•Preoperative diagnosis of PJI is difficult due to the limited availability of diagnostic tests and varying inflammatory host responses [2].

•Identifying an organism preoperatively provides clarity in treatment.

•However, culture-negative PJI occurs between 28 to 46% cases.

•Next generation sequencing (NGS) can universally detect microbes in a sample that meet strict reporting criteria for NGS, and may prove helpful identifying organisms [3-4].

•A current concern regards specificity of NGS, noting improved detection of microbes may over-diagnose PJI [4].

•In this study, we compared NGS results in aspirated synovial fluid samples to paired ICM-18 synovial biomarkers.

a)Evaluating concordance of NGS positivity to biomarkers used in diagnosing PJI.

b)Estimating the performance of NGS as a diagnostic for PJI.

c)Describing the bacterial and fungal composition identified from synovial fluid.

-Synovial fluid was sent to a commercial lab for NGS testing including V1-V2 16S rRNA sequencing for bacteria and ITS 3-4 sequencing for fungi [1, 5].

-Full contents of test panel provided in Table 1

-Cohen’s kappa was used to evaluate the agreement between NGS positivity and PJI.

-Diagnostic performance of synovial markers was evaluated using sensitivity, specificity, accuracy, positive predictive value (PPV), and negative predictive value (NPV), estimated with the epi.test() function, and Wilson 95% confidence intervals.

Results:

2,011 synovial fluid samples studied, the majority being knee samples (85.7%).

22.6% of samples were NGS-positive, predominantly for bacteria (448 vs. 26 fungal).

Totals of 160 unique bacterial and fungal species observed, most abundant in Figure 1.

Eight species were the dominant bacteria in 64% of cases, with remaining third made up of 61 less common species.

Fungal detections generally rare, observed in 2.2% of all specimens led by Candida.

23.2% of samples met the minor criteria for high probability PJI (≥ 4 score; Figure 2a) and NGS positivity had substantial concordance with PJI (k=0.68).

Key Result: NGS positivity correlates with synovial biomarkers

NGS positivity correlated with cumulative biomarker score (R2=0.93, p=0.003, Figure 2b).

Within ICM-negative samples, NGS positivity was significantly associated with both elevated WBC and PMN%, but not CRP (Figure 2c-e).

Across all samples, NGS positivity was significantly associated with elevated results for all biomarkers tested (Figure 2f-h).

The specificity of NGS was greater than CRP but less than WBC and PMN% (Table 2).

Discussion:

Microbial targeted NGS positivity was concordant with ICM minor criteria for PJI

NGS has a low false positive rate (92.3% specificity) comparable to established ICM biomarkers (89.4%–97.9%).

NGS excels in detection of fastidious and atypical pathogens, whereas conventional testing may be unsuited for detection of up to a third of dominant pathogens.

Presented findings suggest NGS offers broad discovery power for pathogens, is specific to PJI, and may warrant guidance as a diagnostic criterion when restructuring future diagnostic criteria defining PJI.

Key Takeaways:

- NGS correlates with clinical biomarkers for diagnosing PJI.

- NGS improves microorganism identification in preoperative evaluations.

- NGS should be considered in future diagnostic classifications.

References:

1. Tipton et al., Microbial next generation DNA sequencing of aspirated synovial fluid shows concordance with ICM criteria biomarkers for diagnosing periprosthetic joint infection in hip and knee arthroplasty. Front. Microbiol., 2026. https://doi.org/10.3389/fmicb.2026.1816780

2. Parvizi, J., et al., The 2018 Definition of Periprosthetic Hip and Knee Infection: An Evidence-Based and Validated Criteria. J Arthroplasty, 2018. 33(5): p. 1309-1314.e2.

3. Kullar, R., et al., Next-Generation Sequencing in Periprosthetic Joint Infections: Clinicians' Guide to Its Diagnostic Role. Infectious Diseases in Clinical Practice, 2025. 33(3): p. e1448.

4. Martinazzi, B.J., et al., 2025 ICM: Diagnostic Techniques: Molecular Tests. J Arthroplasty, 2025. 41(1s1): p. S407-s411.

5. OrthoKEY, MicroGen DX, Lubbock, Texas, US