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Bacteriology

Rapid Molecular Microbiologic Diagnosis of Prosthetic Joint Infection

Charles Cazanave, Kerryl E. Greenwood-Quaintance, Arlen D. Hanssen, Melissa J. Karau, Suzannah M. Schmidt, Eric O. Gomez Urena, Jayawant N. Mandrekar, Douglas R. Osmon, Lindsay E. Lough, Bobbi S. Pritt, James M. Steckelberg, Robin Patel
Charles Cazanave
aDivision of Clinical Microbiology, Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, Minnesota, USA
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Kerryl E. Greenwood-Quaintance
aDivision of Clinical Microbiology, Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, Minnesota, USA
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Arlen D. Hanssen
bDepartment of Orthopedic Surgery, Mayo Clinic, Rochester, Minnesota, USA
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Melissa J. Karau
aDivision of Clinical Microbiology, Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, Minnesota, USA
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Suzannah M. Schmidt
aDivision of Clinical Microbiology, Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, Minnesota, USA
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Eric O. Gomez Urena
aDivision of Clinical Microbiology, Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, Minnesota, USA
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Jayawant N. Mandrekar
dDivision of Biomedical Statistics and Informatics, Department of Health Science Research, Mayo Clinic, Rochester, Minnesota, USA
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Douglas R. Osmon
cDivision of Infectious Diseases, Department of Medicine, Department of Health Science Research, Mayo Clinic, Rochester, Minnesota, USA
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Lindsay E. Lough
aDivision of Clinical Microbiology, Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, Minnesota, USA
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Bobbi S. Pritt
aDivision of Clinical Microbiology, Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, Minnesota, USA
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James M. Steckelberg
cDivision of Infectious Diseases, Department of Medicine, Department of Health Science Research, Mayo Clinic, Rochester, Minnesota, USA
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Robin Patel
aDivision of Clinical Microbiology, Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, Minnesota, USA
cDivision of Infectious Diseases, Department of Medicine, Department of Health Science Research, Mayo Clinic, Rochester, Minnesota, USA
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DOI: 10.1128/JCM.00335-13
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    Fig 1

    Positive microbiologic results (albeit not necessarily concordant with respect to the organism detected) for patients with prosthetic joint infection. Superscript symbol definitions: a, cases 200 (Mycobacterium abscessus), 298 (Staphylococcus aureus), and 369 (polymicrobial, including S. aureus); b, cases 164 (Capnocytophaga canimorsus), 236 (Candida albicans), and 211, 259, and 425 (all three S. aureus); c, cases 214, 250, 280, and 379; d, cases 74, 110, 217, 226, 286, 352, and 430; e, cases 70, 91, 153, 203, 207, 212, 269, 300, 342, 343, 381, 396, 412, and 429.

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  • Table 1

    Real-time PCR assay panel design

    Target organismaTarget geneFragment size (bp)Forward/reverse primer sequences (5′–3′)Probe sequences (5′–3′)bMg2+ concn (mM)Primer concns (forward/reverse, in μM)Cycling parametersLimit of detection (CFU/ml of sonicate fluid)% inclusivityc% cross-reactivityc
    Anneal temp (°C)Anneal time (s)No. of cycles
    Staphylococcus (22)tufd447CAATGCCACAAACTCG/GCTTCAGCGTAGTCTAACGGCCTGTAGCAACAGTAC-FL,e LC640-CGACCAGTGATTGAGAATACGTCCe, GGCGATGCTCAATACGAAGAAAAAATC-FL,f LC705-AGAATTAATGGAAGCTGTAGATACf31/155836550 SCN IDRL-7,371, 325 S. aureus ATCC 43300100 (201/201)0 (0/153)
    StreptococcusrpoBg137CCGGHCGTCACGGWAA/CCATACCAAGRTGAAGYTCCATAGGAACGCCAGTTGATATCATGYTKAAYCC-FL, LC640-CTTGGGGTGCCATCACGKATGAA40.4/4571535110 S. agalactiae, 115 S. pneumoniae100 (15/15)h0 (0/339)
    Enterococcus/Granulicatella/AbiotrophiarpoB143CGYGAAGCYGGCGATGAAT/AWGGCATRTCTTCTTCYGGCCAYGAAGGRGATAARATGGCSGG-FL, LC640-GTCAYGGWAATAAAGGGGTYGTWTC40.4/455835200 E. faecalis100 (14/14)i0 (0/340)
    Enterobacteriaceae other than ProteusrpoB185TCTGCWATYGAAGAAGGCAACT/ATCAGGGAHGCACCRACGCTTGTTCAGCCGCGACC-FL, LC640-GTTGACTACATGGACGTATCCACCCAGC3.50.4/255835190 E. coli83.3 (20/24)j0 (0/330)
    Gram-positive anaerobic cocci16S rRNA162TCGCGTCYSATTAGCTAGT/GCTGCATCAGRGTTYCCCACATTGGRACTGAGAHACGGY-FL, LC640-ARACTCCTACGGGAGGCAGCAG2.50.4/25783510 F. magna100 (12/12)0 (0/342)
    Propionibacterium/Actinomyces16S rRNA243CGGATTTATTGGGCGTAAAGR/AGGGTATCTAAGCCTGTTCGGGCGAAGGCGGTTCTCTG-FL, LC640-CCTTTCCTGACGCTGAGRAGCG2.50.4/255835200 P. acnes100 (52/52)k0.3 (1/302)l
    P. aeruginosa (21)gyrBm222CCTGACCATCCGTCGCCACAAC/CGCAGCAGGATGCCGACGCCGGCGAGACCGATGGCT-FL, LC640-GGCACCGAAGTTCACTTCAAGCCG2.50.4/155835200100 (10/10)0 (0/344)
    Corynebacteriumn (20)Kinasen,o143 (forward/reverse 1), 149 (forward/reverse2)CGRTTGTACCARGARCGGT/GCACCTSAAYCCSCGT (reverse 1), CAACGAGCACCTSAACCC (reverse 2)TAGCGCTGGAAGTACCASGAGGT-FL, LC640-GACTCRCGCGGCGACGG, LC705-GACTCGCGCTCGGAAGG40.4/2571035200 C. tuberculo-stearicum, 440 C. jeikeium80 (4/5)p0 (0/349)
    ProteusrpoB183CTGTCWGCAATTGAAGAAGGTAACT/GGATCAGTGAWGCACCGACTTGCCGTCATARAGGYG-FL, LC705-TCAAGYTTATTTAGTCGTGAT2.50.4/25583552 P. vulgaris100 (4/4)0 (0/350)
    B. fragilis groupleuBq185CGTCCTGAGATAGAACG/GCAGCATTATCCACAAACATATAACTTGCTTCCAGTCGTCTATGGA-FL, LC640-CAGATTGCACAAGAAATGGCGCCGC2.50.4/255835150 B. fragilis92.3 (12/13)r0 (0/341)
    • ↵a References are given in parentheses.

    • ↵b FL, fluorescein; LC640, LightCycler Red 640; LC705, LightCycler Red 705.

    • ↵c Calculated based on 354 isolates.

    • ↵d tuf, elongation factor Tu.

    • ↵e PanStaphHP1 and HP2, probes that detect Staphylococcus aureus and SCN.

    • ↵f SAtufHP1 and HP2, S. aureus-specific probes.

    • ↵g rpoB encodes the β-subunit of RNA polymerase.

    • ↵h S. agalactiae positive, with a Tm of 63°C, compared to Tms of <60°C for other Streptococcus spp.

    • ↵i The G. adiacens (n = 3) and Abiotrophia species isolates had Tms of 58°C and 50°C, respectively, compared to 55°C for the Enterococcus sp. isolate.

    • ↵j The four negative isolates were Morganella morganii, Pantoea agglomerans, Providencia rettgeri, and Serratia liquefaciens.

    • ↵k The Actinomyces spp. had Tms of ≤56°C, compared to 61 to 63°C for Propionibacterium spp.

    • ↵l Corynebacterium amycolatum positive, with a Tm of 58°C.

    • ↵m gyrB encodes DNA gyrase B.

    • ↵n This assay uses one forward and two reverse primers and three probes in the same reaction, primer reverse 1 and probe LC705-GACTCGCGCTCGGAAGG, specific for the resistant species (C. jeikeium and C. urealyticum).

    • ↵o Polyphosphate kinase gene, corresponding to pvdS2 of C. jeikeium K411 (NC_007164).

    • ↵p Corynebacterium group F1 negative.

    • ↵q leuB encodes β-isopropylmalate dehydrogenase.

    • ↵r B. caccae negative.

  • Table 2

    Isolates, from patients with biofilm-associated disease, that were used for analytical assay validationa

    Group and speciesNo. of isolates (total n = 354)
    Staphylococcus spp.201
        S. aureus85
        Coagulase-negative Staphylococcus spp.116
            S. epidermidis80
            S. lugdunensis16
            S. warneri8
            S. capitis3
            S. caprae3
            S. simulans3
            Otherb3
    Propionibacterium spp.41
        P. acnes29
        P. avidum10
        P. granulosum2
    Enterobacteriaceae28
        Escherichia coli5
        Enterobacter cloacae4
        Proteus mirabilis3
        Klebsiella pneumoniae3
        Klebsiella oxytoca2
        Otherc11
    Streptococcus spp.15
        S. agalactiae3
        S. dysgalactiae3
        S. pyogenes2
        S. pneumoniae2
        S. salivarius2
        Otherd3
    Bacteroides fragilis group13
        B. fragilis8
        B. thetaiotaomicron2
        Othere3
    Gram-positive anaerobic cocci12
        Finegoldia magna8
        Otherf4
    Actinomyces spp.g11
    Pseudomonas aeruginosa10
    Enterococcus faecalis9
    Corynebacterium spp.h5
    Granulicatella adiacens3
    Abiotrophia defectiva2
    Otheri4
    • ↵a Categories shown in boldface were targeted with specific assays.

    • ↵b Includes one S. haemolyticus, one S. hominis, and one S. saprophyticus isolate.

    • ↵c Includes one Citrobacter freundii, one C. koseri, one E. aerogenes, one Morganella morganii, one Pantoea agglomerans, one P. vulgaris, one Providencia rettgeri, one Salmonella sp., one Serratia liquifaciens, one S. marcescens, and one Shigella flexneri isolate.

    • ↵d Includes one S. anginosus, one S. mitis, and one S. mutans isolate.

    • ↵e Includes one B. caccae, one B. distasonis, and one B. ovatus isolate.

    • ↵f Includes one Peptoniphilus asaccharolyticus, one P. harei, one Parvimonas micra, and one Anaerococcus prevotii isolate.

    • ↵g Includes three A. neuii, three A. odonolyticus, two A. naeslundii, one A. meyeri, one A. viscosus, and one A. radingae isolate.

    • ↵h Includes one C. amycolatum, one C. simulans, one C. propinquum, one Corynebacterium sp. group F1, and one C. afermentans.

    • ↵i Includes one Candida parapsilosis, one Dermabacter hominis, one P. fluorescens, and one S. saccharolyticus isolate.

  • Table 3

    Characteristics of the 434 patients with or without PJI

    CharacteristicValue for patient groupP value
    Patients with aseptic failure (total n = 290)Patients with prosthetic joint infection (n = 144)
    Age (yrs)0.53
        Median6866
        Range24–9131–92
    Gender [n (%)]0.14
        Male131 (45.2)76 (52.8)
        Female159 (54.8)68 (4..2)
    Reason for primary arthroplasty [n (%)]0.43
        Osteoarthritis208 (71.7)108 (75)
        Bone fracture or trauma44 (15.2)19 (13.2)
        Inflammatory joint disordera20 (6.9)8 (5.5)
        Avascular necrosis12 (4.1)3 (2.1)
        Congenital abnormality2 (0.7)4 (2.8)
        Otherb4 (1.4)2 (1.4)
    Second (or more) revision [n (%)]0.004
        No177 (61.0)67 (46.5)
        Yes113 (39.0)77 (53.5)
    Hardware removal [n (%)]1.00
        Partial14 (4.8)6 (4.2)
        Complete276 (95.2)138 (95.8)
    Risk factor for PJI [n (%)]0.002
        Diabetes mellitus35 (12.1)33 (22.8)0.003
        Long-term use of immunosuppressive therapyc2 (0.7)4 (2.8)0.10
    Site of arthroplasty [n (%)]0.87
        Knee181 (62.4)91 (63.2)
        Hip109 (37.6)53 (36.8)
    Antecedent antimicrobial therapy [n/total no. evaluated (%)]24/284 (8.4)58/134 (43.3)<0.0001
    Diagnostic criterion for PJI
        Acute inflammation [n/total no. evaluated (%)]0/290 (0)90/112 (80.4)
        Purulence [n (%)]0112 (77.8)
        Sinus tract [n (%)]039 (27.1)
    Preoperative laboratory findings [n/total no. evaluated (%)]
        Blood leukocyte count > 10 × 109/literd,e17/231 (7.4)21/123 (17.1)0.005
        Erythrocyte sedimentation rate > 30 mm/hd,f44/266 (16.5)83/135 (61.5)<0.0001
        Serum C-reactive protein > 10 mg/literd,f53/266 (19.9)109/136 (81.2)<0.0001
        Synovial fluid leukocyte count > 1,700/μlf,g33/140 (23.6)63/71 (88.7)<0.0001
        Synovial fluid differential > 65% neutrophilsf,g15/137 (10.9)61/71 (85.9)<0.0001
    • ↵a Includes rheumatoid arthritis, ankylosing spondylitis, and systemic lupus erythematosus.

    • ↵b Includes bone neoplasia, arthrofibrosis, polymyalgia rheumatica, reflex sympathetic dystrophy, and acromegaly.

    • ↵c Includes corticosteroids, methotrexate, and tumor necrosis factor inhibitors.

    • ↵d The cutoff was taken from Bernard et al. (23).

    • ↵e Within one preoperative week.

    • ↵f Within six preoperative months.

    • ↵g The cutoff was taken from Trampuz et al. (24), a study that excluded patients with underlying inflammatory joint diseases or connective tissue diseases and evaluated only knee arthroplasties.

  • Table 4

    Comparison of microbiologic tests for diagnosis of PJI

    TestNo. of patients with positive specimens and:Sensitivity (95% CI)Specificity (95% CI)Positive predictive value (95% CI)Negative predictive value (95% CI)
    Aseptic failure (n = 290)PJI (n = 144)
    Synovial fluid culturea5/16159/8966.3 (55.5–76.0)96.9 (92.9–99.0)92.2 (82.7–97.4)83.9 (77.8–88.8)
    Tissue culture
        Any growth4511982.6 (75.4–88.4)84.5 (79.8–88.5)72.6 (65.1–79.2)90.7 (86.6–93.9)
        ≥2 positive tissues (same organism)610170.1 (62.0–77.5)97.9 (95.6–99.2)94.4 (88.2–97.9)86.9 (82.7–90.3)
    Sonicate fluid culture510572.9 (64.9–80.0)98.3 (96.0–99.4)95.5 (89.7–98.5)88.0 (83.9–91.3)
    Sonicate fluid PCR (10-assay panel)
        Any positive result611177.1 (69.3–83.7)97.9 (95.6–99.2)94.9 (89.2–98.1)89.6 (85.7–92.7)
        Staphylococcus species275
            S. aureus028
            Coagulase-negative staphylococci247
        Streptococcus species311
        Enterococcus/Granulicatella/Abiotrophia species011
        Enterobacteriaceae18
        Gram-positive anaerobic cocci08
        Propionibacterium species08
        P. aeruginosa05
        Corynebacterium species04
            C. jeikeium/C. urealyticum00
            Non-C. jeikeium species04
        Proteus species01
        B. fragilis group00
    • ↵a Denominators are smaller for synovial fluid cultures because samples from fewer patients were submitted for this test.

Additional Files

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  • Files in this Data Supplement:

    • Supplemental file 1 -

      Tables S1 (Characteristics of 33 subjects classified as having prosthetic joint infection who had negative results for sonicate fluid bacterial PCR) and S2 (Discordant results between sonicate fluid PCR and culture)

      PDF, 304K

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Rapid Molecular Microbiologic Diagnosis of Prosthetic Joint Infection
Charles Cazanave, Kerryl E. Greenwood-Quaintance, Arlen D. Hanssen, Melissa J. Karau, Suzannah M. Schmidt, Eric O. Gomez Urena, Jayawant N. Mandrekar, Douglas R. Osmon, Lindsay E. Lough, Bobbi S. Pritt, James M. Steckelberg, Robin Patel
Journal of Clinical Microbiology Jun 2013, 51 (7) 2280-2287; DOI: 10.1128/JCM.00335-13

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Rapid Molecular Microbiologic Diagnosis of Prosthetic Joint Infection
Charles Cazanave, Kerryl E. Greenwood-Quaintance, Arlen D. Hanssen, Melissa J. Karau, Suzannah M. Schmidt, Eric O. Gomez Urena, Jayawant N. Mandrekar, Douglas R. Osmon, Lindsay E. Lough, Bobbi S. Pritt, James M. Steckelberg, Robin Patel
Journal of Clinical Microbiology Jun 2013, 51 (7) 2280-2287; DOI: 10.1128/JCM.00335-13
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