Bead‐beating assay during synovial fluid DNA extraction improves real‐time PCR accuracy for periprosthetic joint infection

摘要

Polymerase chain reaction (PCR)-based genetic diagnosis is a rapid and sensitive method to diagnose periprosthetic joint infection (PJI). DNA extraction using bead beating is an effective method for collecting bacterial genes in Gram-positive bacteria. We compared the detection accuracy between the conventional and bead-beating DNA extraction assay. The detection rate improved from 86.7% using the conventional method to 95.6% using the bead-beating. Our results suggest that bead-beating during DNA extraction can improve the accuracy of PCR-based genetic diagnosis of PJI. The diagnosis of periprosthetic joint infection (PJI) is often difficult because of negative culture results, which complicate selecting appropriate treatment strategies.1 Recent diagnostic criteria suggest multifaceted diagnostic tests to improve the diagnostic accuracy of PJI.2, 3 Despite advancements in test assays, rapid and accurate diagnosis of PJI remains challenging.4, 5 Preoperative PJI diagnosis is necessary to ensure a successful treatment strategy; thus, delayed diagnosis and treatment initiation often result in unfavorable clinical outcomes.6 16S ribosomal ribonucleic acid (rRNA)-targeting polymerase chain reaction (PCR) assays provide rapid and sensitive results for infection detection in patients with suspected PJI.7-10 Although previous studies have demonstrated a highly accurate diagnosis with a specificity of 0.68–1.00 (0.96), the false-negative results remained a major concern.11 This is partly due to the presence of the cell wall of Gram-positive bacteria, which is composed of a thick peptidoglycan layer that prevents effective DNA extraction.12 The bead-beating DNA extraction assay mechanically disrupts the cell walls of Gram-positive bacteria using microglass beads.13-16 This method efficiently increased the yield of DNA extraction, potentially improving PCR assay accuracy for Gram-positive bacterial PJI. However, the effect of this assay on PJI genetic diagnosis has not yet been demonstrated. The purpose of this study was to compare the sensitivity and specificity of conventional and bead-based DNA extraction methods and to examine whether the bead-beating DNA extraction assay can improve the diagnostic accuracy of bacterial PCR assays for Gram-positive bacterial PJI using synovial fluid collections. This retrospective study was approved by our institutional review board, and informed consent was waived and obtained in the form of opt-out on the website. First, 479 samples (256 cases) of joint or puncture fluid were identified that were collected at our hospital between March 2013 and October 2022 for PJI around the hip joint and that were stored at −30°C. Seventy-six samples (48 cases) were selected after excluding 390 (202 cases) culture-negative samples, five (three cases) samples positive for Gram-negative bacteria, and 8 (3 cases) samples positive for fungi (Figure 1). Thirty-one samples were excluded because their volume was <200 µL. Finally, 45 samples (30 cases) with Gram-positive bacterial culture results were enrolled in this study (Table 1). All the cases were diagnosed with PJI.17 Furthermore, synovial fluids from 20 consecutive aseptic cases diagnosed with osteoarthritis from March 2020 to August 2022 were selected as controls. DNA was extracted from 65 samples (45 samples in which Gram-positive bacteria were detected and 20 aseptic samples), using both conventional and bead-beating DNA extraction methods. In the conventional method, extraction was performed at the time the samples were collected, either manually (QIAamp DNA Mini Kit; Qiagen) or automatically using the Bio Robot EZ1 (Qiagen), as previously reported.10, 18 In the bead-beating assay, DNA extraction was performed in 200 µL samples using glass beads for 10 min at 1000 roll/min (Twin Mixer TM-282; AS ONE Corporation) immediately after mixing Proteinase K in the DNA extraction process (Bacterial DNA Extraction Kit; Mitsui Chemicals). The amount of DNA in both grou