How is Cushing syndrome diagnosed using late‑night salivary cortisol testing?

How to Diagnose Cushing Syndrome Using Late-Night Salivary Cortisol Testing

Late-night salivary cortisol (LNSC) collected at bedtime (11 PM–midnight) on 2–3 consecutive nights is the preferred first-line screening test for Cushing syndrome, with a diagnostic threshold >3.6 nmol/L (>0.1 μg/dL) providing >90% sensitivity and the highest specificity (93–100%) among all screening modalities. 1, 2

Pre-Test Requirements

Before ordering any cortisol testing, you must obtain a comprehensive medication history to exclude all sources of exogenous glucocorticoids—oral tablets, inhaled steroids, topical creams, intra-articular or epidural injections, and over-the-counter supplements—because failure to do so leads to unnecessary testing without clinical benefit. 1, 2

Collection Protocol

Timing and Number of Samples

• Collect saliva at the patient's usual bedtime (typically 23:00–00:00 hours) when cortisol should be at its physiologic nadir. 1, 2
• Obtain at least 2–3 samples on consecutive nights to account for intra-individual variability (which can reach 22–51%) and to detect cyclic Cushing syndrome, where patients exhibit weeks to months of normal cortisol interspersed with hypercortisolism episodes. 1, 2, 3
• Collecting at the patient's actual bedtime rather than strictly at midnight may decrease false-positive results because the cortisol nadir is tightly entrained to sleep onset. 2

Sample Collection Technique

• Use a simple, commercially available saliva collection device. 4
• Instruct patients to avoid eating, drinking, or brushing teeth for 1–2 hours before collection to prevent blood contamination from oral trauma. 1
• Warn patients to avoid topical hydrocortisone preparations, which can contaminate samples and cause falsely elevated results, particularly when mass spectrometry is used. 1, 2

Diagnostic Thresholds and Performance

• Abnormal threshold: LNSC >3.6 nmol/L (>0.1 μg/dL) indicates loss of the normal circadian nadir and supports hypercortisolism. 1, 2, 5
• Sensitivity: 92–100% for detecting Cushing syndrome. 1, 2, 6, 4, 5
• Specificity: 91–100%, the highest among all first-line screening tests. 1, 2, 6, 5
• Patients with mild Cushing syndrome may have values just above the upper limit of normal, reinforcing the need for multiple samples. 1

Enhanced Diagnostic Accuracy

• Combined measurement of late-night salivary cortisone (LNS cortisone) with LNSC further improves diagnostic accuracy, with LNS cortisone >14.5 nmol/L providing 95.2% sensitivity and 100% specificity. 6
• Routine immunoassay appears to have better diagnostic performance than liquid chromatography/tandem mass spectrometry, although mass spectrometry can detect both cortisol and cortisone, helping identify samples contaminated with topical hydrocortisone. 7

Absolute Contraindications

• Do NOT perform LNSC in night-shift workers or anyone with disrupted sleep-wake cycles, because the test relies on a normal nocturnal cortisol nadir and will produce false-positive results. 1, 2

Interpretation Algorithm

When LNSC Results Are Abnormal

• If ≥2 LNSC samples are elevated, proceed to measure 9 AM plasma ACTH to differentiate ACTH-dependent (pituitary or ectopic) from ACTH-independent (adrenal) Cushing syndrome. 1, 2
  • Low/undetectable ACTH (<5 ng/L or <1.1 pmol/L) → adrenal imaging (CT or MRI). 1, 2
  • Normal or elevated ACTH (≥5 ng/L or ≥1.1 pmol/L) → pituitary MRI. 1, 2

When Results Are Discordant or Borderline

• Obtain 2–3 additional 24-hour urinary free cortisol (UFC) collections and/or repeat the overnight 1-mg dexamethasone suppression test (DST) to account for intra-patient variability. 1
• Consider cyclic Cushing syndrome if results remain inconsistent; perform extended monitoring with multiple periodic sequential LNSC measurements during symptomatic periods. 1, 2

Complementary Screening Tests

Although LNSC is the preferred initial test, the Endocrine Society recommends performing 2–3 first-line screening tests in patients with intermediate to high clinical suspicion: 1, 2

• 24-hour urinary free cortisol (UFC): Collect 2–3 separate 24-hour specimens; values >100 μg/24 hours are diagnostic. Sensitivity >90%, but lowest among the three screening tests due to 50% random variability. 1, 2
• Overnight 1-mg dexamethasone suppression test (DST): Administer 1 mg dexamethasone at 23:00–00:00 hours; measure serum cortisol at 08:00 hours. Cortisol ≥1.8 μg/dL (≥50 nmol/L) is abnormal; cortisol >5 μg/dL (138 nmol/L) indicates overt Cushing syndrome. Sensitivity >90%. 1, 8
• Measuring dexamethasone levels concomitantly with cortisol reduces false-positive DST results by confirming adequate drug absorption. 1, 8

Critical Pitfalls to Avoid

• Oral contraceptives and estrogen therapy increase cortisol-binding globulin, falsely elevating total cortisol; LNSC is particularly useful in these patients because it measures free cortisol. 1, 7
• Pseudo-Cushing states—severe obesity, major depression, chronic alcoholism, uncontrolled diabetes, and polycystic ovary syndrome—can produce mild hypercortisolism mimicking Cushing syndrome. 1, 2
• CYP3A4 inducers (phenobarbital, carbamazepine, St. John's wort) accelerate dexamethasone metabolism, causing false-positive DST results. 1, 8
• Renal impairment or polyuria can invalidate UFC results, making LNSC a better option. 1
• A single abnormal screening test is insufficient for diagnosis; repeat testing of 1–2 screening modalities is essential to minimize false-positive diagnoses. 2

Advantages of LNSC Over Other Tests

• Non-invasive and can be performed at home, improving patient compliance compared to 24-hour urine collections. 2, 6, 4
• Highest specificity among all first-line screening tests. 1, 2
• Particularly useful for evaluating suspected intermittent hypercortisolism and screening large high-risk populations (e.g., patients with diabetes mellitus). 4
• Excellent correlation with late-night serum cortisol (R = 0.6977) and urinary free cortisol (R = 0.5404) in proven Cushing syndrome. 5

Special Populations

• In children and adolescents with suspected Cushing disease, LNSC has 95% sensitivity and 100% specificity. 1
• In pregnant women, LNSC is preferred because pregnancy increases cortisol-binding globulin, falsely elevating total serum cortisol while free cortisol (measured in saliva) remains accurate. 1