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Towards virological monitoring of HIV-1 drug resistance in resource-limited
settings
Aitken, S.C.
Publication date
2014
Link to publication
Citation for published version (APA):
Aitken, S. C. (2014). Towards virological monitoring of HIV-1 drug resistance in
resource-limited settings.
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UnpublishedSusan C Aitken, Mariette Slabbert, Hugo Tempelman, Peter Schrooders, Rob Schuurman and Annemarie J Wensing
HIV-1 resistance testing on
dried blood spots enables
individual patient management
in rural South African Setting
Abstract
I
n a rural South African clinic over 3600 HIV-positive patients have initiated first-line cART, with high on-treatment suppression rates achieved after one year of follow-up. However, in cases of treatment failure rapid accumulation of resistance has been previously observed upon retrospective resistance testing on EDTA-plasma. Therefore, we implemented a pilot project in which prospective resistance testing on Dried Blood Spots (DBS) was implemented as routine clinical procedure for adult individuals receiving cART for at least one year and experienced viral rebound after an initial response of HIV-RNA <400 copies/ml. DBS were prepared locally and shipped by regular mail to a laboratory in the Netherlands for HIVDR genotyping. The DBS based genotyping results were used to generate individual patient reports including treatment advice, taking into account the observed drug resistance pattern, treatment history and local drug availability. Individual patient reports were sent to the local treating physician by email. A total of 178 DBS were genotyped, with a 94% success rate. All viruses were subtype-C and harboured multiple HIVDR associated polymorphisms. NNRTI (73%) and NRTI (65%) resistance mutations were predominantly observed, PI resistance mutations were less frequent (4%). Turnaround time of the whole procedure was approximately 3 weeks from the moment of shipping the DBS samples, which was largely comparable with the procedure of national centres sending EDTA-plasma to the reference laboratory despite international shipping. Largest drawbacks include time-consuming manual sample processing for nucleic acid extraction. Resistance testing on DBS was successfully implemented as routine clinical procedure in a rural setting.7
DBS for HIV-1 drug resistance testing in RLS
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153Introduction
I
n resource-limited settings (RLS), HIV-1 treatment has expanded greatly, with several treatment programs and initiatives providing care for infected individuals. However, monitoring of antiretroviral (ARV) treatment has not expanded to the same extent. In developed countries HIV-1 treatment success is monitored by means of regular viral load (VL) and CD4 testing. In addition, genotyping is generally performed prior to the initiation of therapy, and following therapy failure. In RLS none of the above are applied on a wide scale, mainly due to the high costs and complexity of these assays. Several low-cost assays for VL screening and HIV-1 drug resistance (HIVDR) genotyping have been developed(1-5), but the costs are still too highfor implementation in standard care. In addition to assay costs, processing, storage and transport of patient plasma samples to laboratories is costly and complex. Dried blood spot (DBS) samples are a popular sampling method overcoming many of the procedural and logistical obstacles and have shown potential application for VL and HIVDR genotyping in RLS.
In a rural South African clinic, Ndlovu medical centre (NMC, www. ndlovucaregroup.com), >3600 HIV-positive patients have initiated combination antiretroviral therapy (cART) since 2003, with high on treatment suppression rates achieved (on-treatment 83%; intention-to-treat 70%) at one year after treatment initiation(6). However, batch-wise retrospective
HIVDR genotyping on plasma of patients with therapy failure has shown rapid accumulation of resistance despite therapy adherence assessments, clinical, immunological, and virological monitoring(7). In attempt to demonstrate drug
resistance genotyping for individual patient management, a pilot evaluation was initiated in which resistance testing on dried blood spot (DBS) samples was initiated in 2009 as routine procedure for patients with therapy failure, log VL >3.0.
Methods
Patient data
All patients were started on a first-line cART consisting of two nucleoside reverse transcriptase inhibitors (NRTI), mostly lamivudine (3TC), plus stavudine (d4T) or zidovudine (AZT), and a non-nucleoside reverse transcriptase inhibitor (NNRTI), either efavirenz (EFV) or nevirapine (NVP). Additional NRTIs available for use included abacavir (ABC), tenofovir (TDF), and didanosine (ddI). Second-line was available for patients failing first-line and consisted of ritonavir boosted lopinavir (LPV/r), combined with two NRTIs. VL testing was routinely performed yearly in all patients, determined using a branched DNA Viral load assay (Bayer AG, Germany), as well as CD4+ T-cell count determination (FACSCount system, Becton Dickinson Biosciences, San Jose, CA).
Sample Collection
Upon determination of virological failure (log10 VL >3.0 copies/ml), patients
were asked to re-visit the clinic and DBS samples were prepared by spotting EDTA whole blood on Whatman 903™ filter cards (GE Healthcare, USA). Each card contained five spots of 50μl Whole Blood per spot. After spotting, cards were air dried for three hours in drying racks in a laminar flow hood. Dried cards were placed in zip-lock bags with Minipax® indicator desiccant (Sigma-Aldrich, Germany). Samples were shipped by standard mail to a laboratory in the Netherlands for HIVDR genotyping. DBS were stored and shipped at room temperature, and further stored at -20°C upon receipt in the Netherlands before processing.
HIV Drug Resistance Genotyping
Two 50ul spots per sample were used as input for nucleic acid (NA) isolation. Viral nucleic acids (NA) were isolated using the NucliSENS MiniMAG magnetic extraction kit (BioMerieux, The Netherlands). Isolated NAs were amplified and genotyped using an assay targeting the protease (PR) and reverse transcriptase (RT) region of HIV-1(3). Samples that failed to amplify for PR-RT
7
DBS for HIV-1 drug resistance testing in RLS
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155were amplified and genotyped using an assay targeting the RT region alone(1).
Generated sequences were analyzed and assembled using SeqScape version 2.6 (Life Technologies). Resistance reports were generated from the consensus sequences using the HIV-GRADE analysis program (http:// www.hiv-grade.de/grade/deployed/grade.pl?program=hivalg). Resistance mutations were assessed according to the 2011 IAS guidelines(8).
Individual patient reports including treatment advice were generated by a clinical virologist based on the drug resistance pattern, treatment history and local drug availability, and reported to the local treating physician by email.
Results
A total of 178 DBS samples were received for genotyping in the Netherlands. Patients were primarily female (62%), age 3-68 (average 37) years, and receiving therapy for 0.6-9.7 years (average 2.7) years. Since initiation of cART, some patients had switched therapy one (n=81), two (n=27) or even three (n=3) times. At the time of DBS sample, 79% of patients were receiving first-line therapy consisting of 3TC/TDF/NVP or EFV (n=64), 3TC/d4T/NVP or EFV (n=44) or 3TC/AZT/NVP or EFV (n=30), and one patient received 3TC/ ABC/NVP. For patients receiving second-line therapy (n=38; 21%), therapy consisted of 3TC/AZT/LPV/r (n=30), 3TC/TDF/LPV/r (n=5), ddI/AZT/LPV/r (n=1), or ddI/TDF/LPV/r (n=1). For one patient no treatment information was available. Mean time after initiation of current therapy was 1.8 years. Mean duration of PI-exposure of in those on a PI containing second-line regimen was 1.9 years. Despite therapy failure, median CD4-count increased from 68 to 192 cells/mm3 since initiation of cART. HIV-RNA at a median of 6 weeks
before DBS sample ranged from 1.00E+03-1.36E+06 (median 1.55E+04) copies/ml.
From a total of 178, 158 (89%) samples were genotyped using the PR-RT assay. A further 10 samples that were negative using the PR-RT assay were genotyped using the RT only assay. Overall, genotyping success rate was
94% (168/178 DBS samples). All individuals harboured HIV-1 subtype-C virus. For the 10 samples that could not be genotyped, two had not been packaged according to instructions, leading to possible sample degradation, two had a repeat sample that was genotyped at another time-point, three samples were not repeated with the RT only assay as this assay was added as an alternative after the start of the study. The three remaining samples had been appropriately packed and had viral loads sufficient for testing, however failed to amplify. Turnaround time of the whole procedure was comparable with the procedure of local centres sending EDTA-plasma to the reference laboratory despite international shipping, with results being returned to the clinic on average three weeks after shipping.
Of the 168 patients with positive genotyping results, 70% (n=118) harboured drug resistance mutations (Figure 1). The overall prevalence of NRTI mutations was 65% (n=109), with M184V (n=99) being the most common mutation (59%). K65R was observed in 37 individuals (22%). NNRTI mutations were slightly more prolific, 73% (n=122), with the most common being K103N (n=61; 36%), Y181C (n=36; 21%), V106M (n=34; 20%), and G190A (n=28; 17%).
PI mutations were limited, found in only six individuals (6/169; 4%), of which only three were receiving a PI-based regimen. PI mutations consisted of M46IL (n=4), I47V (n=1), I54V (n=1), L76V (n=2), N83D (n=1) and I84V (n=2). PI mutation prevalence in patients receiving second-line therapy was 8% (3/38). Two of these patients haboured multiple mutations as indicators for reduced susceptibility to LPV/r. Patient 1 had been on a PI-based regimen for 3.6 years and expressed M46I, 154V, L76V and I84V mutations. Patient 2 had been on a PI-based regimen for 2.4 years and harboured M46I, L76V and I84V mutations.
7
DBS for HIV-1 drug resistance testing in RLS
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1570 10 20 30 40 50 60 70 80 NRT I NNR TI M41L K65R D67N K70R L74V L100I K101E P K103NV106A M Y115F E138A GK Y181CM184I V Y188LH C G190 ASE T210WY215F Y K219Q E M230L
Figure 1. Percentage prevalence of RTI mutations in patients failing first- or second-line cART. NRTI mutations are shown in grey; NNRTI mutations are shown in black; Solid portion of bars represent patients receiving first-line therapy; Striped portion of bar represents patients receiving second-line therapy.
Discussion
Resistance testing on DBS was successfully implemented as routine clinical procedure in a rural setting. NNRTI and NRTI resistance was frequently observed while PI resistance was limited.
Overall, a high genotyping success rate was achieved, demonstrating the use of DBS sampling to improve access to HIVDR genotyping in rural settings. The described study was performed using a PR-RT genotyping, an assay that has previously been shown to be comparable to standard commercial HIVDR genotyping assays(3). This assay has also met the
acceptance criteria of the WHO external QA programs 2013 VQA proficiency panel (GEN002BS). An RT only assay was used as a back-up to the PR-RT and provided comparable data to the PR-PR-RT method. As the majority of resistance was restricted to first-line cART, samples received subsequent to the pilot study are now being initially screened with the RT only assay, and only samples of patients receiving second-line therapy are tested with the full PR-RT assay. Although both assays are significantly cost-reduced compared
to commercially available assays, the RT only assay is the most pragmatic option, providing the necessary data with less processing time and at a lower cost.
Whilst HIVDR genotyping provides valuable information to guide therapy switching, it is still not a viable option for individual patient monitoring in RLS due to costs involved, especially when the money could be used to provide treatment for more people. Although funds do not allow for continued individual patient monitoring, the data obtained from this study also provides important information about the types and prevalence of drug resistance mutations affecting current therapy options within the described population. The largest drawback for this study was the manual nucleic acid isolation procedures for the DBS samples. Excision of the DBS samples is a laborious task and could be improved by using pre-perforated collection cards. Subsequent NA isolation is also laborious. Using the NucliSENS miniMAG takes on average 90 minutes to process 10 samples plus a positive and negative isolation control. An alternative is the automated version of the same method, the EasyMAG, which can handle twice the number of samples in about the same time. However, a higher throughput automated DBS isolation protocol would greatly improve the laboratory capacity for DBS sample processing.
The data in this study have shown that 70% of patients identified by viral load as receiving a failing regimen had drug resistance mutations, which is in line with the findings of the WHO 2012 HIV drug resistance report(9).
This highlights the effective use of viral load testing for monitoring therapy failure, as has been previously shown(10). Availability of genotyping results
to the treating physician could assist in rational decisions on individualized second-line treatment regimens, which may spare drug options. Given the high mutation prevalence, routine genotyping is strongly warranted both for epidemiological use as well as for patient management.
7
DBS for HIV-1 drug resistance testing in RLS
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159Acknowledgements and Funding
We wish to thank the UMCU Molecular Diagnostic laboratory staff for performing the genotyping. This study was conducted as part of the Affordable Resistance Test for Africa (ART-A) program, which is supported by a grant from the Netherlands Organization for Scientific Research for Global Development (NWO/WOTRO), under the Netherlands African Partnership for Capacity Development and Clinical Interventions against Poverty-related Diseases (NACCAP; grant W.07.05.204.00), The Hague, The Netherlands.
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