Table of Contents

Component Description

Human papillomavirus (HPV) infection is the principal cause of a distinct form of oropharyngeal squamous cell carcinoma that is increasing in incidence among men in the United States. However, little is known about the epidemiology of oral HPV infection.

The main objective of this study was to measure oral HPV viral load for the subset of oral rinse samples (ORS) that were positive for high-risk or probably high-risk HPV types from the NHANES 2009-2010 study entitled Prevalence of Oral HPV Infections in the United States Population.

Oral rinse samples previously reported as positive for a high-risk HPV type by Roche Linear Array were evaluated for measurement of HPV viral load by use of HPV type-specific real-time TaqMan PCR assays targeted to the HPV E6 or E7 gene. The number of HPV viral copies was normalized to the number of cells in the PCR reaction as estimated by a real-time PCR assay to a single copy human gene (ERV-3). Real-time PCR assays were designed for the following HPV types: 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 73, 82.

Eligible Sample

Oral rinse samples that were positive for high-risk HPV by use of Roche Linear Array from NHANES 09-10, Prevalence of Oral HPV Infections in the United States Population. Additionally, 40 HPV-negative oral rinse samples were randomly chosen to serve as a negative control for each of the 18 HPV types evaluated. These samples were chosen without replacement, and therefore a total of 720 samples were evaluated.

Description of Laboratory Methodology

Primers and probes were designed to amplify the HPV E6 or E7 gene for HPV types 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 73, 82 as previously described (ref). The primer and probe sequences are listed in the appended table. The Real-time PCR reaction included final concentrations of 1X TaqMan Universal Master Mix, 0.1µM Probe (labeled FAM/Black Hole Quencher-1), 0.4µM forward and reverse primer and 2ul oral rinse DNA in a final reaction volume of 25 ul. The reactions were incubated in a 96-well optical plate at 52°C for 10 min, at 95°C for 12 min, followed by 50 cycles of 95°C for 15 seconds and 60°C for 1 min. For HPV16 Real-time PCR, the 10 min incubation at 52°C was replaced with a 30 min incubation at 37°C. Standard curves were created from serial dilutions of a plasmid containing the E6/E7 regions of each respective type. The cycle threshold (CT) of unknown samples was determined from an equation derived from a linear regression through the log CT of the standard curve. If the 2uL sample was negative, a repeat of 12 uL in 100uL reaction was performed. Lower limit of assay sensitivity was 3-16 copies, depending on the HPV type. All HPV viral loads and ERV3 were subsequently normalized to a 12µLvolume of DNA to be representative of the 12 uL oral rinse DNA sample volume used the Roche Linear Array assay. For line blot positive samples with a viral load of <1 copy per 12ul, this value was set at 1 copy per 12 µL.

Data Processing and Editing

Data were reported after all laboratory testing was complete. The data were not edited.

Data Access: Available on the NHANES website.

Laboratory Quality Assurance and Monitoring

This is a research supplement conducted on residual samples from the NHANES 2009-10 study of the Prevalence of Oral HPV Infections in the United States Population. Please see the NHANES website for quality control and monitoring for that study.

Analytic Notes

Sample ID: Unique ID number assigned to each sample. An ID number may appear more than once in the dataset due to samples that were positive for more than one high-risk HPV type.

HPV type: The HPV type for which the samples was evaluated for viral load analysis, corresponding to the HPV type(s) previously detected in the sample by Roche Linear Array. Includes HPV types 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 73, 82.

Viral load: HPV viral copy number in a 12 uL sample equivalent of oral rinse DNA.

Cell number: The number of human cells evaluated for HPV as estimated by real-time PCR for a single copy human gene, ERV-3.

Norm viral load: HPV viral load per 100,000 human cell equivalents. Calculated as the quotient of HPV copy number and Cell Number times 100,000. For line blot positive samples with a viral load <1 copies per 12ul, this value was set at 1 copy per 12 ul.

References

Codebook and Frequencies

SEQN - Respondent sequence number

Variable Name:
SEQN
SAS Label:
Respondent sequence number
English Text:
Respondent sequence number.
Target:
Both males and females 14 YEARS - 17 YEARS

SSHPTYPE - Oral HPV type

Variable Name:
SSHPTYPE
SAS Label:
Oral HPV type
English Text:
Oral HPV type
Target:
Both males and females 14 YEARS - 17 YEARS
Code or Value Value Description Count Cumulative Skip to Item
16 to 82 Range of Values 91 91
. Missing 0 91

SSHPVLD - Oral HPV viral load

Variable Name:
SSHPVLD
SAS Label:
Oral HPV viral load
English Text:
Oral HPV viral load
Target:
Both males and females 14 YEARS - 17 YEARS
Code or Value Value Description Count Cumulative Skip to Item
0 to 327457 Range of Values 91 91
. Missing 0 91

SSHPVCEL - Oral HPV Cell Number

Variable Name:
SSHPVCEL
SAS Label:
Oral HPV Cell Number
English Text:
Oral HPV Cell Number
Target:
Both males and females 14 YEARS - 17 YEARS
Code or Value Value Description Count Cumulative Skip to Item
911 to 1151856 Range of Values 91 91
. Missing 0 91

SSHPVNLD - Oral HPV normal viral load

Variable Name:
SSHPVNLD
SAS Label:
Oral HPV normal viral load
English Text:
Oral HPV normal viral load
Target:
Both males and females 14 YEARS - 17 YEARS
Code or Value Value Description Count Cumulative Skip to Item
0 to 28429 Range of Values 91 91
. Missing 0 91