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 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 ng/μL, and for HSV-2 at 6.0 × 10 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.
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