sample’s journey in orthopedic implant infections : from the operating room to the microbiology lab

Introduction

Implant-related infections (IRIs) represent one of the most feared complications in orthopedic and trauma surgery [1] Ribeiro M, Monteiro FJ, Ferraz MP. Infection of orthopedic implants with emphasis on bacterial adhesion process and techniques used in studying bacterial-material interactions. Biomatter. 2012;2(4):176-94.. These infections can affect joint prostheses, internal fixation devices, and other biomaterials, often leading to prolonged treatments, multiple surgeries, and poor functional outcomes [2] Benito N, Franco M, Ribera A, Soriano A, Rodriguez-Pardo D, Sorli L, et al. Time trends in the aetiology of prosthetic joint infections: a multicentre cohort study. Clin Microbiol Infect. 2016;22(8):732 e1-8.. One of the main challenges in diagnosing these infections lies in the behavior of the causative microorganisms, which frequently reside within biofilms — structured communities that adhere to implant surfaces and resist both antibiotics and immune responses [3], Rather MA, Gupta K, Mandal M. Microbial biofilm: formation, architecture, antibiotic resistance, and control strategies. Braz J Microbiol. 2021;52(4):1701-18.[4], Anwar H, van Biesen T, Dasgupta M, Lam K, Costerton JW. Interaction of biofilm bacteria with antibiotics in a novel in vitro chemostat system. Antimicrob Agents Chemother. 1989;33(10):1824-6.[5] Hoiby N, Bjarnsholt T, Givskov M, Molin S, Ciofu O. Antibiotic resistance of bacterial biofilms. Int J Antimicrob Agents. 2010;35(4):322-32..

Correct identification of the responsible pathogens is fundamental to plan adequate antimicrobial therapy and surgical strategies [6] Drago L, De Vecchi E. Microbiological Diagnosis of Implant-Related Infections: Scientific Evidence and Cost/Benefit Analysis of Routine Antibiofilm Processing. Adv Exp Med Biol. 2017;971:51-67.. However, microbiological diagnosis is highly dependent on the quality of the sample collection and handling process. The pre-analytical phase — often underestimated — includes all steps from intra-operative sampling to laboratory processing, and each step carries a risk of error that may compromise the final diagnosis [7] Bemer P, Leger J, Tande D, Plouzeau C, Valentin AS, Jolivet-Gougeon A, et al. How Many Samples and How Many Culture Media To Diagnose a Prosthetic Joint Infection: a Clinical and Microbiological Prospective Multicenter Study. J Clin Microbiol. 2016;54(2):385-91..

In this review, we describe the current procedures for intra-operative sampling and biofilm-targeted pretreatment strategies prior to culture analysis. We emphasize the critical impact of false negatives (missed infections) and false positives (contaminants misidentified as pathogens), both of which can lead to inappropriate therapy, extended hospital stays, unnecessary surgeries, and increased costs. Analysis estimated that a single false negative in prosthetic joint infection can incur more than €49.000 in additional patient costs, while a single false positive may generate over €8.500 in unnecessary treatments and follow-up expenses [8] Romano CL, Trentinaglia MT, De Vecchi E, Logoluso N, George DA, Morelli I, et al. Cost-benefit analysis of antibiofilm microbiological techniques for peri-prosthetic joint infection diagnosis. BMC Infect Dis. 2018;18(1):154.. The following sections cover best practices for sampling, handling and transport of explanted materials, chemical biofilm disruption techniques, and workflow optimizations to minimize diagnostic errors. The goal is to provide a clear guide to support clinicians, microbiologists, and healthcare teams in optimizing diagnostic accuracy in implant-related infections.

Sampling: current intra-operative practices

Accurate intra-operative sampling underpins reliable microbiological diagnosis in IRIs by balancing two objectives: maximizing pathogen recovery (high sensitivity) and minimizing contamination (high specificity).

Sampling must follow a strict, stepwise protocol. The World Association against Infection in Orthopaedics and Trauma (WAIOT) “10-rules” procedure, reported in Figure 1, begins with synovial fluid sampling by fine needle joint aspiration prior to surgery and/or, at surgery, immediately after skin incision—thus avoiding the risk of dragging skin flora into the joint—followed by immediate transfer of the fluid into sterile containers or blood culture bottles. Any purulent exudate at the incision site is likewise collected and sent for culture. Next, a biopsy of the synovial membrane or joint capsule is obtained using sterile instruments for each specimen [9], Drago L, Clerici P, Morelli I, Ashok J, Benzakour T, Bozhkova S, et al. The World Association against Infection in Orthopaedics and Trauma (WAIOT) procedures for Microbiological Sampling and Processing for Periprosthetic Joint Infections (PJIs) and other Implant-Related Infections. J Clin Med. 2019;8(7).[10] Bozhkova S, Suardi V, Sharma HK, Tsuchiya H, Del Sel H, Hafez MA, et al. The W.A.I.O.T. Definition of Peri-Prosthetic Joint Infection: A Multi-center, Retrospective Validation Study. J Clin Med. 2020;9(6)..

Once tissue sampling is complete, prosthetic components or osteosynthesis devices are explanted, usually from 3 to 6 samples [7] Bemer P, Leger J, Tande D, Plouzeau C, Valentin AS, Jolivet-Gougeon A, et al. How Many Samples and How Many Culture Media To Diagnose a Prosthetic Joint Infection: a Clinical and Microbiological Prospective Multicenter Study. J Clin Microbiol. 2016;54(2):385-91.. Before handling the device, the surgical team should change gloves and switch to a fresh instrument set. The removed hardware is placed directly—never resting on the operative field or trays—into dedicated sterile, hermetically sealed transport containers [11], Parvizi J, Shohat N, Gehrke T. Prevention of periprosthetic joint infection: new guidelines. Bone Joint J. 2017;99-B(4 Supple B):3-10.[12] Ritter MA, Eitzen H, French ML, Hart JB. The operating room environment as affected by people and the surgical face mask. Clin Orthop Relat Res. 1975(111):147-50.. Finally, periprosthetic tissues, bone sequestra or osteolytic fragments at the implant–bone interface are sampled in the same manner. Each specimen—fluid, soft tissue, implant, bone—must be collected with sterile instruments and immediately sealed [13] Basile G, Gallina M, Passeri A, Gaudio RM, Castelnuovo N, Ferrante P, et al. Prosthetic joint infections and legal disputes: a threat to the future of prosthetic orthopedics. J Orthop Traumatol. 2021;22(1):44. amcli.it.

The Italian Society of Orthopaedics and Traumatology (SIOT) guidelines reinforce these principles, emphasizing gloves and instrument changes between each sampling step and strict avoidance of any intermediate handling or surface contact [https://old.giot.it/wp-content/uploads/2018/04/04_Art_LINEE_Guida-1.pdf]. The Italian Association of Clinical Microbiologists (AMCLI) pathway mirrors this sequence—synovial fluid first, then capsule, then implant components, and finally periprosthetic tissue or bone—providing detailed instructions on container choice, labeling, and rapid transfer to the microbiology laboratory [amcli.it].

Adherence to these recommendations (schematized in Figure 2), dedicated tools per specimen, strict asepsis, immediate sealing, and a clear sampling order, lays the groundwork for reliable downstream transport, storage, pretreatment, and culture, thereby reducing both false negatives and false positives.

Handling, storage, and transport of sample

Proper handling and transport of intra-operative specimens are critical to preserving pathogen viability and preventing overgrowth of contaminants. As soon as each sample is collected, it should be sealed in its dedicated sterile, hermetic container in the operatory room [World Health Organization, 2012. Guidance on Regulations for Transport of Infectious Substances 2013-2014, World Health Organization. Switzerland. Retrieved from https://coilink.org/20.500.12592/crmmz2 on 25 May 2025. COI: 20.500.12592/crmmz2.]. Clear labeling directly on the container—indicating patient identifiers, anatomical site, date and time of collection, and collector’s initials—ensures accurate tracking A standardized request form, detailing relevant clinical information such as recent antibiotic therapy and the patient’s comorbidities, must accompany every specimen [14] Colston J, Atkins B. Bone and joint infection. Clin Med (Lond). 2018;18(2):150-4..

Tissue, bone, and explanted implant components should be kept at 4 °C from collection until they reach the laboratory, if transit exceeds 2 hours. [15], Parvizi J, Della Valle CJ. AAOS Clinical Practice Guideline: diagnosis and treatment of periprosthetic joint infections of the hip and knee. J Am Acad Orthop Surg. 2010;18(12):771-2.[16] Miller JM, Binnicker MJ, Campbell S, Carroll KC, Chapin KC, Gilligan PH, et al. A Guide to Utilization of the Microbiology Laboratory for Diagnosis of Infectious Diseases: 2018 Update by the Infectious Diseases Society of America and the American Society for Microbiology. Clin Infect Dis. 2018;67(6):e1-e94.. If immediate transport is not possible, refrigerated storage at 4°C for up to 24 hours is acceptable, though longer delays increase the risk of bacterial death and false-negative cultures [17], Cuthbertson L, Rogers GB, Walker AW, Oliver A, Hafiz T, Hoffman LR, et al. Time between collection and storage significantly influences bacterial sequence composition in sputum samples from cystic fibrosis respiratory infections. J Clin Microbiol. 2014;52(8):3011-6.[18], Trampuz A, Piper KE, Jacobson MJ, Hanssen AD, Unni KK, Osmon DR, et al. Sonication of removed hip and knee prostheses for diagnosis of infection. N Engl J Med. 2007;357(7):654-63.[19] Van Cauter M, Cornu O, Yombi JC, Rodriguez-Villalobos H, Kaminski L. The effect of storage delay and storage temperature on orthopaedic surgical samples contaminated by Staphylococcus Epidermidis. PLoS One. 2018;13(3):e0192048.. Synovial fluid specimens, when inoculated directly into blood-culture bottles in the operating room, may be held at ambient temperature and processed according to the manufacturer’s incubation protocol. Freezing any clinical material is strictly discouraged, as ice crystal formation damages cellular structures and compromises culture yield [20] Liao CH, Shollenberger LM. Survivability and long-term preservation of bacteria in water and in phosphate-buffered saline. Lett Appl Microbiol. 2003;37(1):45-50..

For transport, specimens must be placed in rigid, secondary container and clearly separated from any non-clinical items. Courier services or laboratory porters should be notified in advance of time-sensitive shipments to prevent unintended delays. Upon arrival at the microbiology laboratory, each batch of samples is checked against its request form, and a chain-of-custody log is signed to document receipt. Any deviations—such as temperature excursions or prolonged transit times—are recorded promptly.

By ensuring immediate sealing and labeling, strict temperature maintenance, rapid delivery, and meticulous documentation, the integrity of the sample “journey” is preserved, maximizing culture sensitivity and reducing diagnostic errors (Figure 3).

Physical or chemical antibiofilm pretreatment techniques

Routine cultures of biofilm- and implant-related infections may fail or yield false culture-negative results in a significant number of patients, with figures ranging from 5% to 42% in Prosthetic Joint Infections. (PJIs) [21] Parvizi J, Tan TL, Goswami K, Higuera C, Della Valle C, Chen AF, et al. The 2018 Definition of Periprosthetic Hip and Knee Infection: An Evidence-Based and Validated Criteria. J Arthroplasty. 2018;33(5):1309-14 e2.; to increase the sensitivity, pretreatment of samples have been proposed to liberate biofilm-embedded bacteria. Validated antibiofilm strategies include sonication and chemical biofilm debonding with dithiothreitol (DTT).

Physical antibiofilm pretreatment with Sonication

Implanted components are submerged in sterile fluid and exposed to low-frequency ultrasound waves (typically 40–50 kHz for 5–10 minutes) [22] Yano MH, Klautau GB, da Silva CB, Nigro S, Avanzi O, Mercadante MT, et al. Improved diagnosis of infection associated with osteosynthesis by use of sonication of fracture fixation implants. J Clin Microbiol. 2014;52(12):4176-82.. Cavitation disrupts the extracellular polymeric matrix, releasing bacteria into suspension for culture [23], Trampuz A, Piper KE, Hanssen AD, Osmon DR, Cockerill FR, Steckelberg JM, et al. Sonication of explanted prosthetic components in bags for diagnosis of prosthetic joint infection is associated with risk of contamination. J Clin Microbiol. 2006;44(2):628-31.[24] Tunney MM, Patrick S, Gorman SP, Nixon JR, Anderson N, Davis RI, et al. Improved detection of infection in hip replacements. A currently underestimated problem. J Bone Joint Surg Br. 1998;80(4):568-72.. The SIOT recommends sonication exclusively for explanted hardware (excluding cement), with quantitative thresholds (≥50 CFU/mL sonicate or ≥200 CFU/mL concentrated sonicate) indicating infection [25] Corvec S, Portillo ME, Pasticci BM, Borens O, Trampuz A. Epidemiology and new developments in the diagnosis of prosthetic joint infection. Int J Artif Organs. 2012;35(10):923-34.. Advantages include wide availability in high volume orthopedic surgery centers and proven efficacy [26] Tsikopoulos K, Christofilos SI, Kitridis D, Sidiropoulos K, Stoikos PN, Gravalidis C, et al. Is sonication superior to dithiothreitol in diagnosis of periprosthetic joint infections? A meta-analysis. Int Orthop. 2022;46(6):1215-24.; limitations are equipment cost, operator dependency, maintenance of the sonicator, potential bacterial damage if parameters are not strictly controlled [27] Monsen T, Lovgren E, Widerstrom M, Wallinder L. In vitro effect of ultrasound on bacteria and suggested protocol for sonication and diagnosis of prosthetic infections. J Clin Microbiol. 2009;47(8):2496-501., and the need to manually sort the sample, a step that can itself introduce cross-contamination. Moreover, phenotypic changes of some pathogen following sonication have been reported [28] Sendi P, Frei R, Maurer TB, Trampuz A, Zimmerli W, Graber P. Escherichia coli variants in periprosthetic joint infection: diagnostic challenges with sessile bacteria and sonication. J Clin Microbiol. 2010;48(5):1720-5.. It is also worth noting that some well-known experts did not find a superiority of sonication over tissue samples collected according to strict rules [29] Dudareva M, Barrett L, Figtree M, Scarborough M, Watanabe M, Newnham R, et al. Sonication versus Tissue Sampling for Diagnosis of Prosthetic Joint and Other Orthopedic Device-Related Infections. J Clin Microbiol. 2018;56(12)..

Chemical antibiofilm pretreatment with Dithiothreitol

Dithiothreitol (DTT) is a reducing agent that cleaves disulfide bonds within the biofilm matrix. Per standard protocols, tissue or explant samples are incubated in 0.1% DTT (≈25 mM) for 15 minutes at room temperature, then vortexed and cultured [30] Drago L, Signori V, De Vecchi E, Vassena C, Palazzi E, Cappelletti L, et al. Use of dithiothreitol to improve the diagnosis of prosthetic joint infections. J Orthop Res. 2013;31(11):1694-9.. According to SIOT guideline, DTT pretreatment can be applied both to prosthetic material and periprosthetic tissue, as shown by various studies [31] De Vecchi E, Bortolin M, Signori V, Romano CL, Drago L. Treatment With Dithiothreitol Improves Bacterial Recovery From Tissue Samples in Osteoarticular and Joint Infections. J Arthroplasty. 2016;31(12):2867-70.. Evidence demonstrates that DTT treatment increases sensitivity—up to 85% in some series—without impairing microbial viability [6] Drago L, De Vecchi E. Microbiological Diagnosis of Implant-Related Infections: Scientific Evidence and Cost/Benefit Analysis of Routine Antibiofilm Processing. Adv Exp Med Biol. 2017;971:51-67.. Advantages include its relative low-cost, the possible implementation in all hospitals without the need for specific equipments, and the applicability to all types of explanted materials (fluids, tissues, metallic and polymeric implants), making the procedure less operator-dependent, more streamlined, and reducing cross-contamination from manual handling. Care must be taken to respect concentration and contact-time parameters, as excessive exposure can have bactericidal effects [32] Karbysheva S, Cabric S, Koliszak A, Bervar M, Kirschbaum S, Hardt S, et al. Clinical evaluation of dithiothreitol in comparison with sonication for biofilm dislodgement in the microbiological diagnosis of periprosthetic joint infection. Diagn Microbiol Infect Dis. 2022;103(2):115679..

To further standardize and secure this workflow and minimize contamination, completely closed systems, like MicroDTTect®, integrate DTT elution within a sterile, single-use, specifically designed closed cartridge : implants or tissues are loaded intra-operatively, sealed, transported and then processed without any open handling, yielding up to 98 % diagnostic sensitivity and specificity while minimizing hands-on time, contamination risk and logistical complexity [8] Romano CL, Trentinaglia MT, De Vecchi E, Logoluso N, George DA, Morelli I, et al. Cost-benefit analysis of antibiofilm microbiological techniques for peri-prosthetic joint infection diagnosis. BMC Infect Dis. 2018;18(1):154..

The 2018 International Consensus Meeting (ICM) on hip and knee PJIs specifically endorses sonication and DTT technologies to minimize culture-negative cases [33] Schwarz EM, Parvizi J, Gehrke T, Aiyer A, Battenberg A, Brown SA, et al. 2018 International Consensus Meeting on Musculoskeletal Infection: Research Priorities from the General Assembly Questions. J Orthop Res. 2019;37(5):997-1006..

Using physical disruption (sonication) or chemical biofilm dissolution (DTT or MicroDTTect®), laboratories can dramatically improve microbial recovery from both tissue and prosthetic materials (Figure 4). In the next section, we will review culture protocols and diagnostic considerations once samples have been pretreated.

Microbiological culture and diagnostic considerations

Optimal sampling, transport, handling and antibiofilm pretreatment are key preliminary steps to cultural examination, that remains the diagnostic cornerstone for implant-related infections. However, accurate interpretation of culture results requires attention to technique, incubation conditions, and the clinical context.

Quantitative and Qualitative Culture

Explanted tissue and synovial fluid should be plated onto solid media and inoculated into blood-culture bottles (BCBs). BCB systems enable larger sample volumes, continuous growth monitoring, and in-built antibiotic neutralization—often yielding faster and more sensitive detection than agar alone [34], Velay A, Schramm F, Gaudias J, Jaulhac B, Riegel P. Culture with BACTEC Peds Plus bottle compared with conventional media for the detection of bacteria in tissue samples from orthopedic surgery. Diagn Microbiol Infect Dis. 2010;68(1):83-5.[35] Peel TN, Dylla BL, Hughes JG, Lynch DT, Greenwood-Quaintance KE, Cheng AC, et al. Improved Diagnosis of Prosthetic Joint Infection by Culturing Periprosthetic Tissue Specimens in Blood Culture Bottles. mBio. 2016;7(1):e01776-15..

Release of bacteria by sonication fluid or DTT eluates must be quantified: recovery of at least 50 CFU/mL of sonicate or DTT eluate is considered significant, whereas lower counts or growth only in enrichment broths demand clinical correlation before labeling an infection. [36], Berbari EF, Kanj SS, Kowalski TJ, Darouiche RO, Widmer AF, Schmitt SK, et al. 2015 Infectious Diseases Society of America (IDSA) Clinical Practice Guidelines for the Diagnosis and Treatment of Native Vertebral Osteomyelitis in Adults. Clin Infect Dis. 2015;61(6):e26-46.[37], McNally M, Sousa R, Wouthuyzen-Bakker M, Chen AF, Soriano A, Vogely HC, et al. The EBJIS definition of periprosthetic joint infection. Bone Joint J. 2021;103-B(1):18-25.[38] Osmon DR, Berbari EF, Berendt AR, Lew D, Zimmerli W, Steckelberg JM, et al. Diagnosis and management of prosthetic joint infection: clinical practice guidelines by the Infectious Diseases Society of America. Clin Infect Dis. 2013;56(1):e1-e25. [https://old.giot.it/wp-content/uploads/2018/04/04_Art_LINEE_Guida-1.pdf ].

To capture slow-growing organisms (e.g., Cutibacterium acnes), incubation should extend to 14 days, with a subculture check at day 5 and a final readout at day 14 [13], Basile G, Gallina M, Passeri A, Gaudio RM, Castelnuovo N, Ferrante P, et al. Prosthetic joint infections and legal disputes: a threat to the future of prosthetic orthopedics. J Orthop Traumatol. 2021;22(1):44.[21], Parvizi J, Tan TL, Goswami K, Higuera C, Della Valle C, Chen AF, et al. The 2018 Definition of Periprosthetic Hip and Knee Infection: An Evidence-Based and Validated Criteria. J Arthroplasty. 2018;33(5):1309-14 e2.[39], Drago L, De Vecchi E, Cappelletti L, Vassena C, Toscano M, Bortolin M, et al. Prolonging culture to 15 days improves bacterial detection in bone and joint infections. Eur J Clin Microbiol Infect Dis. 2015;34(9):1809-13.[40], Schwotzer N, Wahl P, Fracheboud D, Gautier E, Chuard C. Optimal culture incubation time in orthopedic device-associated infections: a retrospective analysis of prolonged 14-day incubation. J Clin Microbiol. 2014;52(1):61-6.[41] Trampuz A, Zimmerli W. Prosthetic joint infections: update in diagnosis and treatment. Swiss Med Wkly. 2005;135(17-18):243-51..

Culture-Negative Scenarios

When microbiological cultures remain negative despite a high clinical and intra-operative suspicion of infection, several steps are strongly recommended. Surface swabs should be avoided to reduce contamination risk and repeat sampling—preferably multiple tissue specimens and synovial fluid—should be performed immediately [42] Calori GM, Colombo M, Navone P, Nobile M, Auxilia F, Toscano M, et al. Comparative evaluation of MicroDTTect device and flocked swabs in the diagnosis of prosthetic and orthopaedic infections. Injury. 2016;47 Suppl 4:S17-S21.. At the time of explantation, all removed materials (hardware, tissue, fluid) should undergo antibiofilm pretreatment—sonication of implants whenever feasible, or chemical elution with dithiothreitol (DTT) for both biotic and abiotic samples—and, where possible, inoculation directly into blood-culture bottles to maximize recovery [43] Portillo ME, Salvado M, Trampuz A, Siverio A, Alier A, Sorli L, et al. Improved diagnosis of orthopedic implant-associated infection by inoculation of sonication fluid into blood culture bottles. J Clin Microbiol. 2015;53(5):1622-7.. Cultures must be incubated for more than 14 days when anaerobic or fastidious organisms are suspected [40] Schwotzer N, Wahl P, Fracheboud D, Gautier E, Chuard C. Optimal culture incubation time in orthopedic device-associated infections: a retrospective analysis of prolonged 14-day incubation. J Clin Microbiol. 2014;52(1):61-6.. Parallel testing using different media and methods can further enhance yield. Finally, if cultures remain sterile, molecular diagnostics—broad-range or targeted PCR and next-generation sequencing (NGS)—may be employed, albeit with caution, since these methods can detect non-viable microbial DNA and require careful interpretation in the clinical context [44] Vandercam B, Jeumont S, Cornu O, Yombi JC, Lecouvet F, Lefevre P, et al. Amplification-based DNA analysis in the diagnosis of prosthetic joint infection. J Mol Diagn. 2008;10(6):537-43..

Molecular and Rapid Diagnostics

Molecular assays can play a crucial adjunct role in culture-negative scenarios, after prior antibiotic exposure, or when fastidious or biofilm-embedded organisms are suspected. Broad-range PCR targeting the bacterial 16S rRNA gene offers an unbiased approach, capable of detecting unexpected or slow-growing pathogens directly from synovial fluid, sonication fluid, or DTT eluate [45] Bemer P, Plouzeau C, Tande D, Leger J, Giraudeau B, Valentin AS, et al. Evaluation of 16S rRNA gene PCR sensitivity and specificity for diagnosis of prosthetic joint infection: a prospective multicenter cross-sectional study. J Clin Microbiol. 2014;52(10):3583-9.. Its main advantages are high sensitivity and the ability to uncover rare organisms; however, it carries a significant risk of false positives from contaminant or non-viable DNA and does not distinguish live from dead bacteria, necessitating rigorous laboratory controls.

Targeted multiplex PCR panels—such as FilmArray or custom real-time qPCR assays—provide results within hours and can simultaneously identify predefined pathogens and key resistance genes [46] Ryu SY, Greenwood-Quaintance KE, Hanssen AD, Mandrekar JN, Patel R. Low sensitivity of periprosthetic tissue PCR for prosthetic knee infection diagnosis. Diagn Microbiol Infect Dis. 2014;79(4):448-53.. This rapid turnaround enables earlier, more focused antimicrobial therapy, but panels are inherently limited to the organisms and resistance markers they include and may miss atypical or emerging pathogens [47], Morgenstern C, Cabric S, Perka C, Trampuz A, Renz N. Synovial fluid multiplex PCR is superior to culture for detection of low-virulent pathogens causing periprosthetic joint infection. Diagn Microbiol Infect Dis. 2018;90(2):115-9.[48] Zegaer BH, Ioannidis A, Babis GC, Ioannidou V, Kossyvakis A, Bersimis S, et al. Detection of Bacteria Bearing Resistant Biofilm Forms, by Using the Universal and Specific PCR is Still Unhelpful in the Diagnosis of Periprosthetic Joint Infections. Front Med (Lausanne). 2014;1:30..

Next-generation sequencing (NGS) and metagenomic techniques hold the promise of comprehensive, culture-independent profiling of bacteria, fungi, and resistance determinants in a single assay [49], Sanabria AM, Janice J, Hjerde E, Simonsen GS, Hanssen AM. Shotgun-metagenomics based prediction of antibiotic resistance and virulence determinants in Staphylococcus aureus from periprosthetic tissue on blood culture bottles. Sci Rep. 2021;11(1):20848.[50] Azad MA, Wolf MJ, Strasburg AP, Daniels ML, Starkey JC, Donadio AD, et al. Comparison of the BioFire Joint Infection Panel to 16S Ribosomal RNA Gene-Based Targeted Metagenomic Sequencing for Testing Synovial Fluid from Patients with Knee Arthroplasty Failure. J Clin Microbiol. 2022;60(12):e0112622.. These methods eliminate panel restrictions and can reveal a full spectrum of pathogens and resistance genes. However, their high cost, need for specialized equipment and bioinformatics expertise, and longer turnaround times currently preclude routine implementation in most clinical laboratories. As these technologies mature, decrease in cost, and become more standardized, they may gradually transition from specialized referral centers to broader diagnostic use [51] Fenollar F, Roux V, Stein A, Drancourt M, Raoult D. Analysis of 525 samples to determine the usefulness of PCR amplification and sequencing of the 16S rRNA gene for diagnosis of bone and joint infections. J Clin Microbiol. 2006;44(3):1018-28..

Reporting and Interpretation

Laboratory reports must strike a balance between speed and accuracy. A preliminary report—issued around day 5—alerts clinicians to any initial growth, even at low levels, with an antibiogram pending. The final report, at the end of the incubation period, should classify isolates as:

• Definitive pathogens (e.g., Staphylococcus aureus, Pseudomonas aeruginosa, Enterobacterales at significant counts)
• Possible contaminants (e.g., coagulase-negative staphylococci, Cutibacterium spp. from a single specimen; annotate “possible contaminant—interpret clinically”)
• No growth

By integrating quantitative culture thresholds, antibiofilm-enhanced methods, extended incubation, and judicious molecular testing, laboratories can deliver nuanced, timely data that directly inform appropriate antimicrobial therapy and surgical decision-making in implant-related infections [9] Drago L, Clerici P, Morelli I, Ashok J, Benzakour T, Bozhkova S, et al. The World Association against Infection in Orthopaedics and Trauma (WAIOT) procedures for Microbiological Sampling and Processing for Periprosthetic Joint Infections (PJIs) and other Implant-Related Infections. J Clin Med. 2019;8(7)..

Critical issues and common pitfalls

Even the best protocols can be undermined by errors at any stage of the diagnostic pathway. Below are the most frequent missteps—and their downstream consequences—that teams must vigilantly guard against:

By anticipating these pitfalls, schematically reported in Figure 5 —and embedding quantitative culture criteria, strict time-and-temperature controls, clear reporting language, and tight coordination between surgeons, microbiologists, and infectious-disease specialists—teams can minimize both false positives and false negatives, ensure appropriate therapy, and curb the rise of antibiotic resistance in implant-related infections.

Illustration of six frequently encountered challenges that can compromise microbiological diagnosis of implant-related infections:

1. inadequate or contaminated sampling leading to false negatives/positives;
2. delays and temperature excursions causing loss of viable pathogens;
3. misinterpretation of low-level growth without quantitative thresholds;
4. inappropriate antimicrobial therapy fueling patient harm and resistance;
5. antibiotic resistance undermining stewardship efforts; and
6. overreliance on molecular tests without clinical correlation.

Conclusions

Successful diagnosis of implant-related infections hinges on rigorous attention to each step of the sample’s journey—from precise, aseptic intra-operative collection through rapid, cold-chain transport, to targeted biofilm disruption and extended culture protocols. When surgeons, microbiologists, and infectious-disease specialists collaborate closely—adhering to consensus-driven guidelines, using sonication or DTT (including closed-system devices), and reserving molecular assays for truly culture-negative cases—they can sharply reduce both false negatives and false positives. This integrated, multidisciplinary approach not only ensures accurate pathogen identification and tailored antimicrobial therapy but also supports antimicrobial stewardship, minimizes patient morbidity, and optimizes clinical outcomes in the management of implant-related infections.