Title: Human Papillomavirus (HPV) DNA Results from Vaginal Swab Samples: Digene Hybrid Capture and Roche Linear Array
Contact Number: 1-866-441-NCHS
Years of Content: 2007 - 2008
First Published: August, 2012
Access Constraints: None
Use Constraints: None
Geographic Coverage: National
Record Source: NHANES 2007 - 2008
Survey Methodology: NHANES 2007 - 2008 is a stratified multistage probability sample of the civilian non-institutionalized population of the U.S.
Medium: NHANES Web site; SAS transport files
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 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. One vaccine is licensed and recommended for use in males (Gardasil). In mid 2006, the Advisory Committee on Immunizations (ACIP), recommended routine vaccination of females aged 11 or 12 years and for those 13-26 years not previously vaccinated. In December, 2011 ACIP recommended routine vaccination of males aged 11 or 12 years and for those aged 13 through 21 years not previously vaccinated. As vaccine becomes more widely used, the national prevalence of HPV infection will be critical for evaluating vaccination strategies in the United States.
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.
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 the tests described below: Digene Hybrid Capture (hc2), and Roche Linear Array.
Digene hc2 HPV DNA Test
LBXH2RL (Hybrid Capture high risk result)
LBXH3RL (Hybrid Capture low risk result)
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.
Roche Linear Array Assay
This assay uses 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.
Read the LABDOC file for detailed data processing and editing protocols.
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 has been CLIA certified since 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 laboratory data must be conducted with the key survey design and basic demographic variables. The Demographic file contains: status variables providing core information on the survey participant including examination status, demographic variables including age, gender, race etc., and interview and examination sample weight variables and survey design variables. The Questionnaire data files contain socio-economic data, health indicators, and other related information collected during household interviews. 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. These files may be linked to the Laboratory data file using the unique survey participant identifier SEQN.
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 require specific testing to confirm the presence of HPV 52.