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.
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
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.
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
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
HPV Vaginal Swab Digene High Risk Laboratory Procedure Manual
Laboratory Quality Assurance and Monitoring
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.
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.
instructions are discussed in the NHANES LPM.
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.
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.
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.
Refer to the 2003-2004
Laboratory Data Overview for general information on NHANES
MEC exam sample
weights should be used for analyses.
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.
data file can be linked to the other NHANES data files using the unique survey
participant identifier (i.e., SEQN).
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
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
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
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=.).
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.