Correct identification of the causative agent from microbiological culture is mandatory for targeted antimicrobial therapy of PPI. However, there is currently no consensus on several preanalytical and analytical aspects such as the most appropriate tissue sample to be cultured, the optimal number of samples studied, the most efficient tissue processing method and finally, media. suitable sensitive culture media that also allow detection of fastidious pathogens. We have already published research results on the latter [2, 3]. In this study, we sought to answer open questions in our collective of patients.
First, we recorded the origin of each tissue sample and its contribution to the detection of infection (Fig. 1). In 25 of the 34 culture-positive cases, samples from the neosynovium were the single best positive location. The value in particular of synovial biopsy in the diagnosis of both hip and knee PIP has also been reported by Fink et al. [7, 8].
Our study specifications for locations and volume are based on an evaluation of several thousand tissue samples that we have analyzed over the past few years (unpublished).
One result of this investigation was that bone biopsies as a whole were found to be less appropriate. This experience is confirmed by Larsen et al. who investigated, among other issues, the contribution of specimen types in the diagnosis of PJI [9].
Second, in our study, four tissue samples were sufficient to confirm the diagnosis of infection. These results are consistent with reports by Bemer et al. and Gandhi et al., who both demonstrated that four samples are optimal, if at least three different media including blood culture bottles (BCB) are used [10, 11]. We agree with the importance of growing media, however it is not the number but the composition of the nutrients that counts. We refer to our publications on this subject, in particular with regard to the limited use of the BCB [12].
Third, although patient samples were sent consecutively regardless of symptom progression, we were able to identify the pathogens of a series of chronic infections that caused patients to live with pain related to prosthesis for an average of 11 (5 –25) months before surgery. Our treatment has thus enabled effective targeted therapy for these cases. This can be taken as an indication of the validity of both procedures, as smaller amounts of bacteria are generally expected with these types of infection.
Moreover, in the AF group, neither procedure resulted in contamination, and therefore they provided an optimal specific result.
Literature on treatment methods is scarce. In 2017, Suren et al. published a prospective analysis of a semi-automated method of tissue homogenization using the ULTRA-TURRAX training workstation with tubes containing ten steel balls [13]. The authors studied 38 total hip and total knee replacements, but their results were inconsistent and no information was given about their routine procedures. Roux et al. published a retrospective analysis in 2011 [14] which included 92 patients undergoing revision surgery. Tissue samples were taken between 2003 and 2006 and examined using vials with added glass beads. The authors found a substantial number of microorganisms associated with PJI, but again they did not compare this data with their routine workflow. Redanz et al. first used an experimental model with an artificially inoculated pig specimen to study the efficiency of the Precellys Evolution ball mill homogenizer. The authors then processed clinical samples using 2 ml tubes and analyzed 22 tissue samples of periprosthetic membranes and synovium recovered from seven patients. Only five samples were positive. Despite this limited amount of data, the authors gave a clear recommendation for the procedure [15]. It should be noted that according to the manufacturer, a load capacity of up to 0.2 g of weight is recommended for 2 ml tubes. In our experience, this volume is too low for a reliable diagnosis, especially if low-grade infections are suspected. In our study, about 90% of the samples had at least 5 to 10 times this weight. It is only recently that Fang et al. demonstrated the superiority of tissue homogenization for the diagnosis of PPI, but for comparison they used methods that have previously been shown to be non-competitive, such as manual techniques or pretreatment of the tissue with ultrasound [16]. Finally, Yusuf et al. evaluated the diagnostic value of tissue samples pretreated with a homogenizer compared to their routine manual procedures. Surprisingly, the authors found no significant difference between the methods. Speculation remains as to whether the selected program was unsuitable for processing these special tissue samples. The authors also did not provide information on the extent to which they carried out preliminary tests and why they selected the program mentioned in the Sect material. [17].
Although we have demonstrated the accuracy of two homogenization techniques, our study has certain limitations. First, we accepted the bias that surgeons could assign the samples themselves, since transfer to another vial in the laboratory, even under laminar airflow, poses a risk of contamination. This freedom of choice could be the reason why, in seven cases, processing under routine conditions revealed additional positive samples compared to processing by the bead crushing method. But even with this method, additional positive samples were found in four cases. However, these differences had no effect on the overall rating. Second, there is no gold standard for processing procedures, which makes investigations very laborious, as all individual steps must be carefully validated. In our study, we first had to determine which bead material (steel, glass or ceramic) was best suited to our needs. Next, we had to identify both the right mix of bead sizes for better homogenization and the right rotation speed without affecting the bacteria. Based on preliminary tests, we opted for ceramic balls. The best ratio of homogenization and bacteria recovery was achieved at 7200 rpm using a 2.8/5.0 mm bead mix. However, at ≥ 8000 rpm, the temperature inside the sample rose to 60°C and impaired bacterial growth.
Another aspect that has not yet been systematically studied is the recording of the volume of tissue samples examined. [4]. Our monitoring showed a dependence between volume and pathogen detection rate, but no difference in the methods used. However, if the weight was
Regardless of this finding, we are working on semi-quantitative PCR assays to allow better integration of the informative power of molecular examination procedures in the diagnosis in case of unexpected negative culture results (not completed).
