Diagnosis of prosthetic joint infections using UMD-Universal Kit and the automated multiplex-PCR Unyvero i60 ITI^® cartridge system: a pilot study

Over the past decade there has been a significant increase in the number of artificial joint replacements worldwide and especially in Germany. Prosthetic joint infections (PJI) represent a serious complication in the era of artificial body implants. PJI is a condition with high morbidity and costly economic side effects for endoprosthetic departments [1, 2]. The complex pathophysiology of PJI is closely related to the unique microbiological properties of biofilms, generated by microorganisms on artificial materials [3]. Biofilm-forming bacteria often remain in a dormant state complicating conventional diagnostic and therapeutic approaches [4]. Recently updated international guidelines on the management of PJI recommend the culture of 3–6 periprosthetic tissue samples as the diagnostic procedure of choice [5]. Various efforts have been made to improve the diagnostic tools for PJI over the past years. Culture samples obtained by sonication of removed artificial material were shown to be more sensitive than conventional tissue culture (78.5 vs. 60.8 %.), especially in patients who were exposed to antiinfective treatment 14 days before surgery (45 vs. 75 %)—the overall specificities were above 98 % [6, 7]. Only few studies have assessed the diagnostic utility of nucleic acid amplification test (NAAT) techniques in this context. The reported findings for different PJI-associated materials indicate a wide range of sensitivity (50–92 %) and specificity (65–94 %) [8‐13]. Results from a recent study comparing 16S rRNA gene PCR and consecutive sequencing of sonication fluid with culture of synovial aspirates, tissue samples and sonication fluids reports that 16S rRNA gene PCR improved sensitivity [14]. An up-to-date large, prospective multicenter trial on 16S rRNA gene PCR applied to periprosthetic tissue samples showed a lack of sensitivity of this broad-spectrum molecular method (sensitivity 73.3 % and specificity 95.5 %) [8]. Almost no data are available on the role of multiplex-PCR assays for periprosthetic tissue samples. Using multiplex-PCR of sonication fluid, sensitivity (96 %) and specificity (100 %) for diagnosing PJI could be further improved and might help to differentiate between the clinical conditions of aseptic failure and PJI [15, 16]. However, the sophisticated infrastructure to obtain and process sonication fluids is not widely accessible, especially for endoprosthetic departments with external microbiological laboratories. To overcome these limitations, automated and easily operable multiplex-PCR systems are being developed, which provide rapid PCR results from periprosthetic tissue and bone samples regarding pathogen identification and genotypic resistance patterns. Here we report on our clinical experience comparing the automated multiplex-PCR Unyvero i60 ITI^® cartridge (U-ITI) system with a 16S rRNA PCR assay and conventional culture in diagnosing PJI.

Samples and clinical information were obtained from 68 patients of whom 14 were excluded because of insufficient clinical information or inappropriate pre-analytical tissue processing (Table 1). A total of 54 patients were included with either septic or aseptic prosthetic joint exchanges or revisions. Conventional culture detected the growth of possible pathogens in 16 patients. Considering the number of culture-positive samples per patient and the pathogenicity of the microorganism, seven patients were classified as culture positive (Table 2). The remainder of nine cases was interpreted as contamination of the conventional culture on the basis of the above mentioned criteria and finally assessed culture-negative (Table 1, 3). Four patients met the criteria for culture positivity regarding coagulase-negative staphylococci: two patients with Staphylococcus lugdunensis and one patient each with Staphylococcus capitis and Staphylococcus epidermidis, respectively. Staphylococcus aureus, Pseudomonas aeruginosa and Enterococcus faecalis were identified by culture and considered true positive in one patient each.

Broad-spectrum 16S rRNA gene real-time PCR was carried out for all samples of the 54 patients. Sequence analysis was successful with species identification in 30 cases (Table 2). Twenty-nine microorganisms identified by 16S rRNA gene sequencing in 23 cases were considered contaminants according to the above mentioned criteria and finally assessed 16S rRNA gene real-time PCR-negative (Table 3). Contaminating DNA originated from Staphylococcus spp. (n = 13), Staphylococcus haemolyticus (n = 2), Staphylococcus epidermidis (n = 1), Staphylococcus hominis (n = 1), Pseudomonas pseudoalcaligenes (n = 6), Comamonas spp. (n = 5) and Pseudomonas spp. (n = 1). Only seven cases met the criteria of the identical pathogen-related sequences in at least two samples. All internal and positive PCR controls yielded the expected results.

In 96 % of all cases (52/54) there was agreement between the results from 16S rRNA gene real-time PCR and conventional culture (Table 3). Six of the seven culture-positive cases gave concordant positive results in the 16S rRNA gene real-time PCR assay. Forty-six of 47 culture-negative samples were also estimated negative by 16S rRNA gene real-time PCR assay and categorized as concordant negative. The one 16S rRNA gene real-time PCR-positive/culture-negative sample (F-035) was interpreted as clinically true positive on the basis of prior culture results and antimicrobial treatment of the patient. In one culture-positive case with growth of Staphylococcus capitis, 16S rRNA gene real-time PCR could not identify the pathogen and was classified as false-negative.

The Unyvero i60 ITI cartridge (U-ITI) was applied to 79 samples of 28 patients (Table 3). In 23 cases three tissue samples and in five cases two samples were processed. In the five cases with only two tissue specimens investigated, the U-ITI result was concordant with culture findings. The overall degree of agreement between U-ITI and culture results in the 28 cases was 82 % (23/28). U-ITI identified 3 of 7 culture-positive cases revealing S. aureus, P. aeruginosa and E. faecalis. Results of the U-ITI molecular resistance typing function in comparison with conventional antimicrobial susceptibility testing are shown in Supplement Table 1. Of the 21 culture-negative samples analysed with U-ITI, 20 gave concordant negative results, including the single 16S rRNA gene real-time PCR-positive/culture-negative specimen (F-035). In this particular case S. aureus was detected by 16S rRNA gene real-time PCR. In one of 21 culture-negative samples U-ITI identified Proteus spp.-DNA (F-043). This finding was clinically assessed as false-positive, and furthermore it was not confirmed by broad-spectrum 16S rRNA gene real-time PCR.

The current gold standard in diagnosing PJI is the conventional culture of periprosthetic tissue samples [19]. However, culture has limited sensitivity especially due to prior antimicrobial use, and its specificity is impaired by contaminating microorganisms of the skin which are often indistinguishable from true pathogens in this setting. Several technical advances have broadened the diagnostic options in recent years [6, 14‐16]. Here, we report on a pilot study comparing conventional tissue culture with two PCR-based methods using prospectively collected tissue samples from patients with either septic or aseptic prosthetic joint replacements or revisions. Overall, we observed an agreement of the U-ITI cartridge results with culture results in about 82 % of all cases. 16S rRNA gene real-time PCR results were in line with the conventional culture results in about 96 % of the studied patient cases. These findings seem to be consistent with previously published data on NAAT techniques, indicating a relatively wide range of sensitivity and specificity [8‐12]. NAATs may reasonably expand the diagnostic panel and contribute to a higher recovery rate especially after antimicrobial use.

However, each of the PCR-based methods has its specific drawbacks. Broad-range PCR is a very sensitive method and, thus, prone to false-positive results from contaminating DNA from different sources [20]. Attempts have been made to reduce the false-positive rates by introducing a detection threshold [21] but this may diminish the sensitivity of the test. In our study, 29 of 162 16S rRNA gene real-time PCR sequencing results, or 23 of 54 patient cases, were classified as contaminated especially by nonfermenters such as Comamonas spp. or Pseudomonas spp. and coagulase-negative staphylococci of the skin. As it is not advised by the manufacturer we did not define a detection limit for 16S rRNA gene real-time PCR. To overcome the contamination problem and as done in literature before [8], an organism identified by 16S rRNA gene real-time PCR was reported as true pathogen when at least two of the three samples investigated showed the same pathogen-related sequence. At this stage, the 16S rRNA gene real-time PCR assay is labour-intensive regarding its DNA extraction procedure, which limits the introduction of this method into the clinical routine.

In contrast to the 16S rRNA gene real-time PCR assay, the U-ITI cartridge system is rapid and easy to operate. The test has an integrated thresholding algorithm. Amplification results reaching an arbitrary cut-off of 250 units are considered positive. Such a threshold can reduce false-positive results due to contamination and therefore might prevent unnecessary antimicrobial therapy. However, this approach poses the risk of false-negative results. In this pilot study we opted to report every single positive result as significant as recommended by the manufacturer. In one culture-negative, antibiotic pretreated and clinically infected case, the U-ITI cartridge amplified Enterobacter cloacae DNA at 198 units which is just below the defined threshold. The clinical significance of this result remained unclear. Compared to broad-range PCR assays, the U-ITI is limited to a fixed panel of pathogens. The U-ITI cartridge includes 94 primers (50 for bacterial or Candida species, and 42 resistance markers) and even though the test covers important gram-positive and gram-negative pathogens, relevant species such as Staphylococcus lugdunensis have not yet been included as a single primer in the latest version. In our study there were two culture-positive cases with Staphylococcus lugdunensis, a clinically important pathogen causing various infections including joint and prosthetic joint infection [22‐24]. U-ITI might benefit from the inclusion of additional organisms into the panel.

Our pilot study has several limitations. First, the overall number and the number of culture-positive patients included is relatively low; therefore, the agreement rate of 82 % is mainly based on concordant negative results. Larger studies with higher number of cases are certainly desirable to determine sensitivity and specificity, but difficult to conduct and, therefore, even smaller sample size studies may give important insights. Second, our “first-experience” trial did not assess the influence of the NAAT results on the management of PJI. The only 4 U-ITI positive patient cases did not permit conclusions regarding the U-ITI molecular resistance typing function. We hypothesize that the automated U-ITI cartridge may facilitate the management of PJI, especially in countries with a high prevalence of multi-drug resistant organisms and may be appropriate to detect polymicrobial PJIs. UMD-Universal assay and the U-ITI cartridge are new complementary technologies in the diagnostic repertoire for PJI.

In conclusion, this pilot study gave no indication of superiority of the used NAAT over conventional culture methods for the diagnosis of PJI. Prospective and controlled trials will be necessary to evaluate the usefulness and the impact of NAATs on the clinical management of PJI.