BRIEF REPORT:BASIC AND TRANSLATIONAL SCIENCE Improved Viral Suppression After Treatment Optimization in HIV-Infected Patients With Persistent Low-Level Viremia Michael J. McConnell, MD, PhD,* Julian Mier-Mota, BS,* Fernando Flor-Parra, BS,* Francisco J. Martinez-Fernandez,* Luis F. Lopez-Cortes, MD, PhD,* Pompeyo Viciana, MD, PhD,* Felipe Fernandez-Cuenca, PhD,† and Pilar Perez-Romero, PhD* Abstract: Optimizing treatment for patients with persistent low- level viremia is complicated because most genotyping tests are validated for viral loads .1000 copies per milliliter. In this study, genotypes of 92 treatment-experienced patients with persistent low- level viremia were determined using an in-house assay. Based on the resistance profiles obtained from genotyping and patient pharmaco- logic history, patients were either maintained on their antiviral reg- imen (n = 51) or received an optimized regimen (n = 41). In the group receiving optimized treatment, undetectable viral loads were achieved in 73.2% at 6 months and at 90.2% at 1 year, indicating that treatment guided by genotyping of patients with low-level viremia is effective in achieving viral suppression. Key Words: genotyping, HIV, persistent low-level viremia, treat- ment optimization (J Acquir Immune Defic Syndr 2011;58:446–449) INTRODUCTION The goal of antiretroviral therapy is to achieve complete viral suppression, as determined by undetectable plasma viral loads. However, it has been reported that a significant proportion of treatment-experienced patients have persistent low-level viremia, which is characterized by sustained viral loads between 20 and 1000 HIV RNA copies per milliliter. 1,2 Patients experiencing persistent low-level viremia have been reported to have higher overall immune activation and are at greater risk for treatment failure, 1,2 suggesting that this patient subset may benefit from treatment optimization. The characterization of resistance mutations in patients with persistent low-level viremia is complicated by the fact that most approved commercially available genotyping assays have been validated for viral loads .1000 HIV RNA copies per milliliter. In the absence of reliable genotyping data, clini- cians can either switch the antiretroviral regimen based on the most frequent mutations that could be selected with a given treatment regimen or continue with the same regimen al- though awaiting an increase in viral load. To address this issue, several assays have recently been developed for geno- typing samples with viral load below 1000 copies per milli- liter. 3–5 A high prevalence of resistance mutations among this patient subset has been reported, supporting the practice of HIV genotyping in patients with low viral loads. 6–9 However, these studies have not addressed the utility of optimizing antiretroviral therapy based on genotyping data obtained from this patient population. The goal of the present study was to use a highly sensitive in-house genotyping assay to characterize resistance mutations in the protease (pr) and reverse transcriptase (rt) genes in patients with persistent low-level viremia and to characterize the virological and immunological effects of treatment optimization based on this genotyping data. METHODS Genotyping Assay Primers were designed for amplifying the rt and pr genes (1911-forward; 59-TACCATAATGATACAGAAAGG CAA-39 and 3602-reverse 59-TGGGCACCCTTCATTCTTG CATA-39) using reference sequences for the subtype B viruses, pNL4.3 and HXB2 (accession numbers: M19921, NC001802). HIV-RNA was isolated from plasma using the QIAmp viral RNA extraction kit (Qiagen, Hilden, Germany) from 560 mL of plasma (2 rounds of extraction with 280 mL) and eluted in 60 mL of distilled water. The SuperScript III one step reverse transcriptase–polymerase chain reaction System (Invitrogen, Carlsbad, CA) was used for reverse transcriptase– polymerase chain reaction according to the manufacturer’s Received for publication April 7, 2011; accepted September 6, 2011. From the *Unit of Infectious Diseases, Microbiology and Preventive Medicine, Instituto de Biomedicina de Sevilla (IBIS)/Hospital Universitario Virgen del Rocío/CSIC/Universidad de Sevilla, Spain; and †Microbiology Service, Hospital Universitario Virgen Macarena de Sevilla, Spain. Supported by the Consejería de Economía, Innovación y Ciencia P06- CTS-01915, Junta de Andalucía; and the Red de Investigación en SIDA (ISCIII RD06/0006), Ministerio de Ciencia e Innovación. M.J.M is sup- ported by the Programme Juan de la Cierva of the Ministerio de Ciencia e Innovación of Spain. P.P.R. was funded by Instituto de Salud Carlos III, Programa Miguel Servet CP05/00226. The authors J.M.M., L.F.L.C., P.V. and P.P.R. are listed as inventors on a patent registered by the Andalusian Foundation for Health Research of Seville with the Spanish Office of Patents and Trademarks covering the method described in this study. M.J.M. and J.M.M. contributed equally to this work. Supplemental digital content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal’s Web site (www.jaids.com). Correspondence to: Pilar Pérez-Romero, PhD, Unit of Infectious Diseases, Microbiology and Preventive Medicine. Instituto de Biomedicina de Sevilla (IBiS). University Hospital Virgen del Rocío/CSIC/University of Sevilla. Avda Manuel Siurot s/n, 41013 Sevilla, Spain (e-mail: mariap.perez. exts@juntadeandalucia.es or mperez-ibis@us.es). Copyright © 2011 by Lippincott Williams & Wilkins 446 | www.jaids.com J Acquir Immune Defic Syndr  Volume 58, Number 5, December 15, 2011