Acute respiratory infections (ARIs) remain a leading cause of clinic visits and the fourth most common cause of mortality worldwide [1]. The main pathogens causing ARIs are primarily viruses (accounting for 80 % of cases), as well as bacteria, fungi, and parasites [2], [3]. The overlapping clinical symptoms of most respiratory infections complicate the diagnosis of ARIs in the absence of laboratory testing. Moreover, co-infections involving multiple pathogens comprise approximately 25 % of cases, contributing to diagnostic delays and worse outcomes [4]. Therefore, rapid and accurate identification of respiratory pathogens is essential for guiding appropriate therapy, monitoring local outbreaks, and implementing effective infection control measures [5].

Current diagnostic methods for infectious diseases include conventional culture, direct fluorescent antibody tests, rapid antigen tests, and molecular assays. Although culture remains the gold standard for laboratory pathogen identification, it is time-intensive and requires substantial technical expertise for sample processing [6]. Direct fluorescent antibody tests offer faster turnaround time and enable simultaneous detection of multiple pathogens, but variable sensitivity and reliance on skilled interpretation restrict their broader clinical utility [7]. Rapid antigen tests are widely used in laboratories because of their high specificity, rapid turnaround, and low cost. However, its poor sensitivity increases the risk of false-negative results, particularly when detecting low-abundance pathogens [8], [9]. Molecular assays, including polymerase chain reaction (PCR)-based tests, isothermal amplification techniques, and nucleic acid sequencing, offer a sensitive and versatile approach for diagnosing respiratory pathogens [10], [11]. Compared with the methods mentioned above, molecular assays provide a unified platform capable of detecting multiple pathogens per reaction with high accuracy, reduce turnaround time, and exhibit the potential to identify novel or unexpected pathogens [12], [13], [14].

In China, the National Medical Products Administration (NMPA) has approved more than ten commercially available respiratory multiplex assays that utilize various technological approaches, including quantitative real-time polymerase chain reaction (qPCR), PCR-capillary electrophoresis, isothermal amplification with multiple-biotin signal amplification (double amplification), and microarray technology, to enable accurate and reliable detection of respiratory pathogens. However, the limited range of target pathogens, covering three to 13 common respiratory pathogens in NMPA-approved assays, restricts their ability in providing comprehensive guidance for clinical decision-making. To overcome this constraint, numerous laboratory-developed tests (LDTs) have been developed, incorporating methods such as qPCR, PCR-melting curve analysis, mass spectrometry, targeted next-generation sequencing, and metagenomic next-generation sequencing. These LDTs enable simultaneous detection of a broader spectrum of respiratory pathogens and are increasingly applied in specific clinical contexts to support ARI diagnosis [15], [16], [17], [18].

Although diverse molecular assays have advanced clinical application, they have also complicated efforts to achieve precise diagnosis [15]. Inter-laboratory inconsistencies in respiratory pathogen testing performance have been reported [19], [20], [21], [22], potentially contributing to inappropriate antimicrobial use and delays in effective treatment [19]. In this context, external quality assessment (EQA) programs are therefore critical for ensuring the quality and reliability of detection. However, no EQA studies evaluating respiratory pathogen testing performance across multiple methodological platforms have been reported so far. To address this gap, we conducted a nationwide EQA study to assess the proficiency of clinical laboratories in China for the molecular detection of multiple respiratory pathogens.