HDV is associated with HBV infection in approximately 5% of HBV surface antigen carriers worldwide. A chronic HBV-HDV mixed infection frequently leads to cirrhosis, and the mortality rate for mixed infections is higher than that for chronic HBV infections alone (34
). The treatment of chronic hepatitis D has made very little progress, although the long-term benefit of high doses of IFN seems to be confirmed (12
). Like the case for chronic hepatitis C (26
), the use of the pegylated form of IFN might improve the treatment efficacy, but this has not yet been evaluated in the case of HDV. To date, there is no consensus for the treatment of chronic hepatitis D. Efficacy is usually evaluated with biochemical, histological, and virological parameters, such as specific anti-HDV IgM or HDV RNA. The detection of HDV RNA in serum or plasma is performed by the use of homemade qualitative RT-PCR techniques and reflects viral replication (8
). During the last decade, the management of various chronic viral infections has become more and more dependent on quantitative molecular approaches. In the case of HCV, the viral load is now used as a predictive marker of treatment efficacy. For HDV, the severity of the infection and the uncertainty of the treatment outcome are incentives for the development of reliable HDV RNA quantification assays to monitor treatment efficacy.
Real-time PCR based on TaqMan technology provides an accurate and sensitive means of quantifying viral genomes, with the major advantage of avoiding post-PCR handling that can be a source of DNA carryover. Several studies have reported the benefit of this technique for the quantification of viral genomes in blood samples (15
). The large dynamic range (10 to 107
copies) of the technique makes it particularly attractive for HDV RNA quantification. Indeed, on the one hand, large viral loads are expected to be detected in the case of immunodeficiency or acute infection (as observed after direct liver inoculation in chimpanzees [2
]), and on the other hand, very small amounts of RNA need to be detected during the follow-up of patients under treatment.
The development of an accurate and sensitive test for HDV RNA quantification in blood samples raises several technical problems. First, the “rod-like” structure of HDV RNA, which is due to intramolecular base pairing of >70% of the sequence (22
), is likely to impair cDNA synthesis and therefore PCR efficiency. Second, the genetic variability of the virus (5
) requires the design of primers and probes to target the most conserved regions of the genome (27
). Indeed, the divergence between HDV types has been shown to be as high as 37% over the entire nucleotide sequence of the genome (30
). As expected (3
), a comparative analysis of HDV sequences of different types indicated that the most conserved regions of the genome are located within the ribozymes (30
). The primers and probe were thus designed for these regions, and despite the strong secondary structures in the primer annealing region, the PCR efficiency was repeatedly over 93%. The quantitative real-time RT-PCR assay that was developed in our laboratory aimed to replace the qualitative assay as a routine diagnosis procedure. This was made possible because of the sensitivity of the assay and its ability to detect HDV RNAs of all known types, thus avoiding false-negative results due to a low copy number or sequence variability. HDV-4 isolates were not available for our study, but sequence alignments indicate that our assay should work for the detection and quantification of HDV-4 genomes. In the case of HDV-3 sequences, the existence of a mismatch in the forward primer led us to propose the use of a specific forward primer when an HDV-3 infection is suspected based on the clinical data, the geographical origin of the patient, or the presence of specific anti-HDV IgM with no detectable HDV RNA. Another problem concerns the standardization of the assay and its comparison to other existing techniques. To date, there is no available international standard or control to calibrate a quantitative assay for HDV. Moreover, apart from the assay described by Yamashiro et al. (41
), which was specifically designed to quantify HDV-2 and HDV-4 isolates (such isolates are only very occasionally involved in HDV infections among European and African patients in our environment, who are usually infected with HDV-1, -5, -6, or -7), there is no reliable test available for HDV RNA quantification in serum. Taken together, our results indicate that the real-time RT-PCR assay reported here is sensitive enough to replace the qualitative assay for the detection of HDV RNA in serum (samples for which the real-time RT-PCR assay detected 100 to 1,000 copies/ml were considered positive even when their values were below the quantification level) and reproducible enough to follow viral load kinetics in infected patients, with the quantification reproducibility being monitored by the use of external controls.
The clinical impact of HDV RNA quantification in serum remains to be fully established, but a recent study already indicated the possibility of an association between the HDV RNA load and liver damage (41
). In the present study, 11 chronically infected patients for whom sequential serum samples were available were evaluated in terms of the HDV viral load. Two patterns of virological response to IFN were distinguished according to the viral load reduction during treatment. Whether the qualification (based on the HDV viral load) of “responder” or “nonresponder” to treatment is appropriate would need further evaluation, since treatment failure could be due to a relapse after the end of therapy. Other factors such as the alaine amino-transferase level may need to be taken into account. However, our results suggest that HDV RNA quantification might help with monitoring the treatment duration. Indeed, in some cases, a viral load decrease might take a long time (as in the case of patient C), and some patients might need to be treated for >1 year, as previously described (24
). In the case of patient D, a significant decrease in the HDV RNA load was observed during the course of PEG-IFN therapy, although the patient had remained resistant to a prior 1-year treatment with IFN. This might indicate more efficacy of PEG-IFN for the treatment of chronic delta hepatitis. A quantitative RT-PCR assay will be useful to evaluate this possibility. Patients E to I maintained high viral loads during PEG-IFN therapy, indicating that other factors might interfere with the treatment efficacy. For example, the initial viral loads were higher for the nonresponder group than for the responders (P
= 0.05), and this could indicate that the baseline viral load might predict treatment efficacy and could be taken into account during the management of treatment in terms of dose and duration.
In summary, we have developed a sensitive assay to quantify HDV RNA in plasma or serum. Most importantly, the assay is consensual in that it performs equally across all known HDV types. Indeed, one should keep in mind that the HDV genome may diverge up to 37% at the nucleotide sequence level, and such variability is usually an impediment to the development of a universally sensitive assay. Our procedure overcomes this difficulty. The medical impact of HDV RNA quantification in serum remains to be established, and studies involving a larger number of patients are currently under way to address this point. However, this assay should help with the management of chronically infected patients, with specifying their evolutionary profiles before, during, and after treatment, and with the study of the natural history of HDV infection. It could also be used in large-scale prospective studies to define treatment guidelines and to evaluate the efficacy of new drugs.