Reducing the prevalence of HPV infection is a Developmental Healthy People 2010 objective: “Reducing the number of new HPV cases can help minimize the overall number of cases of high risk subtypes associated with cervical cancer in females...” Detection and typing of HPV DNA in vaginal swabs (in conjunction with testing of NHANES sera for HPV antibody) will allow evaluation of trends in prevalence of type-specific HPV infection by age, sexual behavior, and race/ethnicity. Two HPV vaccines (Gardasil and Cervarix) are licensed and recommended for use in girls and women. Routine vaccination is recommended for girls 11 or 12 years of age, and catch-up vaccination through 26 years. One vaccine (Gardasil) is licensed and available for boys and men. As vaccine becomes more widely used, the national prevalence of HPV infection will be critical for evaluating vaccination strategies in the United States.
The vaginal swab is extracted to obtain DNA. The DNA extracts are used in the Digene hc2 HPV DNA Test. This test, using Hybrid Capture 2 technology, is a nucleic acid hybridization microplate assay with signal amplification. It uses chemiluminescence for the qualitative detection of eighteen types of human papillomavirus (HPV) DNA in cervical specimens. The hc2 HPV DNA Test can differentiate between two HPV DNA groups: low-risk HPV types (LR) 6, 11, 42, 43, 44; and high/intermediate-risk HPV types (HR)16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68. It cannot determine the specific HPV type present.
Specimens containing the target DNA hybridize with the HR or LR HPV RNA probe cocktail. The resultant RNA: DNA hybrids are captured onto the surface of a microplate well coated with antibodies specific for RNA: DNA hybrids. Immobilized hybrids are then reacted with alkaline phosphatase conjugated antibodies specific for the RNA: DNA hybrids, and detected with a chemiluminescent substrate. As the substrate is cleaved by the bound alkaline phosphatase, light is emitted which is measured as relative light units (RLUs) on a luminometer. The intensity of the light emitted denotes the presence or absence of target DNA in the specimen. An RLU measurement equal to or greater than the Cutoff Value indicates the presence of HPV DNA sequences in the specimen. An RLU measurement less than the Cutoff Value indicates the absence of the specific HPV DNA sequences tested or HPV DNA levels below the detection limit of the assay.
The Digene hc2 HPV DNA Test is approved for clinical testing, but the self-collected vaginal sample does not meet clinical guidelines. The HPV PCR tests are research tests. The HPV laboratory followed strict research QC/QA and was CLIA certified August 2008. Detailed quality control and quality assurance instructions are discussed in the NHANES Laboratory/Medical Technologists Procedures Manual (LPM). Read the LABDOC file for detailed QA/QC protocols. The analytical methods are described in the Description of the Laboratory Methodology section.
Refer to the 2005-2006 Laboratory Data Overview for general information on NHANES laboratory data.
The analysis of NHANES laboratory data must be conducted using the appropriate survey design and demographic variables. The NHANES 2005-2006 Demographics File contains demographic data, health indicators, and other related information collected during household interviews as well as the sample design variables. The recommended procedure for variance estimation requires use of stratum and PSU variables (SDMVSTRA and SDMVPSU, respectively) in the demographic data file.
Variables LBDRHP through LBDRPI are from the RUO Roche Linear Array HPV typing assay, however LBDR52 also includes information from a type-specific assay for HPV 52. The Linear Array typing strip includes an XR probe that hybridizes with HPV 52 as well as HPV types 33, 35 and 58. Samples positive for the XR probe and 33, 35, or 58 require specific testing to confirm the presence of HPV 52.
Steinau M, Swan DC, Unger ER. Type-specific reproducibility of the Roche Linear Array HPV genotyping test. J Clin Virol 42: 412-414, 2008.