We have developed a novel RT-PCR assay for HHV-6. The primers chosen amplify mRNA transcripts corresponding to a major structural antigen of the virus, gp105. This antigen is expressed as a late γ gene and, therefore, should be indicative of productive viral replication (11
). Furthermore, the primers were designed to amplify across well-defined splicing boundaries within gp105 mRNA. Thus, the assay allows one to readily distinguish between amplification of mRNA and amplification of residual DNA contamination.
HHV-6 RT-PCR used on more than 100 clinical specimens was found to be highly sensitive, detecting 95% of primary HHV-6 infections in children, with a 98.8% specificity and negative predictive value. In comparison, a plasma DNA PCR assay developed by Secchiero et al. to diagnose acute HHV-6 infection has a lower sensitivity (13
). In initial studies, plasma DNA PCR for HHV-6 detected primary HHV-6 infection in 6 of 7 infants (13
); subsequent studies demonstrated a sensitivity of only approximately 66% (5
The highly sensitive nature of our assay could explain the single discordant result that we obtained. One positive specimen was RT-PCR positive but DNA PCR negative and tissue culture negative. This could be due to a false-positive RT-PCR. Alternatively, the RT-PCR assay may be sufficiently more sensitive to detect earlier infection than are standard tissue culture and DNA PCR techniques. Our DNA PCR assay can reliably detect 10 copies of the HHV-6 genome (7
). However, total specimen volume, processing, and amplification techniques are distinctly different between the two PCR assays, making comparisons difficult. Additionally, neither PCR is designed to be quantitative.
Our new RT-PCR assay may help in delineating the pathogenesis of HHV-6 by differentiating acute or reactivated infection from past or latent infection. Currently, viral isolation, serology, and DNA PCR are used most frequently to detect HHV-6 infection. Although viral isolation indicates active infection and, combined with serology, can determine primary infection, viral isolation is difficult, costly, and time-consuming. More commonly, DNA PCR and serology are used for diagnosis. These assays, however, cannot differentiate between latent and reactivated infections.
RT-PCR assays have been demonstrated to reliably differentiate latency from reactivation in patients with other herpesvirus infections. The major immediate-early and late mRNA transcripts of HCMV detected by RT-PCR have been linked to viral replication (2
). The assay for HCMV was also compared to viral isolation and HCMV DNA PCR techniques (6
). The RT-PCR had a lower sensitivity but a much higher specificity, suggesting that the RT-PCR assay was a more useful clinical tool (6
Our RT-PCR assay proved useful in determining the pathogenesis of dual HHV-6 and HHV-7 infections in two patients. Although both HHV-6 and HHV-7 were isolated in culture and detected by DNA PCR, the RT-PCR was negative for HHV-6, and serology indicated past infection with HHV-6. Without this conflicting information from the RT-PCR, the recognition of subsequent contamination of exogenous, latent HHV-6 DNA from a CBMC suspension used to refeed the cultures would not have been suspected or detected. Latent HHV-6 has been shown to be reactivated by HHV-7 in vitro (9
). Also, the presence of HHV-6 DNA in CBMCs has been described (4
). Alternatively, reactivation of the patient's own HHV-6 in culture is also a possibility.
In conclusion, this RT-PCR assay appears to reliably differentiate between latent and actively replicating HHV-6. This is a potentially powerful tool for both clinical and basic science investigations and also for greater insight into the pathogenesis of this ubiquitous virus.