• Component Description
• Eligible Sample
• Description of Laboratory Methodology
• Laboratory Quality Assurance and Monitoring
• Data Processing and Editing
• Analytic Notes
• References
• Codebook
  • SEQN - Respondent sequence number
  • WTSSEVD2 - Surplus specimen EV-D68 2 Year Weights
  • SSEVFRM - EV-D68 Fermon strain quantitative
  • SSEVFRMQ - EV-D68 Fermon strain qualitative
  • SSEV953 - EV-D68 14-18953 strain quantitative
  • SSEV953Q - EV-D68 14-18953 strain qualitative
  • SSEV087 - EV-D68 18-23087 strain quantitative
  • SSEV087Q - EV-D68 18-23087 strain qualitative

National Health and Nutrition Examination Survey

2007-2008 Data Documentation, Codebook, and Frequencies

Enterovirus D68 (EV-D68) - Serum (Surplus) (SSEVD_E)

Data File: SSEVD_E.xpt

First Published: November 2024

Last Revised: NA

Component Description

Acute flaccid myelitis (AFM) is a condition affecting the spinal cord and resulting in varying levels of paralysis. AFM can be caused by many different pathogens, but laboratory and surveillance data suggest that enteroviruses (EVs), particularly EV-D68, are a common cause. EV-D68 was associated with increases in AFM cases in 2014, 2016, and 2018 in the United States (U.S.) (McLaren et. al., 2020; Messacar et. al., 2018). AFM is most common in children.

Limited data are available on EV-D68 seroprevalence, and thus, potential population susceptibility to EV-D68 infection and AFM likely exists in the U.S. There are published data from one small study in the U.S. that was conducted using banked sera collected for a poliovirus seroprevalence study in Kansas City, Misssouri in 2012 (n=155) and 2013 (n=281) (Harrison et. al., 2019). The median age of people who contributed sera for this study was 13 years (range 2-81 years), and seropositivity was 89-100% for testing of four reference EV-D68 isolates.

Analysis of NHANES sera would provide the first national estimates of EV-D68 seroprevalence in the U.S. and would add to our understanding of EV-D68 seroprevalence among children over time and by age and birth cohort, as well as how changes in seroprevalence might be associated with the timing of known EV-D68 and AFM outbreaks. Examining EV-D68 seroprevalence in young children aged 1-5 years is of particular interest. The median age of children with EV-D68 respiratory infection is 2.6 years (IQR: 0-15 years) (Ma et. al., 2022), and the median age of AFM patients in the U.S. through 2018 was 5.5 years (IQR: 3.3-9.8 years). Additionally, from the limited data currently available, seropositivity in older age groups is close to 100%, indicating that the majority of children acquire antibodies to EV-D68 prior to age 11. Therefore, exploring EV-D68 seroprevalence in children in age groups 1-5 and 6-11 years is likely to be most informative.

Enterovirus infections are very common; most children will have had multiple infections by the time they reach school age (Oberste et. al., 2014). For enteroviruses, neutralizing antibodies are the primary correlate of protection from disease. Neutralizing antibodies are serotype-specific, allowing measurement of type-specific immunity.

Eligible Sample

All examined participants aged 1 to 11 years were eligible.

Description of Laboratory Methodology

The enterovirus D68 (EV-D68) serology assay measures neutralizing antibody titers to EV-D68 strains using a microneutralization assay. The technical basis of the test is that the presence of anti-EV-D68 antibodies in a sample (e.g., serum) will bind to the virus and block infection of susceptible cells. Since EV-D68 is cytopathic, virus that is not bound by antibody infects and lyses cells. The amount of neutralizing antibody is quantitated as a 50% endpoint titer, or the last dilution of serum that protects susceptible cells from EV-D68 infection and cytopathic effect. The EV-D68 strains tested include: 1) Fermon, the prototypic strain; 2) 14-18953, a 2014 outbreak strain; and 3) 18-23087, a 2018 outbreak strain.

To conserve sample volume, EV-D68 antibodies were tested using a modification of the WHO-standard poliovirus microneutralization assay adapted to 384-well plates. The assay modification requires 50 µL of sample rather than 100 µL used in the standard 96-well plate assay in combination with automated liquid handling (Weldon et. al., 2016). The 96-well polio microneutralization assay is a CLIA-approved test in the laboratory that has been used in a large number of seroprevalence studies and human vaccine clinical trials. The EV-D68 384-well microneutralization assay has been validated against a 96-well microneutralization EV-D68 assay by parallel testing (Rhoden et. al., 2022).

Performance characteristics of qualitative assays

Sensitivity is compared using the lowest concentration of an analyte that can be measured.

Specificity is measured using neutralizing antibodies (nAb) present in the EV-D68 anti-Fermon horse antiserum T2-3420 as a positive nAb control. T2-3420 is effective against each EV-D68 strain. Each assay run includes the positive nAb control T2-3420. A nAb range has been established for T2-3420 against each EV-D68 strain. The assay is repeated with new samples and reagents if values are outside of established ranges. Sera containing non-neutralizing antibodies specific for EV-70 (D70) will not be effective against each EV-D68 strain and will be used as a negative control. Rabbit EV-70 (D70) antisera will test negative for EV-D68 neutralizing antibodies (<3 log₂). Horse anti-Fermon EV-D68 antisera will test positive for EV-D68 neutralizing antibodies (≥3 log₂).

Accuracy measures the closeness of agreement between the neutralization titers for in-house reference sera within a run. This is typically measured as a percent coefficient of variation (%CV). The coefficients of variations, calculated as the standard deviation of titers / mean titer x 100, were 7.35% for EV-D8 strain Fermon, 5.97% for strain 14-18953, and 2.85% for strain 18-23087. All three values are well below the recommended cutoff of 20% CV for accuracy. Human serum samples were used to determine if neutralizing antibodies were present against three EV-D68 strains. A sample is identified as positive or negative, according to the presence of a virus-induced cytopathic effect on RD cells. nAb positive samples are quantified by limiting dilution and used to determine the endpoint titer.

Reproducibility provides a method to yield similar virus titers for the same samples when measured in different runs. Determined by measuring virus titers on 5 different runs. This is typically expressed as percent coefficient of variation (%CV). The average coefficient of variation for each run should not exceed 20% for each serum.

Precision along with measures of reliability and robustness for high-throughput assays (Z-factor) was determined for in-house reference seruThem (IHRS/controls) against each EV-D68 strain. For high-throughput screening (HTS) assays, the Z-factor should be ≥ 0.5 (highest achievable Z-score is 1).

Reportable range is pre-defined based on 2-fold serial dilutions ranging from 2.5 log₂ (2^2.5; lower limit of quantitation, LLOQ) to 10.5 log₂ (2^10.5; upper limit of quantitation, ULOQ). For sera, expect the neutralizing antibody (nAb) titer to fall within the reportable range.

Laboratory Quality Assurance and Monitoring

Specimens tested using either microneutralization assay are stored at -70°C and managed using the CDC-standard laboratory information management system.

The essential parameters required per FDA Guidance for Industry Bioanalytical Method Validation are specificity, sensitivity, accuracy, precision and reproducibility (FDA, 2018). These recommendations were used to validate the automated method used to screen human serum specimens for the presence of EV-D68 neutralizing antibodies.

Data Processing and Editing

Data were received after all analyses were complete. The data were not edited. The data were reviewed. Incomplete data or improbable values were sent to the performing laboratory for confirmation.

Analytic Notes

Refer to the 2007 – 2008 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. Additionally, availability of specimens for surplus projects is lower than for other laboratory tests performed on NHANES participants. The specimen availability can also vary by age or other population characteristics. 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.

Subsample Weights

The analytes included in this dataset were measured in all examined participants aged 1-11 years. Special sample weights are required to analyze these data properly. Specific sample weights for this subsample, WTSSEVD2, are included in this data file and should be used when analyzing these data. The sample weights created for this file used the examination sample weight, i.e., WTMEC2YR, as the base weight. The base weight was adjusted for additional nonresponse to these lab tests and re-poststratified to the population total using sex, age, and race/Hispanic origin. Participants who were part of the eligible population but who did not provide a serum specimen, or did not have sufficient volume of biospecimens, or who did not give consent for their specimens to be used for future research are included in the file, but they have a sample weight assigned “0” in their records.  

Demographic and Other Related Variables

The analysis of NHANES laboratory data must be conducted using the appropriate survey design and demographic variables. The NHANES 2007-2008 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).

Quantitative Result and Qualitative Interpretation

Two variables are provided for each of these analytes. The variable name ending in “Q” (ex., SSEVFRMQ) indicates the qualitative result for seropositivity: the value “1” means that the result was positive, at or above a titer value of 3 log₂ (i.e. ≥2³ or 1:8 dilution), “2” indicates that the result was negative and below the titer value of 3 log₂ (i.e. <2³ or 1:8 dilution). The other variable, (ex., SSEVFRM) provides the quantitative result for that analyte based on the number of 2-fold serial dilutions. For example, a value of 4 would mean a titer value of 4 log₂ (i.e. 2⁴ or 1:16 dilution).

Detection Limits

The detection limit was constant for all of the analytes in the data set.

The lower limit of quantitation (LLOQ) and upper limit of quantitation (ULOQ) for titers, presented as the number of 2-fold serial dilutions, are: 

Variable Name	Analyte Name	LLOQ	ULOQ
SSEVFRM	EV-D68 Fermon strain quantitative titer value	2.5	10.5
SSEV953	EV-D68 14-18953 strain (2014 outbreak strain)	2.5	10.5
SSEV087	EV-D68 18-23087 strain (2018 outbreak strain)	2.5	10.5

References

• Food and Drug Administration. FDA Guidance for Industry: Bioanalytical Method Validation. Rockville, MD: US Department of Health and Human Services, Food and Drug Administration, Center for Drug Evaluation and Research (2018) Available from:  https://www.fda.gov/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/default.htm
• Harrison C.J., Weldon W.C., Pahud B.A., Jackson M.A., Oberste M.S., Selvarangan R. Neutralizing antibody against enterovirus D68 in children and adults before 2014 outbreak, Kansas City, Missouri, USA. Emerg Infect Dis. (2019) 25(3):585-588.
• Ma K.C., Winn A., Moline H.L., et al. Increase in acute respiratory illnesses among children and adolescents associated with rhinoviruses and enteroviruses, including enterovirus D68 — United States, July–September 2022. MMWR Morb Mortal Wkly Rep (2022) 71:1265–1270.
• McLaren N., Lopez A., Kidd S., Zhang J., Nix W.A., Link-Gelles R., et al. Characteristics of patients with acute flaccid myelitis, United States, 2015–2018. Emerg Infect Dis. (2020) Feb;26:212–219.
• Messacar K., Asturias E.J., Hixon A.M., Van Leer-Buter C., Niesters H.G.M., Tyler K.L., et al. Enterovirus D68 and acute flaccid myelitis-evaluating the evidence for causality. Lancet Infect Dis. (2018) 18:e239–247.
• Oberste M.S., Gerber S.I. Enteroviruses and parechoviruses: Echoviruses, coxsackieviruses, and others. In: Viral Infections of Humans: Epidemiology and Control. (2014).
• Rhoden E.E., Mainou B.A., Konopka-Anstadt J.L., Oberste M.S. An automated high-throughput enterovirus D68 microneutralization assay platform. J Virol Methods. (2022) 308:114590.
• Weldon W.C., Oberste M.S., Pallansch M.A. Standardized methods for detection of poliovirus antibodies. In: Methods in Molecular Biology. (2016).

Codebook and Frequencies