ABSTRACTChronic pulmonary aspergillosis (CPA) is a chronic and progressive fungal disease with high morbidity and mortality. Avoiding diagnostic delay and misdiagnosis are concerns for CPA patients. However, diagnostic practice is poorly evaluated, especially in resource-constrained areas whereAspergillusantibody testing tools are lacking. This study aimed to investigate the diagnostic laboratory findings in a retrospective CPA cohort and to evaluate the performance of a novelAspergillusIgG lateral flow assay (LFA; Era Biology, Tianjin, China). During January 2016 and December 2021, suspected CPA patients were screened at the Center for Infectious Diseases at Huashan Hospital. A total of 126 CPA patients were enrolled.AspergillusIgG was positive in 72.1% with chronic cavitary pulmonary aspergillosis, 75.0% with chronic necrotizing pulmonary aspergillosis, 41.7% with simple aspergilloma, and 30.3% withAspergillusnodule(s). The cavitary CPA subtypes had significantly higher levels ofAspergillusIgG.AspergillusIgG was negative in 52 patients, who were finally diagnosed by histopathology, respiratory culture, and metagenomic next-generation sequencing (mNGS). Sputum culture was positive in 39.3% (42/107) of patients andAspergillus fumigatuswas the most common species (69.0%, 29/42). For CPA cohort versus controls, the sensitivity and specificity of the LFA were 55.6% and 92.7%, respectively. In a subgroup analysis, the LFA was highly sensitive forA. fumigatus-associated chronic cavitary pulmonary aspergillosis (CCPA; 96.2%, 26/27). Given the complexity of the disease, a combination of serological and non-serological tests should be considered to avoid misdiagnosis of CPA. The novel LFA has a satisfactory performance and allows earlier screening and diagnosis of CPA patients.IMPORTANCEThere are concerns on avoiding diagnostic delay and misdiagnosis for chronic pulmonary aspergillosis due to its high morbidity and mortality. A proportion of CPA patients test negative forAspergillusIgG. An optimal diagnostic strategy for CPA requires in-depth investigation based on real-world diagnostic practice, which has been rarely discussed. We summarized the clinical and diagnostic laboratory findings of 126 CPA patients with various CPA subtypes.AspergillusIgG was the most sensitive test for diagnosing CPA. However, it was negative in 52 patients, who were finally diagnosed by non-serological tests, including biopsy, respiratory culture, and metagenomic next-generation sequencing. We also evaluated a novelAspergillusIgG lateral flow assay, which showed a satisfactory performance in cavitary CPA patients and was highly specific toAspergillus fumigatus. This study gives a full picture of the diagnostic practice for CPA patients in Chinese context and calls for early diagnosis of CPA with combined approaches.