Validation of a 2-Gene Blood Test for Kawasaki Disease in Febrile Children.

Abstract

Kawasaki disease (KD) remains a clinical diagnosis without an objective molecular test. Early identification is critical to prevent coronary artery complications through timely intravenous immunoglobulin therapy. To validate a 2-gene whole-blood quantitative polymerase chain reaction (qPCR) assay measuring IFI27 and MCEMP1 expression for distinguishing KD from other pediatric febrile illnesses. This multicenter diagnostic study was conducted in Taiwan and Shanghai, China. Patient blood samples were collected prospectively between 2012 and 2023 in Taiwan and between 2022 and 2023 in Shanghai and analyzed retrospectively from children younger than 8 years with KD and febrile controls (FCs) with viral, bacterial, or mixed infections. Data were analyzed from January 2022 to August 2025. Diagnostic accuracy of a prespecified 2-gene KD score derived from change in cycle threshold values normalized to glyceraldehyde 3-phosphate dehydrogenase, assessed by area under the receiver operating characteristic curve (AUC), sensitivity, specificity, predictive values, and likelihood ratios. A total of 541 children (mean [SD] age, 3.7 [1.9] years; 300 [55.5%] male), including 243 children with KD and 298 febrile controls, were analyzed. The KD score achieved an AUC of 0.91 (95% CI, 0.88-0.94), with a sensitivity of 94% (95% CI, 93%-97%) and a specificity of 82% (95% CI, 78%-86%). The positive likelihood ratio was 5.12, and the negative likelihood ratio was 0.05. Performance was consistent across cohorts, including incomplete KD, diverse FC etiologies, and coronary artery phenotypes. The assay was implemented as a laboratory-developed test. Analytical validation demonstrated high linearity (R2 > 0.99), precision (coefficient of variation <5%), and sample stability for up to 6 days at 4 °C or for 24 hours at room temperature. This diagnostic study found that a 2-gene laboratory-developed whole-blood qPCR assay measuring IFI27 and MCEMP1 expression accurately distinguished KD from other febrile illnesses using standard molecular platforms. Prospective evaluation in broader populations is warranted to determine its clinical utility for reducing diagnostic delay and coronary complications.