Clinical Characteristics and Immune Escape Mechanism of Nontuberculous Mycobacterial Lung Disease in South China

1. Background and Rationale NTM-LD presents significant clinical challenges due to its unclear pathogenesis, diagnostic complexity, suboptimal treatment outcomes, and poor prognosis. The unique climatic (hot, humid) and demographic factors in South China suggest potential regional variations in NTM epidemiology and clinical presentation. However, a systematic investigation into its clinical characteristics and underlying mechanisms in this region is lacking, impeding the development of localized precision medicine strategies. 2. Study Objectives Primary Objective: To systematically characterize the epidemiological patterns, clinical phenotypes, and prognostic determinants of NTM-LD in the South China population.

Secondary Objectives:

To analyze the distribution of NTM species and their correlation with disease severity and radiological findings.

To investigate host immune microenvironment alterations post-NTM infection, focusing on inflammatory responses and immune cell functional states.

To explore molecular mechanisms associated with treatment refractoriness in NTM disease. 3. Study Design

This is a hybrid observational study comprising two distinct parts: * Part 1: A multicenter, retrospective, non-interventional survey study. * Part 2: A prospective observational study involving biospecimen collection and laboratory analysis. 4. Study Populations and Sample Size

* Part 1: Approximately 1,500 patients diagnosed with NTM lung disease per standard guidelines between January 2015 and May 2025 across multiple collaborating centers in South China. * Part 2: A subset of 106 consecutively enrolled, confirmed NTM patients from the collaborating network for prospective biospecimen collection. 5. Methods and Procedures

* Part 1 (Retrospective Survey): Data on demographics, clinical symptoms, imaging features (chest CT), microbiology, treatment regimens, and outcomes will be extracted from electronic health records. Statistical analyses will include descriptive summaries, correlation studies, and multivariable regression modeling to identify prognostic factors. * Part 2 (Prospective Study): Peripheral blood and bronchoalveolar lavage fluid (BALF) will be collected. Analyses will include:

Molecular Biology: Real-time quantitative PCR for cytokine expression profiling; Western blot for protein-level assessment.

Single-Cell Sequencing: To delineate immune cell composition, transcriptional states, and cell-cell interaction networks within the pulmonary immune microenvironment. 6. Statistical Analysis For Part 1, appropriate statistical tests (e.g., Chi-square, t-test, ANOVA, Kaplan-Meier survival analysis, Cox regression) will be applied based on data types and distribution. For Part 2, bioinformatics pipelines specific for single-cell RNA sequencing data will be employed for clustering, differential expression, and pathway analysis. A p-value \<0.05 will be considered statistically significant. 7. Ethics and Data Management The study protocol will be submitted for approval to The Eighth Affiliated Hospital of Sun Yat-Sen University Ethics Committee of all participating centers prior to initiation. Patient informed consent will be obtained for the prospective Part 2. All data will be de-identified, coded, and stored on secure, password-protected servers in compliance with data protection regulations. 8. Significance This study is expected to generate a comprehensive clinical profile of NTM lung disease in South China and provide novel insights into its pathogenesis at the immune-microenvironment level. The findings may contribute to more accurate diagnostic criteria, prognostic models, and inform the future development of targeted therapeutic strategies for this refractory infection.

• Spectrum of NTM Species [Time frame: Species distribution will be assessed at baseline, defined as the date of the first positive NTM culture from a respiratory specimen. Retrospective data collection will cover the period from January 2015 to May 2025.]
• Clinical Prognostic Factors [Time frame: Assessed at baseline (within 7 days of enrollment).]
• Immunological Prognostic Factors [Time frame: Serum sample collected at baseline (within 7 days of enrollment) for antibody testing.]