National Health and Nutrition Examination Survey

In 2013, CDC revised its guidelines for Hepatitis C (HCV) testing because of following: 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 This new guidance was adopted beginning in the NHANES 2013-2014 cycle. Among persons aged 6 years and older who participated in NHANES during the 2013–2014 and 2015-2016 cycles, antibody confirmation testing was conducted using a 3rd generation commercial line immunoassay test for the subset of participants whose serum was reactive using a hepatitis C virus (HCV) antibody screening procedure, subsequently found to be negative for HCV RNA. In the 2013-2014 cycle and 2015 only, participants who had stored serum samples were analyzed. In 2016, the regular samples collected for eligible participants were analyzed.

For these reasons, use of 2013 and later data for comparisons or trending with data from 2012 and earlier is not advised.

The new Hepatitis C algorithm flow chart can be found in the laboratory method file and/or by following the MMWR link below:

https://www.cdc.gov/mmwr/pdf/wk/mm62e0507a2.pdf

Beginning in the 2017-2018 cycle, the HCV confirmatory antibody test (INNO-LIA) will be released in the HEPC dataset with HCV RNA and Genotype data.

Examined participants in 2016, aged 6 years and older and were both HCV antibody screening reactive and HCV-RNA negative, were eligible.

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 US. 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.

There were no changes to the lab method, lab equipment, or lab site for this component in the NHANES 2015-2016 cycle.

Hepatitis C Antibody / Hepatitis C Confirmatory Test (Anti-HCV) (January 2018)

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.

The data were reviewed. Incomplete data or improbable values were sent to the performing laboratory for confirmation.

Refer to the 2015-2016 Laboratory Data Overview for general information on NHANES laboratory data.

Demographic and Other Related Variables

The analysis of NHANES laboratory data must be conducted using the appropriate survey design and demographic variables. The NHANES 2015-2016 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.

A special one-year MEC sample weight (WTMEC1YR) is provided for the 2015 Surplus Specimen Hepatitis C antibody (confirmed) dataset (SSHC_I_R), and the 2016 Hepatitis C antibody (confirmed) dataset (HEPC_I_R). This sample weight, along with strata (VSTRA) and PSUs (VPSU) are used to calculate variance estimates based on the Taylor Series Linearization method. In addition, 15 Jackknife replicate weights (WTM1YR01 – WTM1YR15) can be used with VSTRA and VPSU, and are included in the files to obtain variance estimates based on a replication method.

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. 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.