Among the techniques described in recent years for quantifying HBV DNA by PCR (16
), some have been based on semiautomatic assays and were described as providing good sensitivity for the detection of HBV DNA (39
). More recently, introduction of real-time PCR technology for HBV viral load measurement has offered even better characteristics, with an overall larger range of quantification and improved sensitivity (1
). Although these two technologies have been assessed and compared within different single-center laboratories, an evaluation of such techniques through a multicenter quality control study was missing. The strength of such an evaluation is to provide information on what may happen to a patient who may be followed over time in different laboratories using in-house or commercially available techniques. In terms of the technical handling characteristics of the six studied HBV DNA quantification methods, one of the major inconveniences involved the Cobas Monitor PCR system, which has the narrowest dynamic range and requires dilution of samples in cases of high VL to artificially extend the test linearity. This drawback has been pointed out previously (43
). Due to the error rate linked to the dilution process, a weakness is introduced in the accuracy of the technique, especially for active replicative HBV carriers with VLs that may reach over 9 log10
copies/ml. It is likely that the highest CV in interlaboratory comparison observed in our study with the Cobas Monitor for samples with a high VL reflects this phenomenon.
In the performances of the HBV DNA quantification methods, we observed an underestimation of the VLs of genotype F samples with Cobas Monitor. Although genotype F is mainly present in Central and South America (4
), this failure of Cobas Monitor (58
) may have an impact on the monitoring of HBV infection, particularly if the genotype is not determined before VL quantification. Thus, we recommend not using this assay for the monitoring of patients infected by a genotype F strain.
The Artus System tended to provide VL results lower than those of the other assays, especially for the genotype E samples. However, the high CV observed between the results of the two laboratories that used this assay could suggest interlaboratory variability. Further extended studies of the Artus Real Time PCR system (as well as the other real-time PCR methods used by only one participating laboratory) are needed to assess the performances of these new methods of HBV DNA quantification.
Although the observed differences were not significant compared with the other assays, the bDNA assay tended to give higher VL results, as previously reported (25
). Moreover, the reproducibility rate was very high (46
). However, due to its low sensitivity, linked to the principle of this assay (49
), the bDNA assay had a high failure rate for the two samples of the panel with the lowest VLs.
The results obtained here and in other studies have demonstrated that the real-time PCR methods offer several advantages in terms of analytical sensitivity, with lower limits of detection and larger dynamic ranges of quantification. The good performances of real-time PCR methods and the availability of automatic platforms will justify the use of these tests for the diagnosis of HBV infection in the near future, especially for atypical serological patterns, as in occult infection (2
), in HBsAg carriers with a low level of replication (22
), or for treatment monitoring in order to identify resistant mutants that could be difficult to demonstrate with methods exhibiting a low limit of detection.
Although correct results were obtained for 50% of the participants for the entire panel of HBV genotypes, the data analysis revealed a few discrepancies, essentially due to poor amplification sensitivities of the samples with the lowest VLs and sometimes due to misclassification. Indeed, the false-negative results obtained with samples s4 and s5 were probably due to their low VLs rather than to their genotypes. The misclassification of samples s3 and s10, both containing a genotype A strain and classified as G and F/G by two laboratories using an in-house method, may be explained by the close phylogenic proximity of A and G genotypes in the amplified region (3
). However, the possibility that these two samples were really coinfected with the two genotypes cannot be excluded, since that seems to be the case for most subjects infected with genotype G. More surprisingly (and contrary to data provided by three other laboratories using the same technology), laboratory B reported 67% mixed infections with the Inno-Lipa method. This method has been shown to be very sensitive for identification of mixed-genotype infections, but the reported rate of mixed infections is usually between 0.1 and 10% (30
). Clearly, Inno-Lipa allows rapid detection of HBV genotypes in diagnostic laboratories, but extensive comparison studies of this assay with cloning experiments are needed to establish the reality of the mixed infections and to clearly exclude any issue of specificity.
Contrary to the results that we have obtained for HCV genotyping (35
), the fact that no reference genome database was provided to all the participants did not seem to have influenced the genotyping results obtained by direct sequencing. The reason for this finding is probably the absence of subtype definition for HBV, permitting a less precise phylogenic analysis without consequence for the final genotyping classification.
In the precore/core region, the most common naturally occurring HBV mutations are G1896A, which creates a stop codon in the precore gene (9
), and an A1762T-G1764A dual mutation in the core promoter region, which is responsible for a down-regulation of HBcAg production (9
). Some of the participating laboratories did not routinely perform the determination of these mutations (only nine provided results). Besides laboratory B, which gave 75% incorrect results for an unknown reason (a nonspecific hybridization with the line probe assay could be involved), incorrect answers were mainly due to an absence of amplification or an indeterminate result independent of the method used. This is in accordance with the fact that the determination of precore/core mutations is not performed on a routine basis and that only a few laboratories are accustomed to the procedure. Indeed, the usefulness of identification of these HBV mutations is still being debated (10
). Moreover, the relationship between these mutants and the response to antiviral treatment remains controversial (9
) and could be due to other factors, such as genotypes or recruitment bias (21
). Thus, it does not seem crucial to determine the existence of these mutations in order to evaluate the severity of the disease and to manage antiviral treatments.
One may conclude from this multicenter study that the large majority of expert laboratories routinely use commercial assays for HBV VL determinations (8
). The study also showed some drawbacks with two widely used assays: (i) Cobas Monitor has a narrow dynamic range and underestimates genotype F sample VLs and (ii) bDNA shows poor sensitivity and may therefore fail to identify patients with low VLs. With higher performance in terms of analytical sensitivity combined with a larger dynamic range and an ability to quantify the main genotypes equally, the real-time PCR methods appear more appropriate for accurate monitoring of HBV DNA quantification. Furthermore, the clinical implications of HBV genotyping, as well as the determination of precore/core mutants, need to be clearly stated to justify the standardization of these methods. A problem that could justify the intensive use of appropriate diagnostic tools would be the emergence of a high number of drug-resistant mutants.