BACKGROUND: The diagnostic accuracy of 16S rDNA PCR, multiplex PCR (mPCR), and metagenomic next-generation sequencing (mNGS) in periprosthetic joint infections (PJIs) remains unclear. OBJECTIVES: To evaluate the diagnostic accuracy of 16S rDNA PCR, mPCR, and mNGS in PJI. DATA SOURCES: PubMed and EMBASE (January 1, 2000-March 1, 2024), with no language restrictions. STUDY ELIGIBILITY CRITERIA: Studies containing sufficient data to construct a 2×2 contingency table allowing for sensitivity and specificity calculation were considered. PARTICIPANTS: Adults (≥18 years) with PJI and appropriate control groups. TESTS: 16S rDNA PCR, mPCR, and mNGS. REFERENCE STANDARD: Diagnosis required adherence to Musculoskeletal Infection Society, Infectious Diseases Society of America (IDSA), International Consensus Meeting, European Bone and Joint Infection Society criteria. Studies employing alternative author-defined criteria were included only if they did not rely solely on positive cultures to define PJI. ASSESSMENT OF RISK OF BIAS: QUADAS-2 was used. METHODS OF DATA SYNTHESIS: A bivariate model calculated pooled diagnostic odds ratios (DORs), sensitivities, and specificities, each with 95% confidence intervals (CIs). RESULTS: Seventy-nine studies were included, comprising 3,940 PJI cases and 4,700 uninfected controls. Pooled sensitivity/specificity were 80.0% (95% CI: 75.4-84.3%)/94.0% (95% CI: 91-96%) for 16S rDNA PCR
62.2% (52.5-70.9%)/96.2% (93.2-97.9%) for mPCR
and 88.6% (83.3-92.4%)/93.2% (89.5-95.6%) for mNGS. Notably, mNGS had the highest DOR (105.9
95% CI: 60-186.9). A sensitivity analysis excluding lower-quality studies resulted in increased DORs for all methods. DISCUSSION: These molecular techniques display strong diagnostic accuracy for identifying PJI. Although mNGS yielded the highest DOR, numerous technical and practical challenges preclude its routine use for PJI diagnosis. Significant heterogeneity across studies warrants cautious interpretation and underscores the need for future comparative research.