Component Description
The human papillomavirus (HPV) in oral rinse estimates the prevalence and determinants of oral HPV infection in the United States population. Oral HPV infection is newly appreciated as a strong risk factor for a distinct type of oropharyngeal squamous cell carcinoma that is rising in incidences in the United States. It has been estimated that oral HPV16 infection confers an approximate 15-fold increase in risk for oropharyngeal cancer. Despite these strong risks, little is known about the epidemiology of oral HPV infection. No population-based surveys of oral HPV infection, type distribution and determinants, have been performed in any population worldwide.
Eligible Sample
Examined participants, aged 14-69 years, were tested. The public-use data file includes data for persons 18-69 years of age. Please see Analytic Notes about the release of data for adolescents 14-17 years of age.
Description of Laboratory Methodology
Samples will be centrifuged at 4,000 x g for 10 minutes at 4°C; the pellet will be resuspended in 1 ml of Puregene cell lysis solution and incubated at 37°C for 15 min followed by DNase-free RNase a (5µg/ml) digestion for 30 min at 37°C. Proteinase K (20 mg/ml) digestion will be performed overnight at 55°C followed by heat inactivation for 10 min at 95°C. Protein precipitation solution (340 µL) will be added and sample will be further purified according to the Puregene DNA purification kit protocol.
Purified DNA will be analyzed for 37 types of HPV by means of a multiplex polymerase-chain-reaction (PCR) assay targeted to the conserved L1 region of the viral genome, using PGYM09/11 primer pools and primers for ß-globin (33). PCR products will be denatured in 0.13 N NaOH and hybridized to an immobilized HPV probe array (Roche Diagnostics) using an extended line-blot assay for genotyping of 37 HPV types, including HPV-6, -11, -16, -18, -26, -31, -33, -35, -39, -40, -42, -45, -51, -52, -53, -54, -55, -56, -57, -58, -59, -61, -62, -64, -66, -67, -68, -69, -70, -71, -72, -73, -81, -82 (MM4 and IS39 subtypes), -83, -84 and -89, and ß-globin (Roche Molecular Systems, Inc., Alameda, CA). Positive controls, consisting of 10 and 100 HPV-16 (SiHa) or HPV-18 (C4–2)-positive cells diluted in a background of HPV-negative cells (K562), and a negative control (K562 cells), in addition to the manufacturer’s controls, are included in each experiment. Samples positive for ß-globin will be considered of sufficient quality for analysis. The HPV type will be reported for positive samples.
Refer to NHANES 2013-2014 Lab Methods for HPV in oral rinse for detailed description of the laboratory method used.
There were no changes to equipment, lab method, or lab site for this component for the 2013-2014 cycle.
Laboratory Method Files
Laboratory Quality Assurance and Monitoring
Oral rinse specimens were processed, stored and shipped to the Ohio State University Gillison Laboratory, Columbus, Ohio.
Detailed instructions on specimen collection and processing are discussed in the NHANES Laboratory Procedures Manual (LPM). Vials are stored under appropriate frozen (–30°C) conditions until they are shipped to Ohio State University for testing.
The NHANES quality assurance and quality control (QA/QC) protocols meet the 1988 Clinical Laboratory Improvement Act mandates. Detailed QA/QC instructions are discussed in the NHANES LPM.
Mobile Examination Centers (MECs)
Laboratory team performance is monitored using several techniques. NCHS and contract consultants use a structured quality assurance evaluation during unscheduled visits to evaluate both the quality of the laboratory work and the quality control procedures. Each laboratory staff person is observed for equipment operation, specimen collection and preparation; testing procedures and constructive feedback are given to each staff. Formal retraining sessions are conducted annually to ensure that required skill levels were maintained.
Analytical Laboratories
NHANES uses several methods to monitor the quality of the analyses performed by the contract laboratories. In the MEC, these methods include performing blind split samples collected on “dry run” sessions. In addition, contract laboratories randomly perform repeat testing on 2.0% of all specimens.
Progress reports containing any problems encountered during shipping or receipt of specimens, summary statistics for each control pool, QC graphs, instrument calibration, reagents, and any special considerations are submitted to NCHS quarterly. The reports are reviewed for trends or shifts in the data. The laboratories are required to explain any identified areas of concern.
All QC procedures recommended by the manufacturers were followed. Reported results for all assays meet the Division of Laboratory Sciences’ quality control and quality assurance performance criteria for accuracy and precision.
Data Processing and Editing
The data were reviewed. Incomplete data or improbable values were sent to the performing laboratory for confirmation.
Analytic Notes
Refer to the 2013 - 2014 Laboratory Data Overview for general information on NHANES laboratory data.
Demographic and Other Related Variables
The analysis of NHANES 2013 - 2014 laboratory data must be conducted using the appropriate survey design and demographic variables. The NHANES 2013 - 2014 Demographics File contains demographic data, health indicators, and other related information collected during household interviews as well as the sample weight variables. The Fasting Questionnaire File includes auxiliary information such as fasting status, the time of venipuncture, and the conditions precluding venipuncture. The demographics and fasting questionnaire files may be linked to the laboratory data file using the unique survey participant identifier (i.e., SEQN).
The public release data file includes Oral HPV data for participants aged 18–69. Data for youth aged 14–17 years are available through the NCHS Research Data Center (RDC).
Detection Limits
Since this data is reported as qualitative data the use of lower LLODs isn’t applicable.
Exam sample weights should be used for analyses. Please refer to the NHANES Analytic Guidelines and the on-line NHANES Tutorial for further details on the use of sample weights and other analytic issues.