This study describes the first use of the Abbott RealTi
me High-Risk HPV assay for HPV detection in cervical cell samples of women with or without cervical abnormalities from a Gabonese female population. Moreover, this assay was compared with the use of conventional nested PCR coupled to HTS, which identifies the exact hrHPV genotypes revealed with the AbRT assay. The real-time PCR method (AbRT assay) can detect a total of 14 different high-risk HPV genotypes whereas the conventional nested PCR that uses the primers MY09/11 and GP5+/6+ can amplify around 30 different low- and high-risk HPV genotypes [
13,
14]. The genotypes are assayed from DNA extracted from cervical smears whose cervical anomalies have been revealed by a VIA/VILI test and confirmed by cytology. Although the manufacturer recommends using the Abbott automaton (
m200sp, Abbott Molecular) with this assay for HPV detection, extractions were performed manually as described in literature as a practicable alternative. In a recent study, Kocjan et al. (2012) showed that similar results are obtained for HPV detection from biological tissues of head-neck squamous cell cancers regardless of the extraction method used with the AbRT assay [
15,
16]. Moreover, we did not observe any differences in amplification intensity of the human β-globin (a housekeeping gene) between the AbRT kit and a classic qPCR protocol (data not shown).
In this study, the AbRT generally gave consistent genotyping results for the hrHPV screened in HSIL+ cases in comparison with the conventional nested PCR. These data are similar to other studies that compared the AbRT kit with this nested PCR method or with other molecular methods of HPV genotyping [
15,
16]. However, nested PCR appears to be more sensitive than the AbRT kit for the detection of hrHPV in HSIL- cases (i.e. LSIL, ASCUS and normal) even if in most cases, at least one hrHPV genotype was detected by both methods (Table
3). Given that the detection threshold of the AbRT assay is between 500 and 5000 genome copies, genotypes detected using both methods in this study may have a higher number of copies than this threshold [
17], suggesting that cases for which only one genotype was detected by the nested PCR method contain fewer than 500 copies. However, this hypothesis remains to be tested because we have no data on the actual viral loads of our samples. Furthermore, although the correlation between the number of reads and viral load is not perfect, cases with genotypes detected by both methods were generally those with highest numbers of sequenced reads [
10]. Owing to the depth of sequencing with a minimum of several hundred thousand reads per sample, a minor HPV genotype can be detected if it is represented in at least 2% of the total reads [
10]. Therefore, in a given sample, HTS can unambiguously detect several genotypes whose relative proportions potentially vary [
18,
19]. Moreover, although the sequenced amplicon size was only 150 bp, this region of the L1 gene is highly amenable to molecular analysis, which can discriminate between the different HPV genotypes. Finally, the HTS approach for sequencing HPV amplicons may overcome the limit of direct amplicon sequencing using the Sanger method, which is simple to implement from a biopsy, but cannot be performed on DNA extracted from a (LBC) cervical smear, especially when the cytological grade is low or normal, due to the potential simultaneous presence of several HPV genotypes [
20]. Although HPV genotyping assays using consensus or broad-spectrum primers are less sensitive than type-specific or targeted primers [
21], consensus PCR is less affected by competitive primer binding in mixed HPV infections. For instance, the presence of high viral loads of HPV16 or, in some cases, the presence of some hrHPVs can mask the HPV52 genotype in anogenital samples [
22] or inhibit the amplification and detection of HPV31 and 33 [
23]. Moreover, unfavorable amplification kinetics may occur when an HPV genotype belonging to the alpha-9 subgenus is in the presence of other genotypes (Iftner et al. (2016).
Several studies have shown that the sensitivity of the AbRT assay is similar to that of the hc2 test and that the specificity of detection of HPVs associated with the development and/or spread of CIN2+ can reach 92% [
16,
24,
25]. GP5+/6+ PCR has the same clinical performance as the hc2 test [
8]. However, the addition of the first nested PCR step with MY09/11 primers improves the sensitivity of the conventional PCR test, but leads to a decrease in its specificity for the detection of HPV genotypes shown to be associated with the development of HSIL+ into cancer [
26]. Furthermore, the nature of the samples used in this study clearly influences the differences observed between the two methods. In contrast to samples from biopsies, LBC samples recover numerous superficial cells in addition to the cells located in a putative precancerous lesion. However, these superficial cells may be infected by the same hrHPV genotype or other hrHPV genotypes. The use of a sensitive HPV detection method may reveal these supplementary infections and not just the HPV implicated in the (pre)cancerous lesion. In effect, due to this high sensitivity, most studies on the clinical validation of HPV detection tests are carried out on FFPE tissue blocks, not LBC samples, and cytological results are generally confirmed by a histological analysis to confirm the grade of the observed lesion. Therefore, the differences between the two molecular methods for HPV detection are most likely due to the nature of the samples used in this study and may have led to variation in the performance of the test and the specificity of the AbRT compared with the data found in literature [
15,
24,
27‐
29]. However, the use of LBC led to a better preparation of the sample associated with an enhanced quality of the cytological results. Recent studies have shown improved slide reading when LBC systems are used instead of conventional Pap smears in a routine clinical setting. Moreover, LBC using PreservCyt (Thinprep) has been approved by the FDA for molecular HPV tests [
30]. This FDA approval provides the opportunity to carry out two biological analyses from a single sample, a certain advantage in low-income countries or when the population has limited access to healthcare.