Component Description
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, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68) are associated with cervical cancer and the “low-risk” types (e.g., HPV 6, 11, 42, 43, 44) 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 16 antibody) will allow evaluation of trends in prevalence of type-specific HPV infection by age, sexual behavior, and race/ethnicity. HPV vaccine development is underway, and knowledge of the national prevalence of HPV infection will be critical for planning vaccination strategies in the United States.
Eligible Sample
Vaginal Swab - Females aged 14 to 17 years. Data for females aged 18 to 59 will be publically released.
Description of Laboratory Methodology
Vaginal Swab
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.
HPV PCR 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 protype 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.
Data Processing and Editing
Vaginal specimens were processed, stored and shipped to Atlanta, Ga. for analysis. Detailed specimen collection and processing instructions are discussed in the NHANES LPM. Read the LABDOC file for detailed data processing and editing protocols. The analytical methods are described in the Description of the Laboratory Methodology section.
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 but is not CLIA certified. 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.
Analytic Notes
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 for these age groups. 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.
Vaginal Swab
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 (LBDHPCR) indicates if at least one type is positive (LBDHPCR=1), the sample is inadequate (LBDHPCR=3), the sample is negative (LBDHPCR=2), or the sample is missing (LBDPCR=.).
If there is no Beta globin present in the sample and no HPV type is detected either by hybridization or sequencing, the sample is coded as inadequate.
If any of the types on the strips (LBDH06-LBDHP1) are positive, or a type is identified by sequencing, the type is coded as positive. If all of the types on the strip are coded as negative or missing, and LBXNST is missing, the sample is coded as negative.
Data Access
Public data file includes HPV swab data for persons 18–59 years of age. HPV swab data for youth adolescents 14–17 years of age are available through the Research Data Center (RDC) or through special agreement.
Collaborators may obtain the NHANES 2003-2004 Adolescent HPV Swab Special Use Data File through a special agreement. Other interested researchers may access these data through the NCHS RDC.