Hepatitis viruses constitute a major public health problem because of the morbidity and mortality associated with the acute and chronic consequences of these infections. New immunization strategies have been developed to eliminate the spread of hepatitis B virus (HBV) and hepatitis A virus (HAV) in the United States. Recommendations have also been developed for the prevention and control of hepatitis C virus (HCV) infection. Because of the high rate of asymptomatic infection with these viruses, information about the prevalence of these diseases is needed to monitor prevention efforts. By testing a nationally representative sample of the U.S. population, NHANES will provide the most reliable estimates of age-specific prevalence needed to evaluate the effectiveness of the strategies to prevent these infections. In addition, NHANES provides the means to better define the epidemiology of other hepatitis viruses. NHANES testing for markers of infection with hepatitis viruses will be used to determine secular trends in infection rates across most age and racial/ethnic groups, and will provide a national picture of the epidemiologic determinants of these infections.
In 2013, CDC revised its guidelines for Hepatitis C (HCV) testing because of 1) changes in the availability of certain commercial HCV antibody tests; 2) evidence that many persons who are identified as reactive by an HCV antibody test might not subsequently be evaluated to determine if they have current HCV infection; and 3) there have been significant advances in the development of antiviral agents with improved efficacy against HCV.1
The flow chart for the new Hepatitis C algorithm can be found in the laboratory method file or by following the MMWR link below.
https://www.cdc.gov/mmwr/pdf/wk/mm62e0507a2.pdf
Examined participants aged 6 years and older were eligible.
Hepatitis C RNA (HCV-RNA)
The COBAS AMPLICOR HCV MONITOR Test, version 2 0 (v2.0) is an in vitro nucleic acid amplification test for the quantitation of Hepatitis C Virus RNA in human serum or plasma on the COBAS AMPLICOR Analyzer.
Hepatitis C Confirmed Antibody (INNO-LIA)
In 2012 and earlier years, the hepatitis C virus testing algorithm began with an antibody screening test, and antibody screening reactive samples were then tested with a recombinant/synthetic immunoblot assay as the antibody confirmation test. Samples with positive antibody confirmation results were then tested with a nucleic acid amplification test for hepatitis C virus RNA. HCV-RNA positive samples were then tested with a line probe assay to determine the virus genotype from six genotypes, and subtype for genotype 1, the most prevalent genotype in the U.S.
Beginning in 2013, samples reactive to the antibody screening test were first tested for RNA. Then, all RNA positive samples were tested for genotype, and only RNA negative samples were tested with an antibody confirmation test. This change in the testing algorithm was made to align the NHANES HCV testing algorithm with a 2013 update to CDC’s guidance on testing for HCV infection by clinicians and laboratorians. Additionally, the antibody confirmation test changed to a 3rd generation line immunoassay in 2013 because the test used in 2012 and earlier was no longer commercially available and the stored supply of these test kits had been exhausted.
For the antibody confirmation test, the manual 16-hour sample incubation test procedure is used. 10ul of specimens and controls are diluted in 1mL diluent in test troughs to ensure test strips will slide easily and remain submerged during incubation. Specimens and controls are incubated with agitation by rocker at 34 rpm overnight (16±2 hours) at room temperature (18–25°). After a 3X/5min each wash step on microtiter plate shaker, conjugate is added with a 30-minute incubation at room temperature, followed by aspiration, 3X/5min each wash step and 30-minute incubation in substrate solution also on microtiter plate shaker. Finally, strips are agitated with stop solution for 10–30 minutes at room temperature. The microtiter plate shaker is set for 160 rpm and used for all the washes and solution incubations. Strips are then dried completely in a dry incubator at 37°C for 30 minutes then mounted on the line immunoassay score data reporting sheet with inverted tape for reading within one hour using the reading card. The strips have lines for a background control, three reference lines for antibody (level ±, human IgG, weak positive; level 1+ human IgG, moderate positive; and level 3+, anti-human Ig, strong positive), and six lines for HCV antigens (C1, C2, E2, NS3, NS4, and NS5). Samples with a positive antibody confirmation test result are reported as positive. Samples with a negative antibody confirmation test result are reported as negative. Samples with an indeterminate antibody confirmation test result are released as missing. Only specimens insufficient in quantity are deemed indeterminate.
Hepatitis C genotype
The VERSANT ® HCV Genotype 2.0 Assay (LiPA) is a line probe assay designed to identify Hepatitis C virus (HCV) genotypes 1 to 6 in human serum or EDTA plasma samples. Subtype information is available in the majority of cases.
Refer to the Laboratory Method Files section for a detailed description of the laboratory methods used.
There were no changes to the lab method, lab equipment, or lab site for this component in the NHANES 2017-2018 cycle.
Hepatitis C Confirmed Antibody Laboratory Procedure Manual (February 2020)
Hepatitis C Genotype Laboratory Procedure Manual (February 2020)
Hepatitis C RNA Laboratory Procedure Manual (February 2020)
Serum samples were processed, stored, and shipped to the Division of Viral Hepatitis, National Center for HIV/AIDS, Viral Hepatitis, STD, and TB Prevention, Centers for Disease Control and Prevention, Atlanta, GA for analysis.
Detailed instructions on specimen collection and processing are discussed in the NHANES Laboratory Procedures Manual (LPM). Vials are stored under appropriate frozen (–30°C) conditions until they are shipped to Division of Viral Hepatitis, National Center for HIV/AIDS, Viral Hepatitis, STD, and TB Prevention for testing.
The NHANES quality assurance and quality control (QA/QC) protocols meet the 1988 Clinical Laboratory Improvement Act mandates. 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.
The data were reviewed. Incomplete data or improbable values were sent to the performing laboratory for confirmation.
Refer to the 2017-2018 Laboratory Data Overview for general information on NHANES laboratory data.
There are over 800 laboratory tests performed on NHANES participants. However, not all participants provided biospecimens or enough volume for all the tests to be performed. The specimen availability can also vary by age or other population characteristics. For example, in 2017-2018, approximately 80% of children aged 1-17 years who were examined in the MEC provided a blood specimen through phlebotomy, while 95% of examined adults age 18 and older provided a blood specimen. Analysts should evaluate the extent of missing data in the dataset related to the outcome of interest as well as any predictor variables used in the analyses to determine whether additional re-weighting for item non-response is necessary.
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.
Demographic and Other Related Variables
The analysis of NHANES laboratory data must be conducted using the appropriate survey design and demographic variables. The NHANES 2017-2018 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).
Detection Limits
This data is qualitative. The use of lower limits of detection (LLODs) is not applicable.
Exam sample weights should be used for analyses.
| Code or Value | Value Description | Count | Cumulative | Skip to Item |
|---|---|---|---|---|
| 1 | Positive | 51 | 51 | |
| 2 | Negative | 98 | 149 | |
| 3 | Negative Screening HCV Antibody | 6541 | 6690 | |
| . | Missing | 745 | 7435 |
| Code or Value | Value Description | Count | Cumulative | Skip to Item |
|---|---|---|---|---|
| 1 | Positive | 54 | 54 | |
| 2 | Negative | 33 | 87 | |
| 3 | Negative Screening HCV Antibody | 6541 | 6628 | |
| 4 | Positive HCV RNA | 51 | 6679 | |
| . | Missing | 756 | 7435 |
| Code or Value | Value Description | Count | Cumulative | Skip to Item |
|---|---|---|---|---|
| 1 | Genotype 1a | 25 | 25 | |
| 2 | Genotype 1b | 7 | 32 | |
| 3 | Gen 1 other than a/b/not determined | 1 | 33 | |
| 4 | Genotype 2 | 6 | 39 | |
| 5 | Genotype 3 | 6 | 45 | |
| 6 | Genotype 4 | 1 | 46 | |
| 7 | Genotype 5 | 0 | 46 | |
| 8 | Genotype 6 | 1 | 47 | |
| 9 | Genotype undetermined | 2 | 49 | |
| . | Missing | 7386 | 7435 |