Detection and identification of HPV genotypes strongly depend on the accuracy and the precision of the methods used. As vaccine trials include surrogate virological end points (incident and persistent infections) to assess the efficacies of candidate prophylactic HPV vaccines, it is crucial that highly reliable and robust testing methods be used (15
The present study investigated a novel testing algorithm that comprised a combination of broad-spectrum and type-specific PCRs. The results clearly showed that broad-spectrum PCR with a mixture of defined primers followed by reverse hybridization is a very useful tool for the identification of HPV genotypes in clinical samples but may underestimate the prevalence of multiple genotypes compared to the prevalence obtained by type-specific PCR. If two HPV genotypes are present but one genotype is present in great molar excess over the other, it is likely that the minor genotype will be not be detected during broad-spectrum PCR and will remain unidentified. This is clearly illustrated by the results obtained with the plasmid mixtures. When the concentration of the HPV-16 plasmid exceeded a low concentration (100 copies) of the HPV-18 plasmid more than 100-fold, HPV-18 was no longer detected by broad-spectrum PCR, whereas it was still detected by the type-specific PCR. These experiments were not repeated in sufficient replicates to calculate an accurate detection limit for each type in the mixture.
The analytical sensitivity of a broad-spectrum PCR for the detection of multiple genotypes will be influenced by the genotypes present. Since different HPV genotypes contain different nucleotide sequences at the primer target regions, each genotype is preferentially amplified by a subset of PCR primers from the available broad-spectrum primer pool. Thus, the amplification efficacy of every broad-spectrum PCR will be type dependent to some degree (6
). It is noteworthy that this competition phenomenon is observed only in samples that contain multiple HPV genotypes by the LiPA detection system. In samples containing only a single HPV genotype, it is adequately detected by the general PCR primer as well as the type-specific primer sets (32
). More than 1,000 SPF10
PCR-DEIA-negative samples were also tested by TS PCR for HPV-16 and HPV-18 and did not yield any additional positive samples (data not shown). This indicates that the SFP10
PCR has a very high sensitivity for the detection of HPV DNA in general and can be effectively used to screen for HPV positivity. These data again confirm that the SPF10
PCR-DEIA system has a very high level of specificity, and false-positive signals are not observed when only human DNA is used as the target (19
As described here for the SPF10
primer set, the efficacy of any general HPV primer set (such as My09/My11, PGMY, and GP5+/GP6+) will depend on the intrinsic degree of type specificity of the primer set and the competition between multiple genotypes in a complex mixture in the same sample (13
). It has been reported earlier that there are significant differences between the My09/My11 (degenerate primers) and the PGMY (a mixture of defined primers) PCR primer sets, although they target exactly the same region of the HPV genome (6
). Similarly, a comparison between SPF10
LiPA and PGMY PCR-line blot assay showed a very high degree of concordance, but for some genotypes, the two systems showed differences (32
). Therefore, if one is interested only in the detection of a particular subset of HPV genotypes, type-specific PCRs may be more suitable than a broad-spectrum general PCR. However, this requires individual or multiplex testing for each HPV genotype, which is not suitable for routine diagnostic applications and which limits the information obtained for each sample.
Post-PCR detection methods also may yield different detection rates of multiple HPV genotypes (30
). Analysis of broad-spectrum PCR products by direct sequencing lacks sensitivity and specificity for the analysis of complex mixtures of multiple genotypes and identifies only the most predominant types. An individual genotype should constitute at least 20% of the mixture to permit adequate identification by direct sequencing. In contrast, reverse hybridization (such as LiPA) is much more sensitive and allows the identification of minority genotypes, even if their DNA represents less than 1% of the total amount of HPV DNA, as shown by the results of the plasmid mixture experiment.
The specificity of the LiPA genotyping method was further assessed by sequencing SPF10 amplimers from samples that remained negative by the LiPA. A probe was not present in the LiPA for any of the HPV genotypes identified by sequence analysis in this group, confirming the high specificity of this reverse hybridization assay. Combining the genotyping data of the LiPA and sequencing showed a total of more than 50 HPV genotypes, illustrating the truly broad-spectrum amplification by SPF10 primers. Since multiple samples were obtained from the same women over time, the epidemiological information regarding genotype distribution based on these selected analyses is limited.
Within the SPF10 system, the same PCR amplimer is used for both detection of HPV positivity by DEIA and subsequent genotyping by LiPA. The DEIA uses a cocktail of probes that recognize more than 50 different HPV genotypes, whereas the LiPA identifies only the 25 genotypes for which probes are present on the strip. Therefore, if the DEIA were omitted, the overall sensitivity of HPV detection would decrease substantially. Also, use of a reverse hybridization strip assay as a screening method is not economically efficient. Moreover, if a different PCR were used to screen for HPV positivity, reamplification of these HPV-positive samples would be required, which could lead to discrepant results between the two PCRs. Therefore, it is important to use only a single, well-characterized PCR for the initial detection of HPV as well as for subsequent genotyping.
Another important aspect of molecular diagnosis is the effect of sampling variation due to sample heterogeneity. Sampling variation is particularly relevant when nonhomogeneous clinical materials, such as cervical cell suspensions or cervical biopsy specimens, are used (11
). Also, sampling variation may become relevant when only a small volume of the extracted nucleic acid is included in the actual PCR vial, and consequently, the test outcome may not properly represent the true status of the sample. Especially when samples contain low viral loads, sampling variation may lead to false-negative results.
To evaluate the effect of the algorithm testing, samples from a phase II HPV-16 and -18 bivalent candidate HPV-16/HPV-18 vaccine trial were analyzed. The cervical cells had been resuspended in PreservCyt medium for transport and long-term storage. The proportion of cell suspensions that yielded negative β-globin PCR results was very low (<0.1%), indicating that inhibition is a very rare event and confirming the usefulness of the PreservCyt medium for PCR-based analyses.
The comparative results of the SPF10 and the type-specific PCRs clearly confirmed the added value of the combined testing approach. The discrepant results between the SPF10 LiPA and the type-specific PCR could be explained by a combination of the competition effect and the sample heterogeneity effect. Virtually all type-specific PCR-positive but LiPA-negative samples contained multiple genotypes, whereas the proportion of multiple-genotype infections was significantly lower among the type-specific PCR-negative but LiPA-positive samples. For the latter group, sample heterogeneity played a major role. This was further substantiated by quantitative PCR analysis of a subset of the discrepant samples (data not shown). In general, samples with discrepant results (LiPA positive and TS PCR negative or vice versa) contained lower viral loads than samples with concordant positive results (LiPA positive and TS PCR positive), which is consistent with sampling heterogeneity effects.
The benefits of the novel HPV testing algorithm was evaluated with samples from a group of women participating in a trial of a candidate HPV-16/HPV-18 vaccine and clearly revealed that the use of the algorithm during the monitoring of individual women resulted in the earlier and more accurate detection of HPV-16 and/or HPV-18 than when either SPF10 LiPA or TS PCR alone was used. The earliest detection depends on several factors, such as the viral load and the presence of other HPV genotypes.
A higher number of different HPV genotypes was detected in women for whom the TS PCR detected HPV-16 and/or HPV-18 earlier than SPF10 LiPA. This would be concordant with the hypothesis that detection by SPF10 LiPA is influenced by the competition effect. The more genotypes that are present in a woman, the higher the chance that HPV-16 and/or HPV-18 is missed by SPF10 LiPA due to a competition effect.
In the majority of women for whom detection by SPF10
LiPA and TS PCR was simultaneous, HPV-16 and/or HPV-18 was detected at multiple time points. This is compatible with the hypothesis that women with HPV-16 and/or HPV-18 positivity at multiple time points contain higher viral loads, resulting in detection by both SPF10
LiPA and TS PCR. Thus, the testing algorithm will have the biggest impact in detecting incident infections, in which HPV is only transiently detectable at lower viral loads. During persistent infections, HPV is generally present at higher viral loads and is therefore readily detectable by either SPF10
LiPA or TS PCR or by both methods (9
In conclusion, the present study has provided a reliable and highly effective testing algorithm for the molecular diagnosis of HPV infections. This algorithm yields superior results compared to those obtained by the use of any single PCR test. Therefore, the use of the algorithm can substantially contribute to the accuracy of analysis in vaccination trials and epidemiological studies.