Detection of hepatitis C virus in the semen of infected men
Lancet 2000; 356: 42 - 43

Marianne Leruez-Ville, Jean-Marie Kunstmann, Martha De Almeida, Christine Rouzioux, Marie-Laure Chaix

We detected hepatitis C virus (HCV) RNA in the semen of one third of HCV viraemic men. Seminal viral loads were low, but the semen could be

infectious and the role of sexual transmission in the spread of HCV infection should not be underestimated. Sexual transmission of hepatitis C virus (HCV) is still debated and although two isolated cases of HCV detection in semen have been reported, findings from extensive studies of 70 viraemic patients could

not confirm this result.To study the presence of HCV in semen, we developed a reverse transcriptase PCR method that was highly sensitive and adapted to detecting the virus in semen samples. We looked for HCV-RNA in semen samples from 21 untreated HCV viraemic men. Among them 15 were HIV-positive patients treated with antiretroviral therapy. Paired blood and semen samples were obtained.

HCV-RNA detection in blood samples was done with Cobas Amplicor 2.0

(Roche, Meylan, France) with a sensitivity of 100 copies/mL, and quantification of blood viral load was obtained using Amplicor HCV Monitor (Roche) with a sensitivity of 1000 copies/mL. HCV genotypes  were determined by INNO-LIPA HCV II (Innogenetics, Rungis, France). Semen  was fractionated with a Percoll gradient (Sigma-Aldrich, Saint Quentin Fallavier, France) and seminal fluid was frozen at 280C. Seminal  plasma was tested either pure or prediluted 1:1 in a 5 g/L bromelain solution (Sigma-Aldrich). HCV-RNAs were extracted from seminal plasma with Nuclisens kit (Organon Teknica, Fresnel, France) and internal controls

were added to validate the extraction and amplification steps. Amplification of extracted RNA was done with Cobas Amplicor 2.0 and quantification with the Amplicor HCV monitor. Seminal plasma  extractions were done on large volumes (300 to 1000 mL) and extracted RNA was amplified at a high concentration. These modifications of the procedure allowed us to obtain a sensitivity of 20 and 100 copies per mL of seminal fluid for qualitative and quantitative PCR, respectively.

Statistical analysis was done using Mann-Whitney analysis. HCV RNA was detected in the blood of all patients, plasma viral load  and genotype were obtained for 18 patients, with a median viral load of  547 log/mL [227-630], and all genotypes (1 to 4) were represented  (table). Eight seminal plasma samples of 21 (38%) were found to contain HCV-RNA (table). The median blood viral load in patients with positive semen  was higher than in patients with negative semen, but the difference was not significant (figure). Semen viral loads were low. In three samples 172,

149, and 134 copies/mL were detected; in the other five cases HCV-RNA detection was negative using the quantitative method although it was positive with the qualitative PCR and therefore the viral loads could  be estimated between 20 and 100 copies/mL.

Patients   

Number of HCV-positive semen (%)

Median HCV semen viral

(-log/mL)

HCV genotypes (HIV neg, n= 6; HIV pos, n=15; total, n= 21)*

Median HCV blood 600 (520-624)522 (227-582)563 (227-624) viral load (-log/mL)

*Genotypes were available for 18 patients out of the 21, and for seven of eight patients presenting with a positive semen. Number of patients with a positive semen.

AUTHORS COMMENTS

The discrepancy between such a high recovery of positive semen and the negative results of previous studies can be explained by the differences in the method used for HCV detection in semen. First, we were able to overcome PCR inhibition by using both Nuclisens extraction and dilution of samples in bromelain. The presence of PCR inhibitors has been a major hindrance in previo us studies, with PCR inhibition identified in more than half of the semen samples.4 We used bromelain (a non-specific cysteine protease) to efficiently liquefy semen samples, to overcome PCR inhibitors. Indeed, internal control OD values were significantly higher (mean OD value=3018) in diluted semen (n=17) than in undiluted semen (mean OD value=1381, n=15; p=0013). Second, we used a reverse transcriptase PCR, which is more sensitive than those used in previous studies, with a sensitivity of 20 copies per mL of seminal fluid compared with 600 copies/mL.4 The high sensitivity of the method used was critical since HCV viral loads in semen were less than 600 copies/mL.

AUTHOR'S CONCLUSIONS

The presence of HCV-RNA in semen is a strong argument in favour of HCV sexual transmission from men to women. HCV viral loads detected in semen were low, which suggests that the risk of HCV sexual transmission is probably also low. We are aware that only further studies using experimental infection in a cell culture system or an animal model would prove that HCV-RNA positivity in semen reflects the presence of infectious virus. However, our results are consistent with epidemiological data which suggested that HCV sexual transmission may occur with a low risk.5 Finally, these results should be considered in the broader perspective of safety in laboratories and for counselling and management of HCV-serodifferent couples who intend to embark on medically assisted reproduction.

1 Fiore RJ, Potenza D, Monno L, et al. Detection of HCV RNA in serum and seminal fluid from HIV-1 co-infected intraveous drug addicts. J Med Virol 1995; 46: 364-67 [PubMed].

2 McKee TA, Avery S, Majid A, Brinsden PR. Risks for transmission of hepatitis C virus during artifical insemination. Fertil Steril 1996; 66: 161-63 [PubMed].

3 Fried MW, Shindo M, Fong TL, Fox PC, Hoofnagle JH, Di Bisceglie AM. Absence of hepatitis C viral RNA from saliva and semen of patients with chronic hepatitis C. Gastroenterology 1992; 102: 1306-08 [PubMed].

4 Semprini AE, Persico T, Thiers V, et al. Absence of hepatitis C virus and detection of hepatitis G virus/GB virus C RNA sequences in the semen of infected men. J Infect Dis 1998; 177: 848-54 [PubMed].

5 Wejstal R. Sexual transmission of hepatitis C virus. J Hepatol 1999; 13: S92-95.