Exploring the causes of variability in quality of oropharyngeal swab sampling for SARS-CoV-2 nucleic acid testing and proposed improvement measures: a multicenter, double-blind study.

Although coronavirus disease 2019 (COVID-19) has not been considered a public health emergency of international concern since last year, intermittent regional impacts still persist, and accurate testing remains crucial. Ribonuclease P protein subunit P30 (RPP30) RNA, known for its broad and stable expression in tissue cells, was used to evaluate samples from 10 hospitals with over 3,000 negative nucleic acid tests. The results revealed that the overall pass rate for the collected samples was consistently low and exhibited significant heterogeneity. After reassessing the evaluative effectiveness of RPP30 RNA Ct values from the samples of 132 positive individuals under quarantine observation, it was used to identify factors affecting sampling quality. These factors included different stages ranging from sample collection to PCR processing, various characteristics of both samplers and individuals being sampled, as well as sampling season and location. The results indicated that post-sampling handling had minimal impact, winter and fever clinic samples showed higher quality, whereas children's samples had lower quality. The key finding was that the characteristics of samplers were closely related to sampling quality, emphasizing the role of subjectivity. Quality control warnings led to substantial improvements, confirming this finding. Consequently, although there are various factors during the testing process, the most critical aspect is to improve, supervise, and maintain standardized practices among sampling staff.IMPORTANCEThis study further confirmed the reliability of internal references (IRs) in assessing sample quality, and utilized a large sample IR data to comprehensively and multidimensionally identify significant interference factors in nucleic acid test results. By further reminding and intervening in the subjective practices of specimen collectors, good results could be achieved.