Low-Cost Ultra-Wide Genotyping Using Roche/454 Pyrosequencing for Surveillance of HIV Drug Resistance

Author(s)

Dudley, Dawn M. ^[1] ; Chin, Emily N. ^[2] ; Bimber, Benjamin N. ^[1] ; Sanabani, Sabri S. ^[3] ; Tarosso, Leandro F. ^[4] ; Costa, Priscilla R. ^[4] ; Sauer, Mariana M. ^[4] ; Kallas, Esper G. ^[4] ; O'Connor, David H. ^{[1, 5]}

Affiliation

^[1] Univ Wisconsin Madison, Dept Pathol & Lab Med, Madison, WI - USA

^[2] Univ Wisconsin Madison, Dept Cellular & Mol Biol, Madison, WI - USA

^[3] Univ Sao Paulo, Sao Paulo Inst Trop Med, Sao Paulo - Brazil

^[4] Univ Sao Paulo, Div Clin Immunol & Allergy, Sao Paulo - Brazil

^[5] Univ Wisconsin Madison, Wisconsin Natl Primate Res Ctr, Madison, WI - USA

• Document type: Journal article
• Source: PLoS One; v. 7, n. 5 MAY 4 2012.
• Web of Science Citations: 59

Abstract

Background: Great 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/Results: We 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 Sao 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 naive individuals in our cohort, which is similar to frequencies identified using traditional genotyping in Brazilian patient samples. Conclusion: The 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 x 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. (AU)

• FAPESP's process: 04/15856-9 - Prospective analysis of the virological and immunological characteristics in individuals with recent HIV-1 infection in the cities of São Paulo and Santos
• Grantee: Ricardo Sobhie Diaz
• Support Opportunities: Research Projects - Thematic Grants
• FAPESP's process: 06/50096-0 - Characterization of Human Immunodeficiency Virus type 1 (HIV-1) in a cohort of recently infected persons from the State of São Paulo by full genome sequencing
• Grantee: Sabri Saeed Mohammed Ahmed Al-Sanabani
• Support Opportunities: Scholarships in Brazil - Post-Doctoral