Defining detection threshold and improving analytical proficiency of HPV testing in clinical specimens

Fang Yu Chao, Angel Chao, Chu Chuen Huang, Swei Hsueh, Jung Erh Yang, Huei Jean Huang, Lei Chiung Wang, Cheng Tao Lin, Hung Hsueh Chou, Chyong Huey Lai*

*Corresponding author for this work

Research output: Contribution to journalJournal Article peer-review

9 Scopus citations

Abstract

Objective: Previous studies have shown that SPF1/GP6+ PCR followed by HPV Blot has high sensitivity and high reproducibility for the detection of human papillomavirus (HPV). However, there have been reports of false-negative results. In the present study we endeavoured to improve the diagnostic performance of HPV DNA testing in cervical swab specimens. We also aimed to identify viral load thresholds. Methods: We examined DNA from cervical swabs of 7 women with a cervical intraepithelial neoplasia grade 2 or worse (CIN2+) who were negative by HPV DNA testing. For control purposes, 106 cytology negative/HPV-negative samples were included. PCR was performed with either 1 μL of DNA solution or different amounts of input DNA (25, 50, and 100 ng). We tested two different commercial alkaline phosphatases (ALPs) at two distinct reaction times. The overall conclusions were based on HPV Blot, type-specific PCR, direct sequencing, and real-time quantitative PCR (qPCR). Results: All seven patients with CIN2+ disease turned HPV-positive when 100 ng of input DNA and E6 type-specific PCR were used. Discrepant and false-negative results were obtained using different amounts of input DNA. Different commercial ALPs showed a significant impact on detection performance. Analysis of viral load indicated that the detection threshold for HPV infection using SPF1/GP6+ PCR plus HPV Blot was approximately 102-103 copies. Conclusions: Reliable determination of HPV status is crucially dependent on the amount of input genomic DNA.

Original languageEnglish
Pages (from-to)302-307
Number of pages6
JournalGynecologic Oncology
Volume117
Issue number2
DOIs
StatePublished - 05 2010
Externally publishedYes

Keywords

  • Cervical cancer
  • Detection threshold
  • Human papillomavirus
  • Viral load

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