• Component Description
• Eligible Sample
• Description of Laboratory Methodology
• Laboratory Method Files
• Laboratory Quality Assurance and Monitoring
• Data Processing and Editing
• Analytic Notes
• References
• Codebook
  • SEQN - Respondent sequence number
  • LBXH2RL - Hybrid Capture high risk (RLU) result
  • LBXH3RL - Hybrid Capture low risk (RLU) result
  • LBDHPCR - HPV summary variable
  • LBDH06 - HPV type 06
  • LBDH11 - HPV type 11
  • LBDH16 - HPV type 16
  • LBDH18 - HPV type 18
  • LBDH26 - HPV type 26
  • LBDH31 - HPV type 31
  • LBDH33 - HPV type 33
  • LBDH35 - HPV type 35
  • LBDH39 - HPV type 39
  • LBDH40 - HPV type 40
  • LBDH42 - HPV type 42
  • LBDH45 - HPV type 45
  • LBDH51 - HPV type 51
  • LBDH52 - HPV type 52
  • LBDH53 - HPV type 53
  • LBDH54 - HPV type 54
  • LBDH55 - HPV type 55
  • LBDH56 - HPV type 56
  • LBDH58 - HPV type 58
  • LBDH59 - HPV type 59
  • LBDH61 - HPV type 61
  • LBDH62 - HPV type 62
  • LBDH64 - HPV type 64
  • LBDH66 - HPV type 66
  • LBDH67 - HPV type 67
  • LBDH68 - HPV type 68
  • LBDH69 - HPV type 69
  • LBDH70 - HPV type 70
  • LBDH71 - HPV type 71
  • LBDH72 - HPV type 72
  • LBDH73 - HPV type 73
  • LBDH81 - HPV type 81
  • LBDH82 - HPV type 82
  • LBDH83 - HPV type 83
  • LBDH84 - HPV type 84
  • LBDH89 - HPV type 89
  • LBDHPI - HPV type IS39
  • LBXNST - other HPV strip types

National Health and Nutrition Examination Survey

2003-2004 Data Documentation, Codebook, and Frequencies

Human Papillomavirus (HPV) DNA - Vaginal Swab: Digene Hybrid Capture & Prototype Line Blot Assay (L37SWA_C)

Data File: L37SWA_C.xpt

First Published: March 2009

Last Revised: October 2010

Note: 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.

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) 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 (Dataset name: L37swr_c). The LA typing assay is based upon the same assay principles as the prototype, but includes proprietary refinements to improve sensitivity and reproducibility.

Eligible Sample

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.

Description of Laboratory Methodology

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.

Laboratory Method Files

HPV Vaginal Swab Prototype Laboratory Procedure Manual (March 2009)

HPV Vaginal Swab Digene High Risk Laboratory Procedure Manual (March 2009)

Laboratory Quality Assurance and Monitoring

Vaginal swab samples were processed, stored and shipped to the Chronic Viral Diseases Branch, Division of High-Consequence Pathogens and Pathology, National Center for Emerging and Zoonotic Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, GA for analysis.

The Digene hc2 HPV DNA Test is approved for clinical testing, but 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.

Detailed instructions on specimen collection and processing are discussed in the NHANES Laboratory Procedures Manual (LPM). Swabs were stored at room temperature until they were shipped to the National Center for Emerging and Zoonotic Infectious Diseases for testing.

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 competency assessment evaluation during visits to evaluate both the quality of the laboratory work and the quality-control procedures. Each laboratory staff member is observed for equipment operation, specimen collection and preparation; testing procedures and constructive feedback are given to each staff member. 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% 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.

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 2003-2004 Laboratory Data Overview for general information on NHANES laboratory data.

Sample Weights

MEC exam sample weights should be used for analyses.

Demographic and Other Related Variables

The analysis of NHANES laboratory data must be conducted using the appropriate survey design and demographic variables. The NHANES 2003-2004 Demographics File contains demographic data, health indicators, and other related information collected during household interviews as well as the sample design variables. The recommended procedure for variance estimation requires use of stratum and PSU variables (SDMVSTRA and SDMVPSU, respectively) in the demographic data file.

This laboratory data file can be linked to the other NHANES data files using the unique survey participant identifier (i.e., SEQN).

The Questionnaire data files contain socio-economic data, health indicators, and other related information collected during household interviews. Certain sensitive data on participants 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 public release data file includes HPV vaginal swab data for participants aged 18-59. HPV vaginal swab data for youth aged 14-17 years are available through the NCHS Research Data Center (RDC).

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 (LBDHPCR) indicates if at least one type is positive (LBDHPCR=1), the sample is negative (LBDHPCR=2), the sample is inadequate (LBDHPCR=3), or the sample is missing (LBDHPCR=.).

Detection Limits

If data is qualitative, the use of lower limits of detection (LLODs) is not applicable.

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.

References

• Castle PE, Gravitt PE, Solomon D, et al. Comparison of linear array and line blot assay for detection of human papillomavirus and diagnosis of cervical precancer and cancer in the atypical squamous cell of undetermined significance and low-grade squamous intraepithelial lesion triage study. J Clin Microbiol. 2008; 46: 109-117.
• Coutlee F, Rouleau D, Petignat P, et al. Enhanced detection and typing of human papillomavirus (HPV) DNA in anogenital samples with PGMY primers and the linear array HPV genotyping test. J Clin Microbiol. 2006; 44: 1998-2006.
• Gravitt PE, Peyton CL, Alessi TQ, Wheeler CM, Coutlee F, Hildesheim A, Schiffman MH, Scott DR, Apple RJ. Improved Amplification of Genital Human Papillomaviruses. J Clin Microbiol 2000; 38: 357-361
• Steinau M, Swan DC, Unger ER. Type-specific reproducibility of the Roche Linear Array HPV genotyping test. J Clin Virol 42: 412-414, 2008
• Unger ER, Steinau M, Lin JM, Patel SS, Swan DC. Impact of HPV assay on observed population prevalence. Diagn Mol Pathol (in press)

Codebook and Frequencies