Hepatitis C virus RNA detection in different semen fractions of HCV/HIV-1 co-infected men by nested PCR

Abstract

Objective

The aim was to evaluate, by nested PCR, the prevalence of hepatitis C virus (HCV) RNA in seminal plasma in different semen fractions of HCV/HIV-1 co-infected men.

Study design

This study enrolled 16 HCV/HIV-1 infected men. A total of 16 seminal samples and 16 blood samples were tested for the presence of HCV-RNA. HCV-RNA in blood plasma was quantified by Amplicor HCV Monitor Test version 2.0 and HCV-RNA detection in seminal plasma, non-spermatozoa cells (NSCs), spermatozoa pellet and swim-up was investigated by nested PCR.

Results

Thirteen blood plasma samples were positive for HCV-RNA. HCV-RNA was detectable in seminal plasma and in non-sperm cells, but not detectable in spermatozoa samples, neither before nor after swim-up. One of the two patients whose seminal plasma tested positive at nested PCR had undetectable HCV virus in blood plasma.

Conclusions

HCV-RNA can be found in seminal plasma and non-sperm cells but not in spermatozoa before and after swim-up. We observed HCV-RNA in the semen of an aviremic man. According to these findings we suggest that sperm washing should be performed for each semen sample of HCV patients before assisted reproduction techniques.

Introduction

Hepatitis C virus (HCV) transmission is known to occur essentially by the parenteral route [1]. Other potential ways of non-parenteral transmission in HCV-positive-infected patients are through body secretions as saliva, ascites, breast milk, urine and feces [2], [3], [4]. The studies that have analysed the HCV-RNA presence in semen have reported controversial results. Old studies failed to document the presence of HCV-RNA in seminal plasma [5], [6], [7] while some other old studies and all recent studies have indicated its presence [2], [8], [9], [10], [11], [12], [13], [14], [15], [16], [17], [18], [19], [20], [21].

The discrepancy in results on the presence of HCV-RNA in seminal plasma of men chronically infected by this agent is due to various factors, such as the molecular techniques used (Cobas amplicor monitor, nested PCR, real-time PCR, Amplicor HCV amplification kit and detection kit), the sensitivity of the assays designed to detect HCV-RNA, the wide range of protocols used for RNA extraction [9], [22] and eventually the presence in semen of inhibitors of PCR, like lactoferrin, peroxides, and mostly zinc residues that might interfere with the action of Taq polymerases [23].

In order to standardize the methods of detection of HCV-RNA in semen, a multicentre quality control study was performed in 12 French laboratories [22]. All centres used RT-PCR and Amplicor HCV Cobas assay to evaluate the presence of HCV-RNA in semen samples. They concluded that the percentage of correct results ranged from 53.3 to 100 and that the poorest results were obtained when no centrifugation step preceded the Amplicor extraction protocol, due to the negative effects of inhibitors of RT-PCR in seminal plasma. In 2002 Meseguer et al. [12] demonstrated that semen samples tested HCV negative by use of commercial methods (Amplicor Monitor RT-PCR) for HIV/HCV detection, but proved positive at nested PCR examination-data confirmed by Garrido et al. in 2004 [16].

In order to determine whether discrepancies in reported findings on the presence of HCV-RNA in semen are the result of inadequate methodological approaches for the detection of HCV-RNA, or of different seminal fractions, we hypothesized that nested PCR applied to each one of the seminal components – seminal plasma, non-sperm cells (NSCs), washed spermatozoa before swim-up, and washed sperm after swim-up – could overcome these limitations and provide sufficient insight on this issue even in a small series of HCV infected patients.