49  and may not achieve VL suppression despite high levels of adherence currently.50 In 
contrast, drug resistance may take much longer to develop in patients treated with antiretrovirals with a 
high genetic barrier to resistance, such as protease inhibitor (PI) and second-generation integrase strand 
transfer inhibitor (INSTI), with adherence support allowing subsequent VL re-suppression.51,52  
UTRA may help health care workers to elicit reasons for poor adherence: Aim 2 of this study assessed 
patients’ and healthcare workers’ perceptions about integrating the UTRA into adherence support. We 
conducted a series of once-off in-depth qualitative interviews with people living with HIV (PLHIV) (n=25) 
and health workers (n=5) from the Gugulethu CHC. The Aim 1 cohort was purposely sampled to maintain 
diversity in age and gender. Health workers were purposively sampled for ‘rich-cases’ (i.e., people with 
extensive experience in delivering ART services to patients in the setting) and diversity by profession. 

 
 
 
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Many PLHIV were willing to use UTRA because they were interested to know their adherence. Waiting 
for lab-based test results means that informing people about adherence is often long-delayed and 
typically only occurs in the context of a remedial intervention for poor adherence. Contrary to 
expectations that UTRA may be perceived as unwanted additional monitoring, the PLHIV in this 
evaluation were enthused by the opportunity to receive their results in real-time which they believed 
could lead to constructive discussions with their health workers. Participants were comfortable 
providing the urine, as it is a harmless, non-invasive procedure. In addition, PLHIV participants reported 
that the UTRA test would encourage them to take their treatment regularly because they could then 
show off this good adherence at their next clinic visit. Health workers reported that UTRA may offer 
earlier and more accurate assessment of patients’ adherence, so enabling them to prepare earlier 
intervention on patients at risk of virologic failure.  
Low failure rate and low rates of resistance in patients receiving high genetic barrier regimens warrant 
novel approaches to monitoring therapy success: Integrase strand transfer inhibitor (INSTIs) containing 
regimens, have been associated with rapid rates of VL suppression,53–55 and favorable side effect 
profiles. With the use of second generation INSTIs, specifically dolutegravir (DTG), the development of 
INSTI-associated mutations in the face of virologic failure when used as first-line therapy in INSTI-naïve 
patients is rare.56 Therefore, the planned rollout of TDF, lamivudine and DTG (TLD) fixed-dose-
combination therapy as first-line, or possibly second-line, therapy worldwide will require a new 
approach to therapeutic monitoring, as drug resistance will be rare and switching to a second-line 
regimen infrequently warranted.  
Drug concentration monitoring, when applied to drugs with a high genetic barrier to resistance, has 
proven useful: Protease inhibitors have a high genetic barrier to resistance. In a large study of protease 
inhibitor-based 2nd line ART failure from South Africa (the ITREMA study),57 protease inhibitor (PI) drug 
concentrations were measured in plasma at the time of failure to evaluate whether they were predictive 
of viral resistance to the PI (Figure 2) PI viral resistance was shown to be infrequent (26.7%) and 
negative PI concentrations excluded PI-associated mutations with a high degree of certainty (negative 
predictive value 95%). This argues for drug concentration testing when drugs with a higher genetic 
barrier to resistance are used to assess the subsequent necessity of costly resistance testing. A similar 
relationship is expected for DTG-based regimens, as we assess our assay during the roll-out of TLD as 
first- and second-line, given that DTG has a high genetic barrier to resistance like PIs. 
In the third part of this study, AIM 3, we will conduct a randomised study to determine the utility, 
feasibility and acceptability of UTRA as an adherence support tool in people on antiretroviral therapy 
regimens containing high-genetic barrier antiretrovirals e.g. dolutegravir and boosted lopinavir.  

 
 
 
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AIM:  
▪ 
To determine the impact of feedback from the UTRA in an adherence-challenged population 
taking ART with a high genetic barrier to resistance, as compared to standard-of–care (SoC) 
adherence support. 
▪ 
To assess the acceptability and feasibility of integrating the UTRA into adherence support 
amongst patients and healthcare workers. 
HYPOTHESES 
▪ 
Providing real-time information from the UTRA will allow extra opportunities to address poor 
adherence in the intervention arm and will result in improved retention and virological 
outcomes compared to the SoC group.  
▪ 
UTRA will be acceptable and feasible to participants and health care providers.  
STUDY DESIGN 
This is a randomised study, recruiting 200 people taking TDF-based ART. Participants will be randomised 
1:1 to intervention versus standard enhanced adherence counselling. Intervention participants will 
receive an adherence support package informed by feedback from the urine-based test. The study will 
assess the impact of the adherence test on VL suppression rates in each arm (without necessitating a 
regimen switch) at 12 months after enrolment. Enrolling participants with adherence challenges 
increases efficiency and mirrors the population requiring adherence support in our setting.  
STUDY SITE 
The study will take place at the Hannan Crusaid Treatment Centre (HCTC), a primary health care facility 
within the Gugulethu Community Health Centre (CHC) that is broadly representative of the spectrum of 
primary health care services across South Africa. Gugulethu (population 400 000) is located 15 
kilometres (km) outside Cape Town. Residents are predominantly of low socioeconomic status and the 
majority use free, local public sector health services. HCTC is one of the Western Cape’s largest ART 
clinics with more than 18000 patients initiated on ART in the past decade and more than 8500 currently 
enrolled in care (January 2021).  
RECRUITMENT AND ENROLMENT:  
The study will recruit 200 participants attending the HCTC for ART who are at current increased risk of 
virologic failure, due to having a previously recorded viral breakthrough (≥50 copies/ml). 
Individuals will be identified at routine clinic appointments through a folder review conducted by study 
staff. These patients will be approached by study staff and the study will be explained to them. Should 
they be interested, they will be screened for eligibility. Full written informed consent in their chosen 
local language will be obtained prior to any study procedures. If eligible they will be invited to 
participate in the study. The informed consent document will explain the study in detail and briefly 
outline all procedures.  
 

 
 
 
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Participant Inclusion criteria:  
• 
≥18 years old  
• 
Willing and able to provide written informed consent 
• 
HIV-infected, receiving or (re-)starting a tenofovir-based ART regimen  
• 
Current ART regimen includes at least one drug with a high genetic barrier to resistance e.g. 
dolutegravir, atazanavir/ritonavir, darunavir/ritonavir or lopinavir/ritonavir. 
• 
Any previous raised viral load >50 copies/ml (after ART initiation). 
• 
Willing and able to comply with laboratory tests and other study procedures 
Participant Exclusion criteria:  
• 
Not willing or able to provide informed consent in any of the languages provided 
• 
Not receiving a tenofovir-based ART regimen 
• 
Any other clinical condition that in the opinion of an investigator puts the patient at increased risk 
if participating in the study. 
 
We will recruit a sub-sample of ~20 patient participants (2:1 receiving intervention to controls) from the 
above cohort for in-depth interviews. Participants will be selected by sampling for diversity (according to 
levels of adherence), rich-case sampling, and sampling to saturation.  
In addition, we will recruit ~10 health care workers from the study health facility to participate in in-
depth interviews, ideally including the 5 health care workers involved in Aim 2 (see introduction above). 
The health care workers must be willing and able to provide written informed consent; and be 
experienced in delivering adherence support at the study health facility, or knowledgeable about DoH 
ART programmes. 
STUDY PROCEDURES 
SCHEDULE OF EVENTS: 
Study procedures 
Visit 1:  
Recruit-
ment and 
screening 
Visit 2: 
Enrolment 
(with 42 
days of 
screen) 
 
Visit 3: 
Month 3 
Visit 4: 
Month 6 
Visit 5: 
Month 9 
Visit 6: 
Month 12 
Informed consent* 
X 
 
 
 
 
 
Locator information 
X 
X 
X 
X 
X 
X 
Demographic and disease 
data 
X 
 
 
 
 
 
Confirm ART regimen 
X 
X 
X 
X 
X 
X 
Eligibility check 
X 
X 
 
 
 
 
Randomisation  
 
X 
 
 
 
 
Stored blood sample (for 
viral load (VL), plasma 
tenofovir, DBS TFV-DP, 
drug resistance genotype)# 
 
X 
 
X 
 
X 
Table 2: Schedule of events 

 
 
 
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Stored blood sample for 
future analysis$ 
 
X 
 
X 
 
X 
Haematocrit 
 
X 
 
 
 
 
Height 
 
X 
 
 
 
 
Weight, hip 
circumference, waist 
circumference 
 
X 
 
X 
 
X 
Standard of care viral load 
X 
Adherence support (SoC 
or Intervention) 
X 
 
Urine sample for UTRA ** 
with adherence discussion 
- intervention arm only 
 
X 
X 
X 
X 
X 
Self-reported adherence 
assessment 
X 
X 
X 
X 
X 
X 
Pharmacy refill data 
assessment 
X 
X 
X 
X 
X 
X 
In-depth interview*** 
 
 
X 
X 
X 
 
Acceptability 
questionnaire 
 
X 
 
X 
 
X 
* Informed consent will include permission for the study team to review prescribed medication in their 
folder or from the Provincial Health Data Centre (PHDC) database and to access laboratory results via 
NHLS. 
# Plasma will be stored for study viral loads at enrolment, 6- and 12 months, which will be performed at 
the study endpoint. Cases with VL> 400 copies/mL will have genotypic drug resistance testing. DBS will 
be stored at enrolment, 6 and 12 months and plasma at the same time points; in cases with virologic 
failure and controls with suppression a nested case design will compare TFV plasma levels and TFV-DP 
in DBS. 
$ Future assays related to the study objectives will only focus on this study question which include 
investigating the factors associated with antiretroviral treatment failure (e.g. markers of cerebral small 
vessel disease, antiretroviral therapy adverse events) and antiretroviral drug exposure in plasma. 
** UTRA completed for all participants in the intervention arm at visits 2-5. Urine stored for future 
tenofovir concentration analysis. 
***Qualitative interviews completed in a subset of participants.  
 
VISIT PROCEDURES 
 
1. Demographic and disease-status data: age, gender, WHO stage and CD4 cell count as well as current 
disease status and ART history will be collected from the patient and through folder review.  
2. Blood sample for viral load: Plasma will be stored for viral load analysis at the end of the study. 
3.  HIV drug resistance testing: Plasma will be stored for HIV drug resistance testing. HIV drug 
resistance testing will be performed in participants with VF (VL<400 copies/mL) only. HIV drug 
resistance testing after first-line VF is not standard of care in resource-limited settings. In this study, 
HIV drug resistance results will be reported back to the caregivers. 

 
 
 
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4. TFV-DP testing in DBS:  50 µl of whole blood collected by venipuncture into EDTA tubes without 
centrifugation will be accurately pipetted onto Whatman 903 protein saver cards (or equivalent) (five 
50 µl spots per card). The pipetting onto the protein saver cards should be done within 1 hour of 
collection of whole blood into the EDTA. The protein saver card will be air dried at room temperature 
for at least two hours prior to storing in a sealed bag with desiccant sachets and humidity indicators. 
The sealed bags will be stored in cryoboxes at -20°C.  At the laboratory, the TFV-DP concentration will 
be determined using a validated assay. 
5. Urine sample for UTRA: Urine will be collected into a cup at each 
study visit for those in the intervention arm. The urine will be 
analysed by the LFA illustrated in Figure 3 to provide a yes/no value 
for adherence to tenofovir over the past 4 days. A counselor will 
explain and interpret the result of the POC urine tenofovir assay at 
the end of the visit. The urine will be stored to assess the 
appropriateness of UTRA thresholds for quality assurance and future 
assessment of the performance characteristics of the UTRA assay in 
detecting tenofovir in urine in this population. 
6. Self-reported adherence: Participants will be asked for the date and 
time of their last ART dose taken (to correspond with urine-based TFV measure). They will also be 
asked to provide a self-report of their adherence over the past 30 days using a three-item scale (to 
correspond with the TFV-DP in DBS measure).58 
7. Pharmacy refill adherence: Pharmacy refill data over the past calendar year will be collected from 
folder review and from the central pharmacy database associated with the Gugulethu Community 
Health Centre.  
8. Covariates of antiretroviral pharmacokinetic variability: ARV dosing time, DBS sampling time, 
weight, height, waist circumference, hip circumference, most recent creatinine (extracted from 
routine laboratory results) and haematocrit (extracted from DBS sample). 
9. Inclusion and exclusion criteria: will be checked at both screening and enrolment visits.  
10. Sample storage for future analyses: We will store blood for additional markers that may become of 
interest during the study. Future assays related to the study objectives will only focus on this study 
question which includes investigating the factors associated with antiretroviral treatment failure such 
as markers of cerebral small vessel disease, antiretroviral therapy adverse events antiretroviral drug 
exposure in plasma. 
 
 
Figure 3: Negative and positive UTRA test 

 
 
 
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RANDOMISATION: 
100 random numbers between 1 and 200 will be generated in excel and assigned as intervention by the 
data team. Study arm assignments will be placed in sequential sealed envelopes and delivered to site. 
Envelopes will be opened at the time of randomization by the Medical Officer/study coordinator.  
STANDARD OF CARE ADHERENCE SUPPORT:  
If a participant’s VL test shows elevated virus (>50copies/ml), he/she is informed of this result at the 
subsequent clinic visit and referred to adherence support. This support is given via a) a VL driven 
counselling session with a clinician and b) a peer counselling session (as per the MINIMUM PACKAGE OF 
INTERVENTIONS TO SUPPORT LINKAGE TO CARE, ADHERENCE AND RETENTION IN CARE, Standard 
Operation Procedures, published by the SA Department of Health, March 2020).  
 
. 
 
 
Table 3: Standard of Care Adherence Support 

 
 
 
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INTERVENTION ENHANCED ADHERENCE SUPPORT: 
A study counsellor will explain and interpret the result of the POC UTRA and conduct an adherence 
discussion at the end of each visit. Participants with no tenofovir in their urine will be referred for SoC 
adherence support. Participants in the intervention arm will therefore have additional engagement with 
health care providers and an increased chance of receiving adherence support.  
VISIT WINDOWS:  
Enrolment must take place within 6 weeks of the screening visit. If this period is exceeded, the screening 
visit can be repeated. 
Follow up visits will occur in 12-week increments post-enrolment: at month 3, month 6, month 9 and 
month 12; and will be synchronized wherever possible with routine clinic visits. Visit windows will be 
continuous, with each window extending to 6 weeks before and 6 weeks after the visit target date.  
A visit will be considered missed if the participant does not attend during the 12-week visit window. 
RETENTION 
Participants will be called the day before a scheduled visit as a reminder. If the participant is unable to 
attend a scheduled visit, the visit will be rescheduled to a new date within the visit window. 
If a participant does not attend a scheduled visit, study staff will attempt to contact them telephonically. 
If they are unable to contact the participant after three attempts at different times of day, they will 
conduct a home visit if it is feasible to do so.  
FEASIBILITY:   
We will track retention in the study among participants in each arm, missed visits, and the number of 
urine assessments performed in the intervention arm.  
ACCEPTABILITY:  
All participants enrolled will also complete a standardised questionnaire about the acceptability of the 
adherence support they received. This measure of acceptability will be assessed as a secondary outcome 
by arm, along with socio-demographics profiles associated with low/high acceptability described within 
arm.  
QUALITATIVE PROCEDURES: 
1. Participant in-depth interviews: Qualitative data will include in-depth interviews of participants 
(n~20; ~3-4 interviews with each participant over 6-9 months of their treatment) using semi-
structured guides conducted by experienced socio-behavioural scientists. The semi-structured 
interview guide will elicit feelings about the urine adherence metric and counseling messages, 
concerns regarding privacy, advantages/disadvantages of receiving such results, and the likely impact 
of this monitoring test on sustained adherence to ART or just on short-term adherence. Interviews 
will be conducted in participants’ preferred language. 
2. Health care worker interviews: We will additionally assess the feasibility of the intervention by 
interviewing health care providers, who will be administering the UTRA test at the clinical point of 

 
 
 
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care in the future. We will examine provider perceptions of the assay at the end of the study using in-
depth interviews (n~10), assessing perceived usefulness, complexity to use, stigma/social harm, and 
benefits. These interviews will also elicit barriers and facilitators to delivery of the urine assay-
informed counseling messages.  
Interviews will be approximately 45-60 minutes in duration, audio-recorded, transcribed, and (where 
necessary) translated into English. Names and other identifying information will be removed/replaced 
with pseudonyms in all public presentations of data to protect participant confidentiality. 
LAB MANAGEMENT 
1. Viral load assays: Plasma will be stored an enrolment, month 6 and month 12 for viral load assay 
completion after the study. All participants will continue to have viral loads performed as part of 
routine viral load monitoring as per National Health Laboratory Service (NHLS) National Tender 
during the study. Samples are assayed either with the Alinity m HIV-1 assay (Abbott Laboratories. 
Abbott Park, Illinois, U.S.A.); lower limit of detection (LLD) 10 copies/mL or the 
COBAS® AmpliPrep/COBAS® TaqMan® HIV-1 Test, v2.0 (Roche Diagnostics, Basel, Switzerland); LLD 20 
copies/mL.  Whole blood will be centrifuged and plasma then tested on the automated analyzer 
according to national standard operating procedures. 
2. HIV drug resistance testing: Plasma from 5 mL EDTA blood taken at baseline, month 6 and month 12, 
will be stored at -80°C until HIV drug resistance testing. HIV drug resistance testing will only be 
performed in participants with VF (VL>400 copies/mL). Resistance testing is performed by Sanger 
Sequencing of HIV protease, reverse transcriptase and integrase using previously published and 
validated in-house methods.59,60  
3. Plasma for tenofovir quantification: Plasma will be collected for tenofovir quantification at 
enrolment, month 6 and month 12 and stored on site at -20°C. Samples will be shipped to Tygerberg 
weekly on dry ice. This will be determined in cases with virologic failure and controls without and 
compared to urine UTRA and quantitative tenofovir determination.  
4. DBS for TFV-DP:  DBS will be collected at enrolment, month 6 and month 12 and tested in nested 
case control study to compare TFV-DP levels between cases with virologic failure and controls 
without.   
DATA MANAGEMENT 
Each participant will receive a unique participant identification number, which will be used for 
identification for the duration of the study on all source, case report forms (CRFs) and transcript 
documents. The exception is the locator information form that contains their address and contact 
information, which will be kept separately in a locked cupboard at the research office. 
Data will be collected for this study on an open access electronic data capture system using a password 
protected platform, with a limited number of paper-based documents. The study visit CRFs will be 
completed as soon as possible after the study visit. The principal investigators have overall responsibility 
for ensuring the data collected are complete, accurate, and recorded in a timely manner. Confidentiality 
of records will be protected, respecting the privacy and confidentiality rules in accordance with the 
applicable regulatory requirements. 

 
 
 
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Once the electronic case report form (eCRF) is completed data staff will monitor the data for 
completeness and accuracy.  Any discrepancies either manual or automatic will be corrected by site 
staff.  Corrections to eCRFs will only be possible by study personnel with sufficient authorisation to make 
changes. All changes will have an electronic date stamp. Corrections to paper-based forms can only be 
done by study staff and must be signed and dated. All hard copies of source and CRF’s documents will be 
kept in a locked cupboard at the research office or secure long-term storage facility for five years. 
Electronic data will be stored in a password-protected folder on the secure company server. A 
descriptive analysis of participant characteristics and acceptability of the UTRA and will be conducted. 
Qualitative interviews will be digitally recorded, and these data (including any field notes and 
transcripts) managed in ATLAS.ti or similar software. Qualitative data will be kept on a password-
protected server at the Desmond Tutu TB Centre, Stellenbosch University with only the study 
investigators and socio-behavioural team having access.  
STUDY WITHDRAWAL: 
Participants reserve the right to withdraw from the study at any stage, without an obligation to provide 
reasons for their withdrawal. Should a participant voluntarily withdraw their consent, the study team 
will attempt to ascertain the reason for withdrawal, while respecting the participant’s decision.  
If a participant requires a regimen switch for adverse effects, they will remain in the study, provided 
they are still using a tenofovir-based regimen.  If the clinician decides that VF is due to resistance and 
the regimen must be switched, that switch will be adjudicated as an outcome and data collection for 
that participant censored at that point.   
If, in the opinion of the investigator, a participant’s continued participation in the study poses 
unacceptable risks to themselves, the participant may be withdrawn from the study. 
Data from participants who do not complete the study will be used for secondary endpoint analyses. 
ADVERSE EVENT MANAGEMENT 
Not applicable 
 
STATISTICAL ANALYSIS: 
SAMPLE SIZE: 
We assessed sample size based on the primary effectiveness outcome of this pilot RCT e.g. sustained 
virologic suppression with real-time monitoring via the urine assay and adherence counselling in the 
intervention arm compared to the standard-of-care adherence counselling arm. We expect 80% 
suppression (VL<50 copies/mL) in the control group; a sample size of 200 (intervention and control) will 
enable us to detect an increase in suppression to 93% with 80% power. Our actual precision will likely be 
better because of multiple observations per person and the contribution of within-person changes to 
the overall estimate. 
PRIMARY OUTCOME 
The primary outcome is the proportion of participants in each arm achieving viral suppression to <50 
copies/ml by month 12.  

 
 
 
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SECONDARY OUTCOME 
Secondary outcomes include retention in care at month 12; viral suppression (<50 copies/mL) at month 
6;  and feasibility and acceptability measured at enrollment, and months 6 and 12 with standardized 
questionnaires.  
ANALYSIS PLAN 
We will assess the suppression rates (VL< 50 copies/mL) in both study arms using an intention-to-treat 
analysis. Missing values for suppression will be imputed using chained equations which will include the 
history of previous suppression and dispensation and will also include baseline characteristics such as 
sex and age. The suppression and retention analysis will be analyzed by logistic regression extension of 
generalized estimating equations with a robust variance estimate. 
In-depth interviews: 
As this is a qualitative component no statistical analysis applies to this component.  
STUDY MONITORING 
Quality assurance and quality control (QA/QC) systems with procedures to maximise patient safety and 
assure the quality of every aspect of the study will be implemented. 
On-site monitoring will be performed by independent study monitors on a regular basis. The monitors 
will: 
▪ 
verify completeness of the investigator site file 
▪ 
confirm adherence to protocol 
▪ 
review all informed consent documents 
▪ 
review eligibility verification and consent procedures 
▪ 
verify completeness, consistency, and accuracy of data being entered on CRFs  
▪ 
verify completeness, consistency, and accuracy of study samples 
▪ 
provide additional training as needed  
▪ 
document findings in a formal feedback letter (monitoring report) to the site 
ETHICAL CONSIDERATIONS AND INFORMED CONSENT 
ETHICAL CONDUCT 
This study will be conducted in accordance with the ethical principles laid out in the National Statement 
on Ethical Conduct in Research Involving Humans, the Declaration of Helsinki (most current version 
issued, available at www.wma.net), and will be consistent with GCP and applicable regulatory 
requirements.  
The rights, safety, and wellbeing of the study participants are the most important consideration and 
should prevail over the interests of science and society.  All personnel involved in conducting this study 
will be qualified by education, training, and experience to perform their respective task(s). 

 
 
 
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Institutional review board/independent ethics committee (IRB/IEC) and local regulatory approval will be 
documented and kept in the investigator site file, specifying the version number of the protocol and 
informed consent as well as the date of approval. Any amendments will require IRB/IEC and regulatory 
approval.   
The principal investigator will comply with all IRB/EC and regulatory authorities, reporting requirements 
for all safety reporting, annual updates, safety updates, end of study reports and any other important 
information relevant to the conduct of the study.  
COMPLIANCE WITH THE PROTOCOL  
The study will be conducted as described in this protocol.  The principal investigator will not implement 
any deviation or change to the protocol without prior review and documented approval/favourable 
opinion from the IRB/IEC and regulatory authorities of an amendment, except where necessary to 
eliminate an immediate hazard(s) to study participants. Any significant deviation will be reported to 
sponsor, IRB/IEC, and local regulatory authority. 
PROTOCOL AMENDMENTS 
When revisions to the protocol are made by the sponsor, if the revision is an administrative letter, the 
principal investigator will submit this for the information of their IRB/IEC.  Study documents will be 
updated in line with the changes required in the protocol amendment. 
INFORMED CONSENT AND PROCEDURES 
A study specific informed consent will include all elements required by GCP as well as all local ethics and 
regulatory requirements.   
The principal investigator will ensure that participants are clearly and fully informed about the purpose, 
potential risks and other critical issues regarding clinical trials in which they participate and that their 
participation is voluntary.  A copy of the informed consent will be given to the participant in a language 
of their choice.  Informed consent process will be conducted as per the trials site standard operating 
procedures.  A copy of the signed informed consent will be given to the participant.  
If the patient is illiterate, an impartial witness will be present during the entire consent discussion. A 
thumb print may be used as a signature.    
The informed consent document will be updated with any pertinent information that becomes available 
during the study. 
POTENTIAL RISKS  
Participants enrolled in the study may experience the following risks and discomforts. They will be 
informed of these potential risks prior to providing their informed consent. 
The venipuncture procedures to obtain blood have some minor risks. The participant may experience a 
small amount of pain at the needle-prick site, but this should be minimal. A small amount of bleeding 
under the skin may produce a hematoma (bruising), which should resolve in a few days. Risks will be 
minimized by the use of sterile technique by trained, qualified study staff.  
We will take steps to minimize inadvertent disclosure of HIV status through participation in this study 
and will make every effort to protect participant privacy and confidentiality, it is possible that 
participants' involvement in the study, or information collected by the study, could become known to 

 
 
 
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others, and that participants may experience stigmatization or discrimination as a result. We will take 
the following steps to protect against the risk of a confidentiality breach. 
1) All data, including blood samples, assessments, and participant files will be labeled only 
with study identification numbers, and no participant names will be attached to study 
data. 
2) Study documents linking study identification numbers with participant names (e.g. locator 
forms, screening logs) will be kept by the SCO in locked cupboards at the DTHF that can 
only be accessed by study staff. Personal information including participant’s name, 
address and phone number will be stored separately from research data. 
3) Electronic data files will be password protected and stored on a secure study server, 
which requires authentication to access. 
4) All paper research data will be kept in locked cupboards and will be available only to 
research staff directly involved in this project and institutional personnel for the purpose 
of routine audits. 
5) All study staff will receive training on procedures to protect participant confidentiality. 
Participants will be told that all data are confidential within the limits of the law. 
POTENTIAL BENEFITS 
Some participants may avoid virologic failure as a result of receiving adherence counselling and UTRA 
results during study participation.  
The knowledge gained from the study may be of benefit to both the participants and the wider 
population in the future, should the UTRA test become part of routine care.  
CONFIDENTIALITY OF DATA 
The site principal investigator agrees that the Desmond Tutu Health Foundation, University of Cape 
Town and funder, IRB/EC or regulatory authorities may consult and/or copy study documents to verify 
information in the CRF. By signing the consent form the participant agrees to these processes.   
Participant confidentiality will be maintained at all times and no documents containing the participant’s 
name or other identifying information will be collected by the funding organisation.  It may be necessary 
for the funder’s representatives, the IRB/EC and regulatory authority representatives to have direct 
access to the participant’s medical records. If study documents need to be photocopied during the 
process of verifying CRF data, the participant will be identified by a unique code only; full names and 
other identifying information will be masked.  
The principal investigator also agrees to maintain confidentiality with all study information and only 
divulge necessary information to the staff, ethics committee and regulatory authorities.  The data 
generated by this study will be considered confidential, except where it is included in a publication as 
agreed in the publication policy of this protocol. 
 
 

 
 
 
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APPENDIX A: TIMELINE AND GANTT CHART (AIMS 1-3) 
 
Current GANTT – started 1 July 20 
 
 
 
 
Activity 
Aim 
Responsibl
e 
2020 
2021 
2022 
2023 
202
4 
 
 
 
1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 
Training of 
site staff 
1 
CO, GVZ 
X 
Qualitative 
experience of 
UTRA 
2,3 
GH 
X X X X X X X X X X X X X 
Sensitivity of 
UTRA for VF; 
association 
with 
adherence 
measures 
1 
GVZ, CO 
X X X X X 
Training of 
site staff 
3 
CO, GvZ 
X X 
 
 
 
 
 
RCT: Utility 
and feasibility 
of UTRA  
3 
CO, GvZ, 
MG, DG 
X X X X X X X X 
Laboratory 
tests: TFV-DP 
in DBS and 
Drug 
Resistance 
1 
GvZ, ED 
 
X X X X X X X X X X X X 
Final Data 
Analysis, 
write up, 
Finding 
Disseminatio
n  
1,2,
3 
MG, GvZ, 
DG,CO 
X X 

 
 
 
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