Development and evaluation of a qualitative reverse-transcriptase nested polymerase chain reaction protocol for same-day viral validation of human immunodeficiency virus type 1 ribonucleic acid in processed semen

To develop a method for same-day validation of processed semen in the setting of assisted reproductive techniques (ART) with patients who are seropositive for human immunodeficiency virus, type 1 (HIV-1). Laboratory experiments. University hospital. Volunteers who are HIV-1 seronegative and seropositive. Evaluation of the sensitivity of a reverse-transcriptase (RT)–nested polymerase chain reaction (PCR) in HIV-1 RNA–positive blood plasma, in artificially infected blood plasma and semen, and in 85 semen samples of 29 HIV-1–seropositive volunteers. Semen was submitted to gradient separation, followed by swim-up. Qualitative detection of HIV-1 RNA in blood plasma and in different parts of semen preparation by using RT-nested PCR, PCR inhibition control by dilution of samples, and an internal control. The detection limit of our PCR was 20 HIV-1 RNA copies per milliliter. Among seropositive patients, RNA was detected in 25% of fresh semen, 36.5% of seminal plasma, 27.5% of gradient supernatants, and 7.1% of final preparations before the migration–sedimentation stage. Positive final preparations were observed in patients who had blood viral loads of ≥20,000 HIV-1 RNA copies per milliliter. Inhibition was present in 17.6% of seminal plasma and in 20% gradient supernatants and in 2 final preparations among 69 tested. Among 25 preparations tested after the migration-sedimentation stage, 2 were positive (1 patient; 70,000 HIV-1 RNA copies per milliliter). The RT-nested PCR detects low viral load and allows the validation of semen preparations of HIV-1–seropositive patients for ART on the day of sampling. For this purpose, the validation is performed on spermatozoa that are obtained after gradient separation before swim-up. Inhibition of the PCR must be controlled by using an internal control that is well-designed to explore the detection limit of the method.