Comparison of the clinical performances of hrHPV detection by the GP5+/6+-PCR and SPF10
assays showed that both assays had similar overall clinical sensitivities (i.e., 89%) for the prediction of finding CIN 3 lesions (i.e., via detection of hrHPV) in women with cytomorphologically normal smears. It is of note that the actual overall sensitivity values of these assays would be slightly higher (i.e., 93% [95%CI, 86 to 100] for the GP5+/6+-PCR assay and 91% [95%CI, 83 to 99] for the SPF10
assay) in this set when the outcome is based on hrHPV detection using a pool by (D)EIA, since two GP5+/6+-PCR EIA-positive cases and one SPF10
DEIA-positive case were designated as HPV-X and were not included in the final analysis. In practice, in clinical and experimental screening settings, scoring of hrHPV GP5+/6+-PCR assay positivity is based on the EIA readout only, showing a good sensitivity (2
). The positivity rate in controls was significantly low for the GP5+/6+-PCR assay compared to that for the SPF10
assay. The latter could be attributed mainly to the fact that SPF10
scored as positive significantly more samples from control women that had viral load levels apparently falling below the detection level of the GP5+/6+-PCR assays. Consequently, women with normal cytology results and a GP5+/6+-PCR-positive test result exhibited a 4.5 times higher risk of CIN 3 than those with a positive SPF10
test result. Altogether, these data confirm our previous concept (28
) that a too-high analytical sensitivity of an hrHPV test results in a marked decrease in clinical specificity without having an impact on the clinical sensitivity for detecting the ultimate development of ≥CIN 3. This is in line with data from several studies indicating that not hrHPV DNA presence per se but increased viral loads confer an increased risk of ≥CIN 3 (19
It is noteworthy that differences in the population characteristics could influence the relative performance of each assay. However, in this study similar differences in assay performance were found for control women of both the screening and the gynecological outpatient populations. The fact that among the first group of women the median age was significantly higher for controls (i.e., 41 years) than for cases (i.e., 34 years) is unlikely to have contributed markedly to the observed difference between both assays, since in the hospital population the median ages of cases and controls (32 versus 37 years) did not differ significantly. Our data nevertheless seem to contradict those found in a study reported by Safaeian et al. (25
). That study revealed that for women with normal cytology results enrolled for a vaccination trial, there was no difference in hrHPV positivities between the SPF10
and hc2 assays, the latter of which is considered to be compatible with the GP5+/6+-PCR assay (25
). A likely explanation for this apparent discrepancy is that Safaeian et al. studied young women under 30 years of age (median age, 21 years; range, 18 to 25 years). It is therefore tempting to speculate that hrHPV positivity in women under 30 years of age with normal cytology results is often accompanied by relatively high viral loads, giving rise to positivity rates that are equal for assays displaying different analytical sensitivities for hrHPV. This is supported by findings of a different comparison study between SPF10
and hc2 on samples from a population of women visiting a colposcopic clinic (22
). The latter study mainly included women above 30 years of age and displayed for women with normal cytology results a difference between hrHPV positivity by SPF10
versus that by hc2 that was similar to that seen for SPF10
versus GP5+/6+-PCR assays for control samples from our study.
Taken together, these data indicate that the threshold for the positivity of a given hrHPV test should be at such a level that an optimal balance between clinical sensitivity and specificity for identifying women at risk of having or developing ≥CIN 3 is reached. This concept finds support by recent recommendations of the American Society for Colposcopy and Cervical Pathology (ASCCP) (29
), which indicate that in a cervical screening setting, clinical sensitivities for ≥CIN 3 of at least 92% ± 3% seem satisfactory only when clinical specificities are above 85% at the same time. Therefore, the GP5+/6+-PCR assay can be advocated above SPF10
for screening purposes involving women over 30 years of age.
Apart from the difference in clinical performances, both tests showed common characteristics, since there was a high concordance in hrHPV genotyping, especially for samples from case women. We found a relatively high number of infections with HPV52 among the women for whom samples were SPF10
positive and GP5+/6+-PCR negative. This cannot be explained solely by a potentially low sensitivity of the GP5+/6+-PCR assay for HPV52 compared to that for HPV16 and HPV18 (23
). The minimal HPV52 viral load value determined for SPF10
assay-positive but GP5+/6+-PCR assay-negative samples did not differ markedly from those for HPV16 and -18. In fact, only three discordant HPV52 control samples might have resulted from a lower detectability by the GP5+/6+-PCR assay, possibly because these infections involve HPV52 variants. Thus, the majority of HPV52 infections detected by the SPF10
assay alone are due to lower copy numbers of HPV52, which are apparently relatively common among SPF10
-positive women with normal cytology results.
In conclusion, the application of hrHPV detection assays with a too-high analytical sensitivity for hrHPV results in a markedly reduced specificity for CIN 3 without being beneficial for clinical sensitivity. Therefore, hrHPV test requirements, as currently under preparation in The Netherlands, should be incorporated in cervical screening guidelines to prevent overdetection, which would counteract the benefits of implementing hrHPV testing in screening programs.