Gastroesophageal reflux disease (
GERD) is primarily diagnosed based on symptoms and response to a
proton-pump inhibitor (PPI) trial. Gold standard testing requires an invasive endoscopic procedure, often with ambulatory pH monitoring. Salivary
pepsin is a potential noninvasive modality for
GERD diagnosis. This study aimed to assess diagnostic performance of salivary
pepsin thresholds for
GERD and determine optimal collection protocol of saliva in an external validation cohort. Over 10 months, adults with symptoms of
GERD undergoing esophagogastroduodenoscopy with wireless pH-monitoring off PPI were enrolled. Saliva was self-collected by participants over 4 days across three different time points: fasting ante meridiem (AM), post-prandial, and bedtime (PM).
Pepsin levels were calculated via Peptest.
Pepsin variability and agreement were determined using linear mixed effects models and intraclass correlation. Validation of diagnostic threshold and performance characteristics were evaluated by receiver-operator curve analysis. Twenty participants enrolled in the study; 50% with physiologic
acid exposure (
acid exposure time < 4% no
GERD) and 50% with elevated
acid exposure (
GERD). Mean
pepsin concentrations were significantly lower in the AM (22.6 ± 25.2 ng/mL) compared to post-prandial (44.5 ± 36.7 ng/mL) and PM (55.4 ± 47.0 ng/mL). Agreement between
pepsin concentrations across 3 days was substantial for AM samples (kappa 0.61), with lower agreement for post-prandial and PM samples. A single AM
pepsin concentration of 25 ng/mL was 67% accurate for
GERD with 56% sensitivity and 78% specificity. This validation study highlights fair accuracy and performance characteristics of a single fasting AM salivary
pepsin concentration for the diagnosis of
GERD.