Persistent infection of the uterine cervix by the same type of high-risk human papillomavirus(es) (HR-HPV) is related with cervical cancer. It becomes increasingly important to know which HR-HPV type(s) is/are present in the cervical smear. The objective of this study is to evaluate the analytical performance of the PapilloCheck kit for the detection and genotyping of 18 high risk and 6 low risk HPVs.
DNA from patient samples was extracted using the MagNA Pure platform (Roche DNA I High Performance protocol): 1 ml sample was first concentrated by centrifugation (20 min, 20.000 g). 800 µl supernatant was removed and the remaining 200 µl was used for extraction.
Due to the low concentration of cell material of the Quality Control for Molecular Diagnostics (QCMD) panel, the whole sample (5 ml) was concentrated (20 min, 4000g).
DNA was eluted in 110 µl elutionbuffer.
DNA of the WHO Proficiency Panel 2011 (PP) was already extracted.
5 µl DNA was used for the PCR.
The assay was checked for analytical sensitivity, specificity, accuracy and precision following the Belgian guidelines.
Analytical sensitivity: a negative PreservCyt specimen was spiked with the WHO-HPV16 DNA standard to determine the limit of detection (LOD with a 95% hit rate). The lowest concentration was 13.333 international units (IU)/ml, correlating with 120 copies/PCR.
Looking at the results of the WHO PP we can assert that the PapilloCheck detects 50 genome equivalent (GE)/PCR of HPV6, HPV11, HPV33, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, HPV59, HPV66, HPV68. For HPV18, HPV31 and HPV35 the sensitivity was 500 GE (IU)/PCR. HPV16 was detected at 5 IU/PCR. The PP was designed for genotyping needs in HPV vaccinology. The test is not proficient for HPV18 (50 IU/PCR) but according to the manual the LOD for HPV18 is 300 IU/PCR and is in accordance with the clinical needs.
Specificity: the specificity was sufficiently documented by the manufacturer, and was not tested again.
Accuracy: 52 specimens were tested.
WHO PP2011: 5 out of 43 were false negative (lower than LOD).
QCMD 2011 panel: 8 out of 9 were typed correctly. HPV45 in cc10b cells was missed.This cell line does not contain the full target used bij the assay. HPV45 was 4/4 times detected in the WHO PP.
Precision: One sample positive for HPV 31 and HPV51, a second sample with a multiple infection of 3 types: HPV81, HPV33 and HPV73 were extracted in triplicate on 3 different days. All types were detected correctly. This met our validation criteria.
The PapilloCheck method met all our validation criteria and was implemented in our routine diagnostic laboratory.
Raymaekers et al., Acta Clinica Belgica, 2011