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

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).

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.

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.

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.

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.