Affordable Resistance Testing for Africa (ART-A) Michelle Bronze HIV Drug Resistance Laboratory University of the Witwatersrand National Health Laboratory Service Johannesburg, South Africa

Introduction National ARV programs expand to achieve the UN General Assembly health-related Millennium Development Goals by 2015, which include universal access to HAART, there is an urgent need to expand the technology, laboratory capacity, accessibility and affordability to perform HIVDR testing in resource limited settings. ARV roll-out program initiated in April 2004 and has expanded dramatically, with the latest statistics showing that >2.1 million South Africans are accessing the national roll-out programme. NHLS recognized the need to develop an affordable resistance testing platform, and established the HIV Genotyping Laboratory in 2005 in anticipation of future needs. The Affordable Resistance Test for Africa (ART-A) project was founded to address the need to develop an affordable resistance test.

In this project, private, academic and civil society stakeholders joined forces to develop an affordable, comprehensive HIV drug resistance protocol which could be universally applied to individual patients by a network of clinics, hospitals and laboratories in Africa. The public-private consortium launched the ART-A project on the 1st of January 2008.

Introduction HIV-1 RNA viral load (VL) is the preferred tool to identify virological failure (VF) during antiretroviral therapy (ART). The effectiveness and sustainability of ART is threatened by the emergence of HIV-1 drug resistance (HIVDR). Timely detection of VF can reduce the complexity of HIVDR mutation patterns. The work conducted assessed: a novel affordable two-step approach to identify Virological Failure and detect key HIVDR mutations in order to improve quality of HIV care in resource-limited settings. Moreover, to ensure results could be reproduced in different centre’s, field evaluations were conducted in Uganda where subtype A and D are prevalent, and South Africa & Zimbabwe (high subtype C prevalence) by the ARTA team

Introduction South Africa is better equipped to manage the local epidemic having significantly more laboratory capacity for toxicity tests, CD4 and viral load assays than many other resource-limited countries. This country has a three-tier laboratory system: regional tertiary reference laboratories (21% of laboratories): VL’s , CD4’s, HIVDR (handle large volumes of work, and for the most part possess appropriate technical resources, including electricity, freezer capacity, dedicated laboratory space, skilled personnel, training programs, reliable internet connections and appropriate quality control systems secondary or district facilities (10%): medium volume laboratories that also perform CD4 and viral load assays but with a reduced repertoire of tests, and do not have specialized facilities primary health care laboratories (69%): technically non-demanding assays, which typically include rapid and point-of-care tests The high burden of disease and ever-increasing testing volumes is placing an enormous workload on the current public sector laboratory networks and innovative diagnostic solutions are needed for the future as HAART coverage expands.

The following approach is how this algorithm would be applied in a clinical setting:

Virological Failure Assay (ARTA-VFA) A simplified and affordable approach using a qualitative viral load assay with a pre-determined cut-off that gives a threshold above which VF could be confirmed and below, treatment success was likely. RNA extraction from plasma/DBS  cDNA synthesis  real-time PCR amplification of a short sequence of the HIV-1 LTR region. Its applicability using either plasma or DBS was confirmed, with virological failure being qualitatively classified as a viral load >1000 RNA copies/ml in plasma samples, and >5000 RNA copies/ml in DBS samples. The ARTA-VFA identified all major HIV-1 group M subtypes with equal specificity. Accurate virological failure determination for therapy-switching in approximately 93% of clinical cases tested.

ARTA-VFA Performance of VFA – linearity and classifications around 5000 and 1000 copies/ml cut-off’s Bronze et al. (2013) Journal of Virological Methods

Performance of VFA (South Africa) – Performance characteristics around the 5000 and 1000 copies/ml cut-off’s Comparison 5000 copies/ml cut-off 1000 copies/ml cut-off Sensitivity (%) Specificity (%) PPV (%) NPV (%) Plasma VFA vs. EasyQ v1.2 95 93 92 96 90 98 86 DBS VFA vs. EasyQ v1.2 91 100 70 72 DBS VFA vs. Plasma VFA 76 Sensitivity: True virological failure/ (True virological failure + False non-virological failure) Specificity: True non-virological failure/ (True non-virological failure + False virological failure) Positive predictive value: True virological failure/ (True virological failure + False virological failure) Negative predictive value: True non-virological failure/ (True non-virological failure + False non-virological failure) Bronze et al. (2013) Journal of Virological Methods

Performance of VFA on DBS (South Africa) Bronze et al. (2013) Journal of Virological Methods

Performance of VFA across three study sites in Uganda 5000 cp/ml cut-off Sensitivity (%) Specificity (%) PPV (%) NPV (%) Kampala 78 92 Fort Portal 80 94 74 96 Mbale 95 86 Combined 83 87 Balinda et al. (2012) ASLM Performance of VFA at Newlands Clinic, Zimbabwe Comparison 5000 copies/ml cut-off 1000 copies/ml cut-off Sensitivity (%) Specificity (%) PPV (%) NPV (%) Plasma VFA vs. TaqMan 85 97 95 93 88 98 96 76 DBS VFA vs. TaqMan 33 99 53 100 78 Ondoa et al. (Unpublished)

Overall Performance of VFA on DBS in RSA, Uganda and Zimbabwe Comparison 5000 copies/ml cut-off Sensitivity (%) Specificity (%) PPV (%) NPV (%) RSA 91 95 96 Uganda 83 93 87 92 Zimbabwe 33 99 97 76 Bronze et al. (2013) Journal of Virological Methods Balinda et al. (2012) ASLM Ondoa et al. (Unpublished) Ondoa et al. (Unpublished)

Short RT HIV Drug Resistance Assay (ARTA-HIVDRultralight) The ARTA-HIVDRultralight assay targets amino acids 41 – 238 of RT (harboring all major RT inhibitor resistance mutation positions, thus providing all relevant susceptibility data for first-line regimen failures) The aimed specifications for this assay: to be practical and affordable flexible with respect to equipment choices (open platform) include the option of DBS or plasma amplify and sequence a smaller amplicon (RT)

ViroSeqTM Assay PR amino acids 1 99 1 335 RT amino acids In-house assay PR amino acids 1 99 1 353 RT amino acids Short RT assay 41 238 RT amino acids

Performance of ARTA-HIVDRultralight Aitken et al. (2013) Journal of Clinical Microbiology

ARTA-HIVDRultralight amplification and sequencing rates (South Africa) Aitken et al. (2013) Journal of Clinical Microbiology ARTA-HIVDRultralight amplification and sequencing rates (South Africa) Bronze et al. (2013) Journal of Virological Methods Sample Size (n) Viral Load (copies/ml) Amplification Success Rate (%) Sequencing Success Rate (%) 41 >5000 98 (39/40) 85 (33/39) 25 <5000* 44 (11/25) 55 (6/11) * The <5000 copies/ml DBS group will be further assessed to assess success rates of assay between the 1000 and 5000 copies/ml range

Cost of compared HIV-1 viral load and HIV-1 drug resistance genotyping assays (South Africa) Bronze et al. (2013) Journal of Virological Methods

Conclusion Plasma and DBS sampling extends the access of testing to more remote settings. These assays were designed to either be used as a testing strategy of initially assessing virological failure, and once confirmed performing an HIVDR assay, or alternatively to be used separately as stand-alone, or within different laboratory tiers. Since large scale commercial assays are commonly used in reference laboratory service areas with a large burden of HIV-1, and primary health care centres ideally use rapid or point-of-care assays, the ARTA-VFA could potentially be used in the middle laboratory tier. This would involve screening patients for virological failure with a qualitative outcome (a >1000 RNA copies/ml cut-off for plasma samples, and a >5000 RNA copies/ml cut-off for DBS), followed by referral to a Tier 1 laboratory with the available infrastructure for HIVDR testing using the ARTA-HIVDRultralight if indicated.

Some lessons learnt This study highlights the importance of validating assays where they are to be used. Costing in one country is not equivalent to all countries Assays originally designed to be used on plasma require further “tweaking” to increase assay sensitivity for use on DBS