Received: 18 July 2017 | Accepted: 5 December 2017 DOI: 10.1002/jmv.25003 RESEARCH ARTICLE Real time PCR to evaluate HSV-2 shedding from anal and genital samples among men who have sex with men, living with HIV Dayana N. Vergara-Ortega 1 | Edgar E. Sevilla-Reyes 2 | Antonia Herrera-Ortiz 1 | Leticia Torres-Ibarra 3 | Jorge Salmerón 4 | Eduardo Lazcano-Ponce 3 | Miguel A. Sánchez-Alemán 1 1 Centro de Investigación sobre Enfermedades Infecciosas, Instituto Nacional de Salud Pública, Cuernavaca, Morelos 2 Centro de Investigación en Enfermedades Infecciosas, Instituto Nacional de Enfermedades Respiratorias, Mexico 3 Centro de Investigación en Salud Poblacional, Instituto Nacional de Salud Pública, Cuernavaca, Morelos 4 Universidad Nacional Autónoma de México, Ciudad de Mexico, Mexico Correspondence Miguel Angel Sánchez-Alemán, Instituto Nacional de Salud Pública. Universidad 655, Col. Santa María Ahuacatitlán, Cerrada Los Pinos y Caminera. CP 62100, Cuernavaca, Morelos, México. Email: msanchez@insp.mx This study shows the relative quantification of HSV-2 by qPCR, using the MIQE Guidelines. The reaction efficiency was evaluated, and the relative quantification used the R =2 −ΔCq method. The relative quantification of HSV-2 was conducted with anal and genital samples from men who have sex with men (MSM), living with HIV. The presence of a single amplification product was validated with a dissociation curves profile and the determination of the melting temperature. The limit of detection for β- globin was determined as 3.3 × 10 −5 ng/μL, and for HSV-2 at 6.0 × 10 −6 ng/μL. The efficiency for β-globin was 100.2% and for HSV-2 was 106.8%. From 336 MSM, 2.1% and 3.9% individuals presented anal or genital HSV-2 shedding, respectively. The HSV- 2 viral load was 9.2 RU, individuals with fewer CD4 + presented higher HSV-2 viral load. The qPCR method is reproducible and has optimal reaction efficiency. KEYWORDS herpes genitalis, human herpesvirus 2, real-time polymerase chain reaction, reproducibility of results 1 | INTRODUCTION Viral load is a relevant marker for diagnosis and clinical follow up, in relation to human immunodeficiency virus, hepatitis C virus, and hepatitis B virus. Viral load has been used to evaluate the treatment, to change therapy or to search for resistant viral variants and has been proposed as a biomarker to predict the outcome of infection (persistance or elimination) as is the case with Human Papillomavi- rus. 1–4 Herpes Simplex Virus type 2 (HSV-2) is a DNA virus ascribed to the Herpesviridae family, and the main etiological agent of genital ulcers.Viral quantification of HSV-2 is used to study asymptomatic infections, monitoring neonatal and encephalitis herpes cases, as well as the risk of acquisition and transmission of HIV. 5–8 The quantitative real time PCR (qPCR) used to measure viral loads, because of its high sensitivity, can be applied to different biological samples such as serum, plasma, urine, genital secretions, and anal exudates. 9 The standardization and validation of a methodology for viral quantification is critical to have reliable and comparable results with other studies. There is published work that reported the use of qPCR for HSV-2 quantification but, without application of the parameters proposed in Minimum Information for Publication of Quantitative Real-Time PCR Experiments guidelines (MIQE). 10 In 2011, about 5% of the publications that used qPCR cited the MIQE guides, increasing to 11% in 2013 11 ; however, not all papers that mention the guide provided the information for qPCR. The current study shows the conditions of standardization, validation and quality control for the relative quantification of HSV-2 by qPCR in anal and genital samples to evaluate HSV-2 shedding among a population of men who have sex with men (MSM), living with HIV. J Med Virol. 2018;90:745–752. wileyonlinelibrary.com/journal/jmv © 2017 Wiley Periodicals, Inc. | 745