Evaluation of the Modified Two-Tiered Testing Method for Diagnosis of Lyme Disease in Children

J Clin Microbiol. 2019 Sep 24;57(10):e00547-19. doi: 10.1128/JCM.00547-19. Print 2019 Oct.


Conventional two-tiered testing (CTTT) for Lyme disease includes a first-tier enzyme immunoassay (EIA) followed by a supplemental immunoblot, and modified two-tiered testing (MTTT) relies on two different sequential EIAs without the inclusion of an immunoblot. MTTT has shown promising results as an alternative strategy for the diagnosis of Lyme disease in adults but has not yet been evaluated in children. We performed a cross-sectional study of children and adolescents ≤21 years of age undergoing clinical investigation for suspected Lyme disease. Serum specimens were analyzed with both a whole-cell sonicate (WCS) and a C6 EIA, with a supplemental immunoblot if either EIA was positive or equivocal. We compared CTTT (WCS EIA followed by supplemental immunoblot) to MTTT (WCS EIA followed by C6 EIA) using McNemar's test to evaluate for agreement beyond chance alone. We then used a kappa statistic to measure level of the agreement between testing strategies. We included 1,066 serum specimens, of which 156 (14.6%) had a positive CTTT and 165 (15.5%) had a positive MTTT. There were no significant differences between MTTT and CTTT (P = 0.16). Although the overall agreement between MTTT and CTTT was high (kappa, 0.88; 95% confidence interval, 0.84 to 0.92), 33 children had discordant test results. In a cohort of children and adolescents undergoing investigation for suspected Lyme disease, CTTT and MTTT results agreed in most cases. Since immunoblots are time-consuming, laborious, and challenging to interpret, MTTT provides a promising alternate Lyme disease testing strategy for children.

Keywords: Borrelia burgdorferi; Lyme disease; enzyme immunoassay; pediatrics; serologic testing.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adolescent
  • Algorithms
  • Antibodies, Bacterial / blood
  • Antibodies, Bacterial / immunology
  • Borrelia burgdorferi / immunology
  • Child
  • Child, Preschool
  • Female
  • Humans
  • Immunoblotting*
  • Immunoenzyme Techniques*
  • Immunoglobulin G / blood
  • Immunoglobulin G / immunology
  • Immunoglobulin M / blood
  • Immunoglobulin M / immunology
  • Infant
  • Lyme Disease / diagnosis*
  • Lyme Disease / immunology
  • Lyme Disease / microbiology
  • Male
  • Sensitivity and Specificity
  • Serologic Tests
  • Young Adult


  • Antibodies, Bacterial
  • Immunoglobulin G
  • Immunoglobulin M