Elevated carbon dioxide in exhaled breath induces false-positive DOB results during Helicobacter pylori detection with ¹³C-urea breath test using infrared spectroscopy

The urea breath test (UBT) detects Helicobacter pylori ( H. pylori ) by measuring 13 CO₂ in exhaled air after the ingestion of a 13 C-urea metabolized by bacterial urease. However, the influence of CO₂ concentrations in exhaled breath on UBT results remains unclear. This study aimed to evaluate the effect of varying exhaled CO₂ levels on the outcomes of infrared spectroscopy-based ¹³C-UBT. Six volunteers confirmed to be H. pylori -negative via ¹³C-UBT and stool antigen assay were enrolled. All participants completed overnight fasting and oral rinsing before specimen collection, followed by standardized breath sampling with three breath-holding durations (5 s, 10 s, and 20 s). Baseline samples were collected at 0 min, followed by a 30-min rest without ¹³C-urea administered, then test samples were collected. Following each sampling cycle, participants rested with spontaneous quiet breathing for 20–30 s before performing the next breath-holding procedure. Each participant completed 30 sampling cycles, yielding 30 paired baseline–test samples per individual. CO₂ concentrations and delta over baseline (DOB) values were measured using three independent infrared spectrometers under calibration settings of 5.0 and 6.0. A DOB value ≥ 4 was defined as a positive result. High CO₂ was defined as a test-sample CO₂ concentration greater than or equal to the corresponding calibration concentration plus 0.2. A total of 180 paired baseline–test datasets were included in the analysis. At calibration concentrations of 5.0, positive results were observed in 104 of 118 high CO₂ datasets (88.14%; 95% exact CI, 80.90%–93.36%), whereas none of the 62 low CO₂ datasets showed positive result (0.00%; 95% exact CI, 0.00%–5.78%). At a calibration concentration of 6.0, positive results occurred in 20 of 26 high-CO₂ datasets (76.92%; 95% exact CI, 56.35%–91.03%), whereas all 154 low CO₂ datasets tested negative (0.00%; 95% exact CI, 0.00%–2.37%). Sample-level Fisher’s exact tests were significant at both settings (both P < 0.001). Elevated CO₂ concentrations may be associated with false-positive DOB results in infrared spectroscopy-based ¹³C-UBT, particularly when test-bag CO₂ exceeds the calibration value by more than 0.2 units. Monitoring of exhaled CO₂ during specimen collection is essential to guarantee accurate results.