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Background <p>The urea breath test (UBT) detects <i>Helicobacter pylori</i> (<i>H. pylori</i>) by measuring <sup>13</sup>CO₂ in exhaled air after the ingestion of a <sup>13</sup>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.</p> Methods <p><?tk 2?>Six volunteers confirmed to be <i>H. pylori</i>-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&#xa0;s, 10&#xa0;s, and 20&#xa0;s). Baseline samples were collected at 0&#xa0;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&#xa0;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.</p> Results <p>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 <i>P</i> &lt; 0.001).</p> Conclusion <p>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.</p> Graphical abstract <p></p>

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

  • 期刊论文

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  • 发表日期：

    2026年7月24日

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  • Wentao Fan,
  • Yuwen Tao,
  • Jinjin Shi,
  • Zixiang Huang,
  • MeiHong Chen,
  • Guoxin Zhang,
  • Bo Hao,
  • Feng Ye

摘要

Background

The urea breath test (UBT) detects Helicobacter pylori (H. pylori) by measuring 13CO₂ in exhaled air after the ingestion of a 13C-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.

Methods

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.

Results

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).

Conclusion

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.

Graphical abstract