{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,1]],"date-time":"2026-09-01T13:23:37Z","timestamp":1788269017983,"version":"build-2803163510"},"reference-count":37,"publisher":"American Society for Microbiology","issue":"9","license":[{"start":{"date-parts":[[2026,9,1]],"date-time":"2026-09-01T00:00:00Z","timestamp":1788220800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"},{"start":{"date-parts":[[2026,9,1]],"date-time":"2026-09-01T00:00:00Z","timestamp":1788220800000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/journals.asm.org\/non-commercial-tdm-license"}],"funder":[{"DOI":"10.13039\/501100001691","name":"Japan Society for the Promotion of Science","doi-asserted-by":"publisher","award":["17K16707"],"award-info":[{"award-number":["17K16707"]}],"id":[{"id":"10.13039\/501100001691","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001691","name":"Japan Society for the Promotion of Science","doi-asserted-by":"publisher","award":["20K18045"],"award-info":[{"award-number":["20K18045"]}],"id":[{"id":"10.13039\/501100001691","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001691","name":"Japan Society for the Promotion of Science","doi-asserted-by":"publisher","award":["23K08641"],"award-info":[{"award-number":["23K08641"]}],"id":[{"id":"10.13039\/501100001691","id-type":"DOI","asserted-by":"publisher"}]},{"name":"Japanese Society for Replacement Arthroplasty Foundation","award":["2026-RP04"],"award-info":[{"award-number":["2026-RP04"]}]}],"content-domain":{"domain":["journals.asm.org"],"crossmark-restriction":true},"short-container-title":["Microbiol Spectr"],"published-print":{"date-parts":[[2026,9,1]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                  <jats:sec>\n                    <jats:title\/>\n                    <jats:p>\n                      Next-generation sequencing (NGS) enables the detection of specific pathogens unidentifiable by conventional cultures, but its application in orthopedics remains inconsistent due to background contamination and irreproducible findings. This study evaluated the diagnostic performance of a novel workflow combining broad-range 16S rRNA gene quantitative PCR (qPCR) screening with downstream NGS, focusing on bacterial biomass thresholds. The qPCR assay demonstrated excellent intrarater reliability, with an intraclass correlation coefficient (ICC) of 0.961 (95% confidence interval, 0.881 to 0.997). Based on serially diluted positive controls, a quantitative threshold of 10\u2075 CFU\/mL was established as the minimum concentration required for the consistent detection of fastidious taxa, such as\n                      <jats:italic toggle=\"yes\">Escherichia coli<\/jats:italic>\n                      . When evaluated against conventional cultures using 95 sonicate fluid and 276 pre\/intraoperative tissue samples, the qPCR assay achieved a sensitivity of 80% and a specificity of 72%. Subsequent NGS sequencing of 26 clinical samples and 9 controls showed concordance in 4 of 6 culture-positive infected cases with NGS taxonomy, whereas the remaining discrepancies were likely attributable to culture-based phenotypic misidentification. Notably, among the qPCR-positive cases, three were culture-negative, including two hip prosthesis loosening cases exhibiting polymicrobial profiles, and one post-traumatic osteoarthritis case harboring low-level\n                      <jats:italic toggle=\"yes\">Staphylococcus<\/jats:italic>\n                      . Crucially, this post-traumatic patient developed delayed periprosthetic joint infection (PJI) 2 years post-surgery, with cultures identifying\n                      <jats:italic toggle=\"yes\">Staphylococcus<\/jats:italic>\n                      previously detected by the initial NGS analysis. Integrating qPCR screening with targeted NGS effectively refines pathogen identification, filters environmental artifacts, and overcomes the diagnostic limitations of culture-negative infections in orthopedic practice.\n                    <\/jats:p>\n                    <jats:sec>\n                      <jats:title>IMPORTANCE<\/jats:title>\n                      <jats:p>Next-generation sequencing (NGS) enables the detection of specific pathogens in clinical samples that are not identifiable by conventional methods. However, NGS applications in orthopedics have not been quantitatively evaluated, and findings have been inconsistent owing to contaminants and the presence of non-credible causative organisms. These factors primarily stem from the failure to evaluate low-biomass samples and the absence of proper controls, such as negative controls or mock community DNA samples. This study demonstrates that interpreting results from low-biomass samples requires careful consideration because NGS relies on relative bacterial abundances; distinguishing likely pathogens from contaminants is particularly challenging when bacterial loads are low. We demonstrated that combining NGS with quantitative PCR (qPCR) and applying a Cq cutoff can reduce false positives.<\/jats:p>\n                    <\/jats:sec>\n                  <\/jats:sec>","DOI":"10.1128\/spectrum.04128-25","type":"journal-article","created":{"date-parts":[[2026,7,28]],"date-time":"2026-07-28T13:00:06Z","timestamp":1785243606000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":0,"title":["The diagnostic potential of combined quantitative polymerase chain reaction and next-generation sequencing using the same primers for periprosthetic joint infection"],"prefix":"10.1128","volume":"14","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-8123-5121","authenticated-orcid":true,"given":"Narumi","family":"Ueda","sequence":"first","affiliation":[{"name":"Department of Orthopedic Surgery, Japanese Red Cross Wakayama Medical Center, Komatsubara-dori","place":["Wakayama City, Japan"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jun","family":"Inoue","sequence":"additional","affiliation":[{"name":"Division of Gastroenterology, Department of Internal Medicine, Kobe University Graduate School of Medicine, Chuo-ku, Kusunoki-cho","place":["Kobe, Japan"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kazuyuki","family":"Okuda","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/001xjdh50","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Genome Analysis Center, Kansai Medical University Medical Center, Kansai Medical University, Fumizono\u2010cho","place":["Moriguchi, Japan"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hirokazu","family":"Iida","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/001xjdh50","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Rehabilitation, Kansai Medical University, Uyamahigashi-cho","place":["Hirakata, Japan"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"235","reference":[{"key":"e_1_3_5_2_2","doi-asserted-by":"publisher","DOI":"10.3109\/17453674.2010.525201"},{"key":"e_1_3_5_3_2","doi-asserted-by":"publisher","DOI":"10.3109\/17453674.2015.1086942"},{"key":"e_1_3_5_4_2","doi-asserted-by":"publisher","DOI":"10.1302\/2058-5241.6.200099"},{"key":"e_1_3_5_5_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.jse.2019.07.039"},{"key":"e_1_3_5_6_2","doi-asserted-by":"publisher","DOI":"10.1128\/JCM.00462-17"},{"key":"e_1_3_5_7_2","doi-asserted-by":"publisher","DOI":"10.2106\/JBJS.17.00434"},{"key":"e_1_3_5_8_2","doi-asserted-by":"publisher","DOI":"10.1302\/0301-620X.100B2.BJJ-2017-0531.R2"},{"key":"e_1_3_5_9_2","doi-asserted-by":"publisher","DOI":"10.1128\/jcm.02156-21"},{"key":"e_1_3_5_10_2","doi-asserted-by":"publisher","DOI":"10.1007\/s10096-019-03520-3"},{"key":"e_1_3_5_11_2","doi-asserted-by":"publisher","DOI":"10.1038\/s41586-022-05546-8"},{"key":"e_1_3_5_12_2","doi-asserted-by":"publisher","DOI":"10.1186\/s12915-014-0087-z"},{"key":"e_1_3_5_13_2","doi-asserted-by":"publisher","DOI":"10.11477\/mf.1408202275"},{"key":"e_1_3_5_14_2","doi-asserted-by":"publisher","DOI":"10.15106\/j_besei81_79"},{"key":"e_1_3_5_15_2","unstructured":"Ueda N. 2024. Method for predicting sample quality a method for predicting accuracy of nucleic acid sequence analysis device for predicting sample quality and device for predicting accuracy of nucleic acid sequence analysis. 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Ion amplicon library preparation (fusion method) for use with ion torrent personal genome machine system. Publication No.4468326 Rev. C. Available from: https:\/\/assets.thermofisher.com\/TFS-Assets\/LSG\/manuals\/4468326_IonAmpliconLibraryPrep_FusionMethod_UG.pdf. Retrieved Jun 2024."},{"key":"e_1_3_5_28_2","doi-asserted-by":"publisher","DOI":"10.1038\/nmeth.f.303"},{"key":"e_1_3_5_29_2","doi-asserted-by":"publisher","DOI":"10.1002\/0471445428.ch18"},{"key":"e_1_3_5_30_2","unstructured":"Lowry R. 2026. Clinical calculator 1. vassarstats Available from: http:\/\/vassarstats.net\/clin1.html"},{"key":"e_1_3_5_31_2","doi-asserted-by":"publisher","unstructured":"Ueda N Inoue J Okuda K Hashiyada M Iida H Saito T. 2023. Verification of a new pathogen detection method by postsonication next-generation sequencing with quantitative real-time PCR for the diagnosis of orthopedic biofilm infection. 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