Serum Anti-enteric Neuronal Antibodies in Patients With Achalasia and Their Association With Clinical Profiles

Achalasia is a rare esophageal motility disorder characterized by impaired relaxation of the lower esophageal sphincter and progressive esophageal dilation. Increasing evidence suggests that autoimmune mechanisms may contribute to its development. Serum anti-enteric neuronal antibodies (AENA) have been reported more frequently in patients with achalasia than in healthy individuals.

This study aims to evaluate the association between AENA levels and disease severity in patients with achalasia. Specifically, it aims to determine whether the intensity of AENA positivity is associated with endoscopic severity (measured by the CARS score), esophageal dilation, and integrated relaxation pressure. The study also aims to assess whether AENA status is associated with symptomatic outcomes following peroral endoscopic myotomy (POEM).

The objective is to determine whether AENA may serve as a potential biomarker for identifying patients with a more severe disease phenotype and less favorable treatment response.

Study Overview

• Status: Completed
• Conditions: Achalasia Cardia
• Intervention / Treatment: Procedure: peroral endoscopic myotomy (POEM)

• Study Type: Observational
• Enrollment (Actual): 195

Contacts and Locations

Study Locations

• China
  • Beijing Municipality
    • Beijing, Beijing Municipality, China, 100730
      • Peking Union Medical College Hospital

Participation Criteria

Eligibility Criteria

• Ages Eligible for Study: Adult; Older Adult
• Accepts Healthy Volunteers: N/A

• Sampling Method: Non-Probability Sample

Study Population

This prospective cohort study included 97 patients with AC diagnosed according to the Chicago Classification version 4.0 and 98 age- and sex-matched healthy subjects (HS).

Description

Inclusion Criteria:

• Age between 18 and 75 years;
• Fulfilment of the diagnostic criteria for AC according to the Chicago Classification version 4.0
• Completion of symptomatic assessment, esophagogastroduodenoscopy, high-resolution manometry (HRM), and barium esophagram.

Exclusion Criteria:

• pregnant
• lactating
• with coexisting major gastrointestinal disorders

Study Plan

How is the study designed?

Number of groups / cohorts

2

Cohorts and Interventions

Group / Cohort	Intervention / Treatment
healthy subjects; HS without gastrointestinal symptoms were recruited from the health examination center during the same period. All HS had normal physical and laboratory examination results. They were matched to patients with AC by age (± 2 years) and sex. Serum samples from HS were collected as part of routine health assessments, and both ANA and AENA testing were performed for all enrolled subjects.
Achalasia patients; Patients diagnosed with AC were consecutively enrolled. Inclusion criteria were: (1) Age between 18 and 75 years; (2) Fulfilment of the diagnostic criteria for AC according to the Chicago Classification version 4.0; and (3) Completion of symptomatic assessment, esophagogastroduodenoscopy, high-resolution manometry (HRM), and barium esophagram at PUMCH. Individuals who were pregnant, lactating, or those with coexisting major gastrointestinal disorders were excluded.	Procedure: peroral endoscopic myotomy (POEM); A subset of eligible patients subsequently underwent POEM at our institution using the standardized technique described by Inoue et al. Postoperative follow-up assessments were conducted at 1, 3, and 6 months and included evaluation of Eckardt scores. At the 6-month follow-up, repeat esophagogastroduodenoscopy (with grading of erosive esophagitis according to the Los Angeles classification esophagitis), HRM, and barium esophagogram were performed.

What is the study measuring?

Primary Outcome Measures

Outcome Measure	Measure Description	Time Frame
Proportion of Participants with Positive Serum Anti-Enteric Neuronal Antibodies (AENA)	Serum AENA is assessed by indirect immunofluorescence using guinea pig intestinal submucosal whole-mount preparations. Patient sera are incubated with neuronal tissue, followed by fluorescein-conjugated secondary antibodies. Mouse anti-Hu antibody serves as a positive control and PBS as a negative control. Fluorescence intensity is graded by independent investigators as: 0 (negative), 1 (moderate positive), or 2 (strong positive). This measure reports the percentage of participants graded as 1 or 2 (AENA-positive rate).	Baseline

Secondary Outcome Measures

Outcome Measure	Measure Description	Time Frame
The Eckardt score	The Eckardt score was used to evaluate symptom severity in patients with AC. This composite score includes four components: dysphagia, retrosternal pain, regurgitation, and weight loss. Dysphagia, retrosternal pain, and regurgitation are each graded from 0 to 3 (0 = none; 1 = occasional; 2 = daily; 3 = at each meal). Weight loss is also scored from 0 to 3 (0 = none; 1 = < 5 kg; 2 = 5-10 kg; 3 = > 10 kg). The total score ranges from 0 to 12, with higher scores indicating greater symptom severity.	Baseline, 1-month follow-up after the procedure, 6-month follow-up after the procedure
Endoscopic evaluation	Endoscopic assessment was conducted using the CARS scoring system, a validated tool developed through video-based reliability testing and shown to correlate with disease severity. The CARS system allocates 0-2 points each for the domains of Contents, Anatomy, and Lower Esophageal Sphincter (LES) Resistance, and 1 point for each Stasis component. Erosive esophagitis was graded according to the Los Angeles classification into four severity grades.	Baseline, 6-month follow-up
High-resolution manometry (HRM)	HRM was conducted per standard protocol, and key parameters were analyzed following the Chicago Classification version 4.0 criteria, including AC subtype, integrated relaxation pressure (IRP), basal LES pressure (BLESP), and distal contractile integral (DCI); and Radiologic assessment: A barium esophagogram was used to measure the maximum barium column width and assess esophageal tortuosity.	Baseline, 6-month follow-up
The degree of esophageal distortion on barium esophagogram	The degree of esophageal distortion on barium esophagogram was classified into four grades: None, mild, moderate, and severe. Mild distortion was defined as slight dilation or localized tortuosity with mild distal narrowing. Moderate distortion was characterized by a "C"-shaped or partial "S"-shaped configuration, whereas severe distortion included "L"-type or sigmoid-type esophagus. Senior gastroenterologists and radiologists with over 10 years of experience determined the grading.	Baseline, 6-month follow-up

Collaborators and Investigators

• Sponsor: Peking Union Medical College Hospital

Study record dates

Study Major Dates

• Study Start (Actual): February 1, 2019
• Primary Completion (Actual): March 1, 2025
• Study Completion (Actual): November 1, 2025

Study Registration Dates

• First Submitted: February 26, 2026
• First Submitted That Met QC Criteria: March 3, 2026
• First Posted (Actual): March 5, 2026

Study Record Updates

• Last Update Posted (Actual): March 5, 2026
• Last Update Submitted That Met QC Criteria: March 3, 2026
• Last Verified: March 1, 2026

More Information

Terms related to this study

• Keywords: Treatment Outcome; Enteric nervous system; Disease Severity; Anti-enteric neuronal antibody; Achalasia Cardia
• Additional Relevant MeSH Terms: Digestive System Diseases; Gastrointestinal Diseases; Esophageal Diseases; Esophageal Motility Disorders; Deglutition Disorders; Esophageal Achalasia
• Other Study ID Numbers: I-25PJ0734