A Pragmatic Approach to HIV-1 Drug Resistance Determination in Resource-Limited Settings by Use of a Novel Genotyping Assay Targeting the Reverse Transcriptase-Encoding Region Only

Author:

Aitken Susan C.1,Bronze Michelle2,Wallis Carole L.3,Stuyver Lieven4,Steegen Kim25,Balinda Sheila6,Kityo Cissy6,Stevens Wendy25,Rinke de Wit Tobias F.78,Schuurman Rob1

Affiliation:

1. University Medical Centre, Utrecht, The Netherlands

2. University of the Witwatersrand, Johannesburg, South Africa

3. Lancet Laboratories, Johannesburg, South Africa

4. Janssen Diagnostics BVBA, Beerse, Belgium

5. National Health Laboratory Services, Johannesburg, South Africa

6. Joint Clinical Research Centre, Kampala, Uganda

7. Department of Global Health, Amsterdam Institute for Global Health and Development (AIGHD), Academic Medical Center (AMC), Amsterdam, The Netherlands

8. PharmAccess International, Amsterdam, The Netherlands

Abstract

ABSTRACT In resource-limited settings (RLS), reverse transcriptase (RT) inhibitors form the backbone of first-line treatment regimens. We have developed a simplified HIV-1 drug resistance genotyping assay targeting the region of RT harboring all major RT inhibitor resistance mutation positions, thus providing all relevant susceptibility data for first-line failures, coupled with minimal cost and labor. The assay comprises a one-step RT-PCR amplification reaction, followed by sequencing using one forward and one reverse primer, generating double-stranded coverage of RT amino acids (aa) 41 to 238. The assay was optimized for all major HIV-1 group M subtypes in plasma and dried blood spot (DBS) samples using a panel of reference viruses for HIV-1 subtypes A to D, F to H, and circulating recombinant form 01_AE (CRF01_AE) and applied to 212 clinical plasma samples and 25 DBS samples from HIV-1-infected individuals from Africa and Europe. The assay was subsequently transferred to Uganda and applied locally on clinical plasma samples. All major HIV-1 subtypes could be detected with an analytical sensitivity of 5.00E+3 RNA copies/ml for plasma and DBS. Application of the assay on 212 clinical samples from African subjects comprising subtypes A to D, F to H (rare), CRF01_AE, and CRF02_AG at a viral load (VL) range of 6.71E+2 to 1.00E+7 (median, 1.48E+5) RNA copies/ml was 94.8% ( n = 201) successful. Application on clinical samples in Uganda demonstrated a comparable success rate. Genotyping of clinical DBS samples, all subtype C with a VL range of 1.02E+3 to 4.49E+5 (median, 1.42E+4) RNA copies/ml, was 84.0% successful. The described assay greatly reduces hands-on time and the costs required for genotyping and is ideal for use in RLS, as demonstrated in a reference laboratory in Uganda and its successful application on DBS samples.

Publisher

American Society for Microbiology

Subject

Microbiology (medical)

Reference19 articles.

1. WHO/UNAIDS/UNICEF. 2010. Towards universal access: scaling up priority HIV/AIDS interventions in the health sector: progress report 2010. http://whqlibdoc.who.int/publications/2010/9789241500395_eng.pdf.

2. HIV-1 drug resistance among antiretroviral-naive individuals in sub-Saharan Africa after rollout of antiretroviral therapy: multicentre observational study;Hamers RL;Lancet Infect. Dis,2011

3. WHO. 2010. Antiretroviral therapy for HIV infection in adults and adolescents: recommendations for a public health approach 2010 revision. http://whqlibdoc.who.int/publications/2010/9789241599764_eng.pdf.

4. AitkenSC TempelmanH SchroodersP van BusselE SlabbertM SchuurmanR WensingAM. 2011. Limited PI resistance after failure of second-line therapy in rural South African setting. Antivir. Ther. 16:A143. http://www.aegis.org/DisaplayConf/abstList.aspx?Conf=75.

5. Protease Inhibitor Resistance Is Uncommon in HIV-1 Subtype C Infected Patients on Failing Second-Line Lopinavir/r-Containing Antiretroviral Therapy in South Africa

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