An accurate identification of bacteria in PJI is essential for a good outcome. Even if the diagnosis is set without an isolated causative organism, a significant roadblock is created on the choice of antibiotic used by the surgeon. The cost of antibiotic resistance averages $107,264 per case versus $68,053 in each PJI case caused by nonresistant strains [3].

Culture is considered a gold standard in the diagnosis of PJI. According to the recent definition of PJI as proposed by the MSIS, the sole isolation of a pathogen by culture from two separate tissue or fluid samples obtained from the affected prosthetic joint is a strong indication for PJI [1]. Tissue and fluid cultures may be negative even in the presence of other clear indicators of infection in 7–11 % of cases [4, 5].

False-negative cultures in a patient with PJI may be due to a variety of factors. Preoperative antibiotic prophylaxis can result in negative cultures. Appropriate collection of samples, the number of samples obtained, and optimum incubation period have been topics of debate. Any use of antibiotic in the preceding 2 weeks before diagnosis was associated with a lower yield of cultures in one study [6]. In another report of 60 patients with culture-negative PJI, 61 % of patients received antimicrobial therapy before culture samples were obtained from the affected joint [7]. Further, the administration of antibiotics prior to making the diagnosis and obtaining synovial fluid from the joint may affect the accuracy of the synovial fluid WBC count and differential, which are powerful tests commonly used to diagnose PJI.

Formation of complex biofilms on prosthetic surfaces decreases the quantity of free-floating bacteria, consequently leading to a greater likelihood of a negative. Another cause of culture-negative PJI is the inability of traditional tissue cultures to recover infecting organisms encapsulated in a biofilm. The organisms in a biofilm are encapsulated in a complex structure composed of macromolecules such as glycocalyx that allow the organisms to evade the host’s immune system (macrophages and neutrophils). It has been estimated that these sessile bacteria are 1,000 times more resistant to antibiotics than their planktonic (free-floating) counterparts [8]. In chronic PJI most of the microorganisms exist in a biofilm form attached to the implant surface and surrounding tissues. Additionally, the absence of free-floating bacteria explains the paucity of inflammatory signs in chronic PJI.

Second, slow-growing pathogens may not be identified within the standard incubation period. Prolonged periods of incubation (around 14 days) are known to be an important factor in isolation of Propionibacterium species [9]. Additionally, extending the incubation period up to 14 days significantly increases the culture sensitivity without increasing the risk of false-positive results caused by contaminants. However, although 14 days of incubation has been recommended for all routine culture samples by some authorities, we feel that this exercise should be reserved for patients with suspected PJI, culture-negative cases, and patients who may be infected with low-virulence organisms. It is unnecessary to lengthen the incubation period for which the infecting organism has been isolated preoperatively.

A minimum of three or more independent intraoperative tissue or fluid samples from a representative area should be taken, and each sample should be taken with an unused instrument [10]. Infection with atypical organisms, such as fungi and mycobacteria that do not grow on routine aerobic or anaerobic media and require a specialized culture medium for growth and isolation in the lab, is another reason for the existence of culture-negative cases of PJI.

The method of obtaining and transferring culture samples to the laboratory can also affect the ability of the microbiology lab to isolate the infecting organisms. Samples taken from nonrepresentative areas of the joint, delaying the transport of the samples, allowing the tissue to dehydrate, or exposure to extremes of temperature can further affect the yield of cultures. Swab cultures from wound or periarticular tissues are found to have higher false-positive and false-negative results [11]. In addition, it has been shown that the introduction of bacteriostatic compounds such as saline or local anesthetic into the joint cavity at the time of aspiration increases the probability of false-negative culture results. Irrigation of the wound with bacteriostatic agents prior to obtaining the culture samples can also interfere with isolation of the infecting pathogen [12].

The use of sonication to dislodge adherent bacteria from removed total joint prostheses increases the number of bacteria isolated on culture or other techniques enabling the detection of bacteria [13, 14]. Sonicate cultures were found to have 83.3 % sensitivity as compared to single positive tissue cultures, with 72.2 % sensitivity. In patients receiving antibiotic therapy, the sensitivity was 65.9 and 57.5 %, respectively, suggesting that sonication cultures are less affected by prior antibiotic usage [14]. Sonication is mainly recommended when there is implant failure with no clear signs of infection and in patients with delayed implant failure. Implant sonication increases the diagnostic accuracy of infection in patients with delayed, but not early, orthopedic infected arthroplasty; therefore, it is not recommended as a routine diagnostic test in these patients [15]. Prolonged incubation time has not been found to be beneficial in increasing sensitivity for the diagnosis of implant-related infection using samples prepared by sonication of the implants [16].

Analysis of ESR and CRP is useful in all cases of suspected infection and is strongly recommended by the American Academy of Orthopaedic Surgeons (AAOS) as an initial step in establishing PJI diagnosis. ESR and CRP tests should be used together, and when both tests are positive, PJI must be considered with a positive likelihood ratio of 4.3/12.1. When both tests are negative, PJI is unlikely with a negative likelihood ratio of 0/0.06. However, ESR and CRP are elevated in some other diseases such as inflammatory conditions or infections in locations other than the prosthesis. They also remain elevated for 3–8 weeks after an arthroplasty [17–19]. This decreases the reliability of both markers as an indicator of infection remission after the first stage of a two-stage exchange. The threshold for ESR and CRP is not clear for reimplantation, but it is said that after the first stage, the trend should be downward. Cessation of antibiotics should occur before the second step, but values may not necessarily return to normal.

The reliable threshold for ESR is similar for PJI diagnosis in hips and knees. However, the optimal threshold for CRP is reported to be different for hips and knees. Moreover, different thresholds should be implemented for CRP in the early postoperative and late chronic PJI settings, and while ESR thresholds are probably similar in both conditions [19], CRP has been found to be more reliable in the diagnosis of PJI compared to ESR [20].

Although the threshold for ESR and CRP may change according to many factors such as age, sex, and medical comorbidities of the patient as well as measuring method, the MSIS suggests that a serum ESR of greater than 30 mm/h and a CRP greater than 10 mg/L represent elevated levels [1]. However, this threshold value is revised by the International Consensus Meeting and accepted as only the cutoff value for chronic cases. For acute cases the ESR is not found useful and the threshold should be >100 mg/L [2].