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 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.
Eligible Sample
Examined female
participants aged 14-59 years were eligible. This public data file includes
data for examined participants aged 18-59 years. Please see Analytic Notes about
the release of data for adolescents aged 14-17 years.
Description of Laboratory Methodology
Digene hc2 HPV DNA
Test
LBXH2RL (Hybrid Capture high 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 human papillomavirus (HPV)
DNA in cervical specimens. The high-risk (HR) hc2 HPV DNA Test detects 13 HR
HPV types: 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 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.
Laboratory Method Files
HPV Vaginal Swab Digene High Risk Laboratory Procedure Manual
(November 2018)
HPV Vaginal Swab Linear Array Laboratory Procedure Manual
(November 2018)
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 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.
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 2009-2010
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 2009-2010
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).
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 beta-globin is
not 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 (Onyekwuluje et al., 2012).
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.