Results from the plasmid standards revealed differences between the HPV typing assays. The titration of HPV16 in different amounts against constant HPV18 template numbers highlighted the robustness of the assays to competing types with different concentrations. Copy numbers of more than 5 × 103
or a >100-fold-larger template amount of HPV16 suppressed amplification of HPV18 in LiPA and RH PCRs. These observations are in line with assessments by van Doorn et al. (9
), who found that 100 copies of HPV18 were outcompeted by a 1,000-fold-higher concentration of HPV16. Only the LA was able to detect both types even at 100,000-times-higher HPV16 concentrations. The larger reaction volume (100 μl in LA versus 50 μl in the others) might be responsible for the superior tolerance to competition. The extreme differences in copy number are unlikely to occur in actual biologic samples; however, pushing the technical limits of the assays allowed for evaluation of robustness to a variety of challenges. All three LBAs generally could detect 50 IU or GE of the single high-risk HPV types. Only HPV68b was not detected by either RH duplicate, which is not surprising since the limit of detection (LOD) is stated as 105
viral copies in the RH detection kit handbook. The sporadic lack of reproducibility may indicate that this input amount is in the range of the lowest LOD for at least some types ().
Significant deficiencies were seen in samples that included more than one type. LiPA failed to detect 18% of the types included in the 36 multiplasmid challenges. The 22 missed types consisted exclusively of HPV39, -58, -59, and -69. In some instances, types 39 and 68b were identified as “possible” types due to the LiPA's multiprobe set (). Nevertheless, both HPV39 and -68b were also missed in other samples that were free of this ambiguity, suggesting unequal amplification efficiency or competition. Disregarding HPV6 and -11, the RH failed to detect 20.5% of the types in this subset. Besides HPV68b, types 52, 59, and 39 were missed in several instances. A critical limitation of the RH might result from the large discrepancy in detection sensitivities for different HPV types. According to the handbook, LODs differ 25,000-fold, ranging from four copies for HPV16 to 100,000 copies for HPV53 and -68.
Reproducibility of results for the plasmid challenges was greatest for the LA but was generally good in all assays. Among the results derived from the patient samples, the RH had the highest positive reproducibility (90.2%), but the denominator was also lowered to 51 types since HPV6 and -11 are not detected by this test. It was rather surprising that no significant differences were found between STM and FFPE extracts, as fractured and poor-quality DNA from archived tissues should favor shorter amplicon lengths as targeted by the SPF primers (8
). The sample size may not have been sufficient to allow differences in assay performance to be identified. Generally, results from the patient samples were comparable between the assays, with at least partial agreement in 87.5%. Performance differences found with the plasmid samples might reflect rather extreme situations which are not relevant for the majority of real clinical specimens.
False-positive results were a particular concern. Two instances were observed among LiPA results in plasmid samples. In both cases, the algorithm for identifying the expected type (HPV18 or -58) requires that more than one probe hybridizes with the amplicon. As only one of the required probes hybridized, another type (HPV39 or -52) was falsely indicated. It is likely that individual LiPA probes differ in their affinity to the same amplicon and generate an incomplete band pattern at small target amounts, which would consequently lead to attributing the result to an incorrect type. Detection of HPV52 by LiPA might be particularly vulnerable to this problem. In the LiPA, the HPV52 amplicon hybridizes to a single probe. However, that probe also hybridizes to amplicons of five additional HPV types that are distinguished by combinations with other probes. In this regard, it is noteworthy that HPV52 was detected in 9 of the 15 types found exclusively by LiPA among the patient samples (), and two of three patient samples that were exclusively HPV positive by LiPA had only type 52. It seems likely that at least some of these cases are false positives.
Conversely, some HPV52 may have been missed by the LA, as it has a similar shortcoming and detects HPV52 only through the cross-binding XR probe. While this ambiguity occurred in 28 cases among the LA results, only two of these samples tested positive for HPV52 by LiPA.
Every laboratory test is also influenced by the accuracy of human handling. The samples prepared from plasmid DNA yielded one inadequate result each by LA and LiPA, and the (single) HPV types not detected in these results were also counted as “missed.” Although the tests were performed with utmost care in a clinically certified laboratory, operator errors cannot be ruled out as a cause and may have lowered the real performances of the tests. However, this possible distortion should be minimal.
Analysis for this comparison study was restricted to the most relevant high-risk types as well as HPV6 and -11. It should be considered, however, that LA detected 50, LiPA 16, and RH 4 additional HPV types in the 60 patient samples. Depending on the number of types covered by each assay, the scope of HPV detection will always be limited and does not directly allow an “HPV-negative” interpretation.
For the majority of samples, HPV typing results will be very similar and comparable for the three LBAs evaluated. In difficult situations such as multiple infections, low copy numbers, or large difference in viral copies, LA has an advantage. Some limitations of LiPA are due to its multiple-probe detection system. RH is disadvantaged by low sensitivity for some types, particularly HPV53, -68, and -82. LA performed nearly perfectly and is hampered only by the cross-reacting XR probe for HPV52 detection.