Joint aspiration in acute cases should be performed before antibiotic treatment is started; however, in the presence of sepsis antibiotic treatment should not be delayed [1]. In chronic cases aspiration can be performed with ultrasound guidance. The aspirate should be sent for culture and sensitivity and WBC count with PMN percentage. This is a well-established method for diagnosis of septic arthritis and PJI [26, 30–34]. The sensitivity of synovial fluid culture is variable and is reported between 12 and 100 % [1–35]. Della Valle et al. [14] reviewed 105 consecutive painful TKAs and concluded that a synovial fluid WBC count of greater than 3,000 was the most precise test in predicting chronic infection with a sensitivity of 100 % and specificity of 98 %. Dry tap is a diagnostic challenge and some authors have suggested saline joint washout [36]. There is significant variation in the cut-off value for the diagnosis of PJI. These variations may be due to the joint involved (hip, knee or shoulder), different definitions of PJI utilized in these studies [37, 38], variance in laboratory results, traumatic aspiration and presence of metal-on-metal reactions [2, 38]. Meermans and Haddad suggested a combination of tissue biopsy and joint aspiration to improve sensitivity and specificity [39]. The work group of the international consensus calculated the thresholds (Table 17.1) with the new definition of PJI and similar laboratory techniques when available. Ghanem et al. [40] showed that a corrective formula (adjusting the synovial WBC for the ratio of red blood cell count in the synovial fluid and blood) can detect false-positive joint aspirations without jeopardizing the diagnosis of periprosthetic infection.

Leucocyte esterase is an enzyme secreted by neutrophils that have been recruited to the site of an infection. The test has been used for diagnosis of urinary tract infections. It is a simple test that can be used in a point-of-care assessment of suspected PJI in theatre [15]. Two studies have shown this to be an accurate marker of joint infection [15, 41]. Aggarwal et al. [42] showed that centrifuging the blood-stained aspirates for 2–3 min helps in using the leucocyte esterase test for diagnosis of PJI. Future studies are focused on using automated readers to better standardize the use of this test.

Specimen culture is considered the gold standard for diagnosing infection. Identification of an organism is not only important in making the diagnosis but also crucially important in planning an appropriate antibiotic regimen [1]. Two positive culture results from phenotypically identical organisms are considered diagnostic for infection. A single positive culture however, whilst suggestive of PJI, can represent a false positive [17, 43, 44] and hence considered a minor criterion. The use of preoperative antibiotics, the number of samples, the sites of sampling, length of time of culturing, use of sonication and when to use culture for acid fast organisms and fungi are discussed below.

Both inflammatory markers are raised after arthroplasty. The peak levels are reached on the third day after hip arthroplasties and on the second day after knee arthroplasties. CRP levels usually dropped to less than 10 mg/L within 21 days after surgery [64]. ESR has limited diagnostic utility in the acute postoperative phase (less than 6 weeks since the most recent surgery) [30]. The cut-off values for CRP and ESR have been studied in total knee arthroplasty (TKA) and total hip replacement (THR) patients [65, 66]. Greidanus et al. [67] studied a group of 151 TKA patients and using the ROC analysis found that the optimal positivity criterion was 22.5 mm/h ESR and 13.5 mg/L for the CRP. Ghanem et al. [68] studied a group of 479 patients who underwent revision THR for diagnosis of infection. Using ROC analysis, they found the predictive cut-off levels of 31 mm/h for ESR and 20.5 mg/L for CRP to have the highest combined sensitivity and specificity. Both studies used the current major criteria (i.e. sinus tract or two positive cultures) for the diagnosis of infection and hence may have overestimated the optima cut-off values for the inflammatory markers. Multiple factors may determine the difference in cut-off points.

It is important to note several limitations with regard to ESR and CRP. The joint operated on is an important factor. It has been suggested that TKA is a more traumatic procedure and therefore induces a higher level of CRP [64–69]. Different values may be produced at different laboratories, and the results of ROC curves must be interpreted in view of different sample sizes and distributions.

The international consensus reached 81 % agreement for the following cut-off levels for acute and chronic infections (Table 17.1) and also agreed that the cut-off values would be the same in the presence of inflammatory arthropathies [31]. It is important to note that failed metal-on-metal bearings may result in a marked variability of the synovial fluid WBC count and differential due to misinterpretation of monocytes as PMNs by automated instruments [70], so for these cases manual counting should be performed.

The number of polymorphonuclear cells per high-power field (MN/HPF) in microscopic examination is another method shown by multiple authors to be useful in making a diagnosis of PJI [13, 71–77]. In a review of 26 studies, Tsaras et al. [13] concluded that intraoperative frozen tissue examination performed well in predicting a diagnosis of culture-positive PJI but had moderate accuracy in ruling out this diagnosis. They also could not find a significant difference in the diagnostic accuracy of frozen section histopathology when the threshold of five or ten PMN/HPF was used. Future research studies could focus on defining the threshold number of PMN/HPF that is most predictive of PJI. Samples obtained by sharp dissection (rather than cautery) should be used to help limit false-positive diagnoses due to thermal artefacts.

The gold standard for diagnosis of PJI is culture of synovial fluid and multiple intraoperative-periprosthetic tissue samples, but standard cultures have limited sensitivity. This is especially problematic in patients who have received antibiotics prior to sampling. Several clinical studies have shown increased sensitivity of cultures after sonication of the prostheses without compromising specificity [2, 54, 55, 78–83]. However, it is a costly and time-consuming procedure that is not justified in presumed aseptic cases and the equipment is not widely available. The recommendation of the international consensus work group is to use sonication only for cases of suspected or proven PJI (based on presentation and other testing) in which preoperative aspiration does not yield positive culture and when antibiotics have been administered within the previous 2 weeks [2, 55].

PCR techniques have been shown to be more sensitive than standard tissue culture for detecting pathogens [2, 53, 79, 84–92]. However, the number of false-positive results [2] and a wide specificity range (0 and 100 % [2, 53, 86–89, 93]) preclude its use as a screening tool (with the types of molecular techniques currently available). Molecular techniques are not as useful as culture in providing information about antibiotic susceptibility of organisms. The cost and availability of this technology limit its broad application and hence it is not considered a standard tool in the workup of PJI. Criteria have been established to aid in distinguishing pathogen from contaminant for culture results. Although Rak et al. [94] suggested that two positive PCR results for the same organism should be used as a criterion for infection (with a sensitivity of 83.2 % and specificity of 100 %), such a criterion has not been established, which is in part responsible for confusion about the reliability of PCR for PJI diagnostics. An advantage of PCR is that it can be used in the detection of organisms with recent antibiotic use [2, 79, 93]. The international consensus work group is not currently recommending PCR as a routine diagnostic test for PJI. In cases with high clinical suspicion but negative cultures or other diagnostic tests, molecular techniques with or without sonication may help identify the unknown pathogens or antibiotic sensitivity for targeting antimicrobial therapies. Table 17.2 shows the sensitivity, specificity, cut-off levels, cost and source/type of different tests.