Verification of a New Pathogen Detection Method by PostSonication Next-Generation Sequencing with Quantitative Real-Time PCR for the Diagnosis of Orthopedic Biofilm Infection

Abstract

Next-generation sequencing (NGS), are recommended for the unconfirmed diagnosis of periprosthetic joint infection (PJI), but initial assessment using quantitative methods is required to determine whether microorganisms are present, before embarking on sequencing approaches. This study aimed to validate a new diagnostic approach for orthopedic biofilm infections that combines quantitative methods as quantitative real-time polymerase chain reaction (qPCR) with NGS. This study sequenced 16 clinical samples from one patient with PJI and 16 quantitative positive controls. The detection rate of multiple Staphylococcus species with NGS was approximately ten times higher than with culture. The read counts of Staphylococci detected in peri-implant samples were negatively correlated with the qPCR Cq value, while the three most commonly detected contaminant bacteria were positively correlated with the qPCR Cq values. The results were similar to those of the quantitative positive control study. In conclusion, In addition, pathogens and contaminants can also be discriminated by correlation with qPCR Cq values. These results of NGS with qPCR method can be used to determine the cause of clinically problematic infections in NGS that are difficult to confirm because false positive results in diagnosing infection are expected to cause misdiagnosis and misidentification of the causative organism, causing inappropriate therapy.