Human papillomavirus (HPV) infection is one of the most common sexually transmitted infections in the United States. Cervical infection with certain types of HPV is a major risk factor for cervical cancer in women. The “high-risk” types of HPV (e.g., HPV 16, 18) are associated with cervical cancer and the “low-risk” types (e.g., HPV 6, 11) with genital warts. No national surveillance system exists to measure the full burden of HPV infection, and no reliable national population estimate of HPV exists. NHANES offers a unique opportunity to assess the prevalence of HPV infection in the general population.
Reducing the prevalence of HPV infection is a 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 planning vaccination strategies in the United States.
Results of the Digene hc2 HPV DNA Test and HPV typing based on the Roche prototype line blot assay have been previously released. The current data release adds HPV typing results based on the Roche research use only Linear Array (LA) HPV Genotyping kit. The LA typing assay is based upon the same assay principles as the prototype, but includes proprietary refinements to improve sensitivity and reproducibility.
Vaginal Swab - Females aged 14 to 59 years.
Public data file includes data for persons 18-59 years of age. Please see Analytic Notes for Data Users about the release of data for adolescents 14-17 years of age.
Description of Laboratory Methodology
Vaginal specimens were processed, stored and shipped to Atlanta, Ga. for analysis. Detailed specimen collection and processing instructions are discussed in the NHANES LPM. DNA from the vaginal swab is extracted for use in three tests described below: Digene Hybrid Capture (hc2), Prototype Line Blot Assay, and Roche Linear Array.
Digene hc2 HPV DNA Test
LBXH2RL (Hybrid Capture high risk result)
LBXH3RL (Hybrid Capture low risk result)
The Digene hc2 HPV DNA 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.
Prototype Line Blot Assay
This assay uses HPV L1 consensus polymerase chain reaction (PCR) with biotinylated PGMY09/11 primer sets and β-globin as an internal control for sample amplification. The primer mix amplifies essentially all HPV types found in the genital tract. The amplicons are evaluated by gel electrophoresis for the presence of the 450 bp HPV amplicon. Positive samples are typed by hybridization to the Roche prototype line probe typing strips followed by colorimetric detection. The strip is a linear array of probes specific for 37 HPV types (6, 11, 16, 18, 26, 31, 33, 35, 39, 40, 42, 45, 51, 52, 53, 54, 55, 56, 58, 59, 61, 62, 64, 66, 67, 68, 69, 70, 71, 72, 73, 81, 82, 83, 84, IS39, and 89) and for the positive β-globin control as well. Types are read by comparing the reaction pattern to the typing template. Samples that do not hybridize to the typing strip are sequenced to determine the HPV type.
Roche Linear Array Assay
This assay uses the Research Use Only (RUO) Roche Linear Array HPV Genotyping test that is based on HPV L1 consensus polymerase chain reaction (PCR) with biotinylated PGMY09/11 primer sets. It also includes biotinylated β-globin primers as an internal control for sample amplification. The primer mix amplifies essentially all HPV types found in the genital tract along with the human β-globin gene. After amplification the samples are typed by hybridization to the typing strips followed by colorimetric detection. The strip is a linear array of probes specific for 37 HPV types (6, 11, 16, 18, 26, 31, 33, 35, 39, 40, 42, 45, 51, 52, 53, 54, 55, 56, 58, 59, 61, 62, 64, 66, 67, 68, 69, 70, 71, 72, 73, 81, 82, 83, 84, IS39, and 89) and for the positive β-globin control as well. Types are read by comparing the reaction pattern to the typing template. Samples that are negative for HPV and the β-globin control indicate lack of a suitable sample and are considered inadequate for interpretation.
Data Processing and Editing
Read the LABDOC file for detailed data processing and editing protocols.
Laboratory Quality Assurance and Monitoring
While HC2 is approved for clinical testing, 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.
The analysis of NHANES 2003–2004 laboratory data must be conducted with the key survey design and basic demographic variables. The NHANES 2003–2004 Household Questionnaire Data Files contain demographic data, health indicators, and other related information collected during household interviews. They also contain all survey design variables and sample weights . Certain sensitive data on respondents under 18 years of age (e.g. HPV typing results, sexual behavior variables) are not included in the public use files. These data may be requested as described in the NHANES guidelines. The phlebotomy file includes auxiliary information such as the conditions precluding venipuncture. The household questionnaire and phlebotomy files may be linked to the laboratory data file using the unique survey participant identifier SEQN.
HPV DNA detection and typing results from two assays are provided: the Roche prototype line blot assay (previously released, Data set name: l37SWA_C) and the Roche research use only Linear Array (LA) HPV Genotyping kit (Data set name: l37SWR_C). The Roche prototype reagents were discontinued when the LA assay was released and will not be available for further use in future NHANES cycles. While based on identical principles, the LA was refined for commercialization as a research use only kit. The two assays were comparable, although LA was found to be more sensitive and detected more types per sample (3, 4, 5). To assure comparability of results in the ongoing NHANES sampling, residual extracts from 2003-2004 were retested with LA.
• We recommend all analysis of HPV DNA PCR be conducted using the Roche LA results (Data set name: l37SWR_C) in order to provide the most accurate longitudinal information on HPV detection and typing by PCR.
Note: The two data sets can also be distinguished by the variable names. The data for the Roche prototype line blot assay has a fourth letter of the variable name, H. The Roche LA assay has the fourth letter of the variable name, R.
Digene hc2 HPV DNA Test
An RLU measurement equal to or greater than the Cutoff Value of 1.0 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.
HPV PCR Assay
HPV Summary Variable
The HPV PCR Summary variable (LBDRPCR) indicates if at least one type is positive (LBDRPCR=1), the sample is negative (LBDRPCR=2), the sample is inadequate (LBDRPCR=3), or the sample is missing (LBDRPCR=.).
If there is no Beta globin present (both LBDRHP and LBDRLP are negative) in the sample and no HPV type is detected, the sample is coded as inadequate.
If any of the types on the strips (LBDR06-LBDRPI) are positive, the sample is coded as positive. If all of the types on the strip are coded as negative, and beta-globin is detected (either LBDRHP or LBDRLP is positive) the sample is coded as negative.
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 requires specific testing to confirm the presence of HPV 52.