Significance of “Not Detected but Amplified” Results by Real-Time PCR Method for HPV DNA Detection

Abstract

Human papillomavirus (HPV) infection is an important etiologic factor in cervical carcinogenesis. Various HPV DNA detection methods have been evaluated for clinicopathological level. For the specimens with normal cytological finding, discrepancies among the detection methods were frequently found and adequate interpretation can be difficult. 6,322 clinical specimens were submitted and evaluated for real-time PCR and Hybrid Capture 2 (HC2). 573 positive or “Not Detected but Amplified” (NDBA) specimens by real-time PCR were additionally tested using genetic analyzer. For the reliability of real-time PCR, 325 retests were performed. Optimal cut-off cycle threshold (C T) value was evaluated also. 78.7% of submitted specimens showed normal or nonspecific cytological finding. The distributions of HPV types by real-time PCR were not different between positive and NDBA cases. For positive cases by fragment analysis, concordance rates with real-time PCR and HC2 were 94.2% and 84.2%. In NDBA cases, fragment analysis and real-time PCR showed identical results in 77.0% and HC2 revealed 27.6% of concordance with fragment analysis. Optimal cut-off C T value was different for HPV types. NDBA results in real-time PCR should be regarded as equivocal, not negative. The adjustment of cut-off C T value for HPV types will be helpful for the appropriate result interpretation.

Comparison between the Roche Cobas 4800 Human Papillomavirus (HPV), Abbott RealTime High-Risk HPV, Seegene Anyplex II HPV28, and Novel Seegene Allplex HPV28 Assays for High-Risk HPV Detection and Genotyping in Mocked Self-Samples

“…Importantly, the not detected but amplified (NDBA) phenomenon for the Abbott RealTime assay, i.e., the event in which the assay reports samples to be negative despite showing an amplification curve with a C q value higher than the manufacturer’s positivity cutoff (32.00), as described by earlier research of Kim and colleagues ( 21 ), also occurred frequently within our sample selection. Kim and colleagues ( 21 ) suggested that samples NDBA should be regarded as equivocal, not negative, considering that they could be a sign of a low-copy presence of hrHPV. Consequently, a comparison between the four HPV assays was also performed using a less stringent C q cutoff range for positivity on the Abbott RealTime assay, being 32.00 to 36.00.…”

“…For all HPV assays, concordance results were analyzed based on the manufacturer’s recommended C q cutoff for positivity. However, as described in earlier research ( 21 ), the Abbott RealTime assay often reports samples as HPV negative, while showing an amplification curve with a C q value above the manufacturer-defined cutoff for positivity (<32.00) (NDBA [not detected but amplified]). Therefore, in the current study, concordance and agreement between the different HPV assays was determined both by using the Abbott RealTime manufacturer’s recommended C q cutoff for positivity (<32.00) and by using a less stringent C q range for positivity on NDBA samples (32.00 to 36.00).…”