Abstract

BackgroundGreat efforts have been made to increase accessibility of HIV antiretroviral therapy (ART) in low and middle-income countries. The threat of wide-scale emergence of drug resistance could severely hamper ART scale-up efforts. Population-based surveillance of transmitted HIV drug resistance ensures the use of appropriate first-line regimens to maximize efficacy of ART programs where drug options are limited. However, traditional HIV genotyping is extremely expensive, providing a cost barrier to wide-scale and frequent HIV drug resistance surveillance.Methods/ResultsWe have developed a low-cost laboratory-scale next-generation sequencing-based genotyping method to monitor drug resistance. We designed primers specifically to amplify protease and reverse transcriptase from Brazilian HIV subtypes and developed a multiplexing scheme using multiplex identifier tags to minimize cost while providing more robust data than traditional genotyping techniques. Using this approach, we characterized drug resistance from plasma in 81 HIV infected individuals collected in São Paulo, Brazil. We describe the complexities of analyzing next-generation sequencing data and present a simplified open-source workflow to analyze drug resistance data. From this data, we identified drug resistance mutations in 20% of treatment naïve individuals in our cohort, which is similar to frequencies identified using traditional genotyping in Brazilian patient samples.ConclusionThe developed ultra-wide sequencing approach described here allows multiplexing of at least 48 patient samples per sequencing run, 4 times more than the current genotyping method. This method is also 4-fold more sensitive (5% minimal detection frequency vs. 20%) at a cost 3–5× less than the traditional Sanger-based genotyping method. Lastly, by using a benchtop next-generation sequencer (Roche/454 GS Junior), this approach can be more easily implemented in low-resource settings. This data provides proof-of-concept that next-generation HIV drug resistance genotyping is a feasible and low-cost alternative to current genotyping methods and may be particularly beneficial for in-country surveillance of transmitted drug resistance.

Highlights

  • Availability of antiretroviral therapy (ART) is increasing in low and middle-income countries [1]

  • By using a benchtop next-generation sequencer (Roche/454 GS Junior), this approach can be more implemented in low-resource settings

  • Transmitted drug resistance may thwart current efforts to scale-up treatment in low and middle-income settings where few treatment options are available. It is highly recommended by the World Health Organization (WHO) that surveillance of drug resistance occur in conjunction with scale-up efforts to ensure appropriate first-line therapy is offered relative to the resistance that exists [5]

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Summary

Introduction

Availability of antiretroviral therapy (ART) is increasing in low and middle-income countries [1]. Transmitted drug resistance may thwart current efforts to scale-up treatment in low and middle-income settings where few treatment options are available. It is highly recommended by the World Health Organization (WHO) that surveillance of drug resistance occur in conjunction with scale-up efforts to ensure appropriate first-line therapy is offered relative to the resistance that exists [5]. It is believed that surveillance will maximize the utility of first-line therapy and help minimize the cost of providing ART thereby sustaining current antiretroviral drug programs. Traditional HIV genotyping is extremely expensive, providing a cost barrier to wide-scale and frequent HIV drug resistance surveillance

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