Microbiologic analysis of 140 patients undergoing revision total knee arthroplasty due to periprosthetic joint infection and aseptic loosening: droplet digital PCR analysis

Abstract

Objective

The primary reasons for postoperative joint prosthesis revision after total knee arthroplasty (TKA) are periprosthetic aseptic prosthesis loosening and joint infection (PJI). The objective of this study is to analyze the predominant microbial species responsible for PJI while also discussing was related.

Methods

A retrospective analysis was conducted on patients who underwent TKA revision surgery for the treatment of PJI and aseptic loosening of joint prostheses at our institution. Patients with aseptic loosening of the prosthesis are group A (n = 68), and patients with PJI are group B (n = 72). The fluid obtained after ultrasonic treatment was then subjected to microbial culture. Droplet Digital PCR (ddPCR) analysis was employed for DNA detection.

Results

In our study, we included a total of 140 patients divided into groups A (patients with aseptic loosening of the prosthesis) and B (patients with PJI). The microbial culture results showed that 30 patients (45.45%) had Staphylococcus aureus infections, and 24 patients (36.36%) had methicillin-resistant Staphylococcus aureus (MRSA) infections in group B. The DNA testing results revealed that 54.41% (n = 37) of Group A patients still had microbial infections, including both Gram-positive and Gram-negative species. Notably, these patients showed negative microbial cultures but positive DNA detection. Among them, 83.78% (n = 31) had Gram-negative infections, while 16.22% (n = 6) had Gram-positive infections. In Group B, 66 patients (91.67%) had predominantly Gram-positive infections.

Conclusion

This study innovatively employed ddPCR for DNA detection and microbiologic analysis in 140 patients undergoing revision total knee arthroplasty due to periprosthetic joint infection and aseptic loosening, showed that most patients with PJI mainly affected by infections caused by high-virulence Gram-positive microbe, especially Staphylococcus aureus and MRSA. On the other hand, patients with aseptic loosening of the prosthesis may be associated with a history of multiple low-virulence microbial infections, primarily involving Gram-negative bacteria.

Background

Total knee arthroplasty (TKA) is a standard orthopedic procedure used to relieve pain and restore mobility in patients with end-stage joint diseases such as osteoarthritis and rheumatoid arthritis (RA) [1, 2]. Research shows that the incidence rate of TKA will continue to rise year by year, posing a huge challenge to the future health care system [3, 4]. While the majority of patients report satisfaction after total knee arthroplasty, existing evidence suggests that approximately 10–20% may achieve poor results [5]. The leading causes of TKA failure are periprosthetic joint infection (PJI) and aseptic prosthesis loosening [6, 7]. It is worth noting that the in-hospital mortality rate of PJI is about 3.5%, and in cases with concomitant systemic inflammatory response syndrome (SIRS) or septic shock, the in-hospital mortality rate sharply increases, highlighting the necessity of timely diagnosis and treatment [8, 9]. Periprosthetic bone destruction is their primary mechanism for TKA failure. Inflammatory osteolysis after uncemented TKA can be induced by microbial products or implant-derived wear particles, which activate immune cells to produce pro-osteoclastogenic cytokines that enhance osteoclast recruitment and activity adjacent to and surrounding the bone-implant interface, leading to localized bone destruction [10–12]. The distinction between PJI and aseptic failure in orthopedic practice is crucial to making preoperative treatment decisions. Still, it can be clinically challenging due to the low virulence and biofilm-forming ability of the pathogens [13, 14]. The diagnostic criteria for prosthetic joint infections follow the ICM (International Consensus Meeting) standard, which comprehensively evaluates clinical manifestations, laboratory tests, and microbiological evidence, providing a more standardized and reliable diagnostic framework compared to previous criteria [15]. If a patient diagnosed clinically with aseptic loosening of the prosthesis has a history of low virulence microbial infection, all of these indicators may be negative.

Routine cultures of periprosthetic tissue and synovial fluid are the standard method for microbiologic diagnosis of PJI, but false-negative results occur in up to 30% of cases [16–18]. Although culturing methods are the foundation of pathogen detection in PJI, false-negative results often occur [19, 20]. While DNA testing, though not as accurate as traditional microbial cultures in some aspects, offers much faster results and excels in species-level identification and detection of polymicrobial infections compared to culture studies [21, 22].

This study innovatively incorporates droplet digital PCR (ddPCR), an advanced molecular diagnostic tool that surpasses traditional PCR and microbial culture methods. ddPCR provides absolute quantification of target DNA, even at extremely low concentrations, enabling the detection of low-abundance or fastidious pathogens that conventional methods might overlook. Its ability to simultaneously detect multiple microbial species with high specificity significantly reduces false-negative results, making it particularly valuable for identifying the complex polymicrobial infections often seen in PJI and aseptic loosening scenarios. This approach not only addresses the limitations of existing diagnostic methods but also fills a critical gap in the current literature by offering a more sensitive and accurate diagnostic strategy for these challenging conditions. We hypothesized that the application of ddPCR in the diagnosis of PJI and aseptic loosening of prostheses will significantly improve the detection rate of pathogens, especially low-virulence microbe, and provide more accurate guidance for clinical antibiotic use and treatment decisions [24–26].

Patients and methods

Participants

Patients who underwent revision TKA at our institution due to PJI and aseptic loosening of the articular prosthesis were enrolled in this retrospective study. The included patients were categorized into two groups, A and B. Group A comprised of patients with aseptic loosening of the prosthesis, while group B comprised of patients with PJI.

The study protocol was approved by the Ethical Review Committee (Approval No. QYFY WZLL 28857) and was in accordance with the Declaration of Helsinki. Informed consent was waived because the research exclusively utilized de-identified patient data, which presents no risk of harm or impact on patient care. This waiver was granted in compliance with the regulatory and ethical guidelines relevant to retrospective research studies. The clinical trial registration number of the study was NCT06716883.

Eligibility criteria

The diagnosis of PJI ​​strictly adhered​​ to the ICM standard, which comprehensively evaluates multiple aspects. (1)Clinical Manifestations: Presence of local symptoms including redness, swelling, pain, and fistula formation, along with systemic signs such as fever. (2) Laboratory Diagnosis: Elevated levels of C - reactive protein (CRP), erythrocyte sedimentation rate, and white blood cell count. (3) Microbiological Criteria: Positive results from tissue sample cultures (usually 1–3 samples). In cases where cultures were negative, the presence of purulent fluid, positive histological examination, or positive molecular diagnostic evidence (such as positive DNA testing) could also meet the requirement. Only patients who meet al.l the above - mentioned diagnostic criteria defined by the ICM standard were included in the study [15].

For Patients with Aseptic Loosening: (1) Clinical Symptoms: Experienced of unexplained pain, recurrent dislocation, stiffness, and premature joint failure. (2) Imaging Evaluations: Conventional radiography was used as the initial imaging method. Depending on the clinical situation and disease progression, computed tomography and nuclear medicine imaging were carried out to exclude infection and accurately assess the degree of loosening [23, 27].

Patients with PJI were excluded if they had infections not related to PJI such as systemic infections or infections at other anatomical sites, ensuring the study focuses solely on PJI. Patients who had undergone previous revision surgeries and those with significant concurrent medical conditions that could affect the diagnosis or treatment of PJI, such as severe immunodeficiency, malignancy, or chronic inflammatory diseases, were also excluded. Furthermore, patients whose tissue samples or ultrasonically treated fluids were of insufficient quality for reliable DNA testing or microbial cultures, were excluded. For patients with aseptic loosening, exclusion criteria included having infections not related to aseptic loosening, incomplete imaging evaluations such as missing conventional radiography or advanced imaging studies like computed tomography and nuclear medicine imaging, and significant concurrent medical conditions that could affect the assessment of aseptic loosening (Fig. 1).

Fig. 1.

Flow chat

Laboratory processing and droplet digital PCR (ddPCR) analysis

Previous studies have demonstrated that the microbial culture of the ultrasonically treated solution from the exoskeleton is more sensitive in detecting PJI compared to culturing periprosthetic tissue [25, 28].Therefore, prosthesis samples taken during revision surgery were carefully placed in sterile plastic containers and transported to the laboratory. One hundred milliliters of Ringer’s solution was added to each container. The containers were then vortexed for 30 s using a Vortex-Genie vortexer and sonicated in an ultrasound bath (40 ± 2 kHz; power density 0.22 ± 0.04 W/cm2, supplied by a calibrated hydrophone; J.P. Selectra, Spain) for 5 min, followed by vortexing for another 30 s. We inoculated the sonicated extracts onto aerobic and anaerobic sheep blood agar plates (0.5 ml of liquid) and incubated them in the same way as for tissue culture [29]. Similarly, we used the sonicated solution obtained by the above method for DNA detection.

DdPCR analysis was employed for DNA detection in this study. The sonicated solution (1 Ml) was subjected to DNA extraction using the QIAamp DNA Mini Kit (Qiagen, Germany) following the manufacturer’s instructions. The extracted DNA was eluted in 50 µL of AE buffer and quantified using a NanoDrop spectrophotometer (Thermo Fisher Scientific). The DNA concentration was adjusted to a working concentration suitable for ddPCR. The ddPCR assays were performed using the QX200 Droplet Digital PCR System (Bio-Rad Laboratories, USA). For each target microbe, a 20 µL reaction mix was prepared. The 20 µL reaction mix was loaded into droplet generator cartridges, and droplets were generated using the QX200 Droplet Generator. The resulting droplets were transferred to a 96-well PCR plate and sealed with pierceable foil seals. Thermal cycling was performed on a C1000 Touch Thermal Cycler (Bio-Rad). After amplification, the droplets were read using the QX200 Droplet Reader. Positive and negative droplets were counted, and the presence or absence of target DNA was determined using QuantaSoft software (Bio-Rad). Each sample was run in duplicate, and a no-template control (NTC) was included in each run to monitor for contamination. The results were categorized as positive or negative for each microbial target based on the detection of target DNA in the droplets.

Assessment of knee symptoms

The Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) was utilized to evaluate knee symptoms in patients. This index comprises three subscales: Pain, Stiffness, and Function. Higher scores on these subscales indicate more severe symptomatology. The internal consistency, as measured by Cronbach’s alpha, for the three WOMAC subscales was 0.84 for Pain, 0.86 for Stiffness, and 0.96 for Function [30].

Assessment of life quality

The quality of life (QOL) of patients was assessed using the Medical Outcomes Study 36-Item Short Form Health Survey (SF-36). This survey evaluates eight health-related domains: Physical Functioning (PF), Role-Physical (RP), Bodily Pain (BP), General Health (GH), Vitality (VT), Social Functioning (SF), Role-Emotional (RE), and Mental Health (MH). Each domain score ranges from 0 to 100, with higher scores indicating better QOL. The Cronbach’s alpha was 0.700 [31].

Statistical analysis

Patient information, including age, gender, prosthesis side, microbial testing results, surgical details, WOMAC scores and SF-36 scores. was collected through the case management system. Variations between groups were computed using a t-test for continuous variables and chi-square tests for categorical variables with the Statistical Package for Social Sciences (SPSS 27, IBM Corp, Armonk, NY, USA). The significance level was determined as p < 0.05. In this study, we did not perform any corrections for multiple comparisons since no such analyses were conducted. Additionally, there were no missing data points in our dataset; all patient records were complete and included in the analysis. Furthermore, we examined the outcomes of DNA testing and microbial cultures of ultrasonically treated fluids from articular prostheses to argue our point of view through the microbial species obtained and their percentage.

Result

Clinical data

In our study, we included a total of 140 patients who underwent revision surgery after arthroplasty. Among them, 68 patients had aseptic loosening of the articular prosthesis and were assigned to group A, while 72 patients had PJI and were assigned to group B. Detailed statistics regarding baseline patient information, DNA testing, and microbial culture results are provided in Table 1. Based on our analysis, there was no significant difference in the baseline data between the two groups of patients (p > 0.05) (Table 1).

Table 1.

Comparison of preoperative baseline data among the two groups of patients

Classification		Group A(n = 68)	Group B(n = 72)	t/χ2	P
Age (years, ±s)		64.42 ± 3.56	63.53 ± 3.01	1.603	0.111
Gender (n (%))	Male	37 (54.41%)	30 (41.67%)	2.276	0.131
	Female	31 (45.59%)	42 (58.33%)
Surgical side (n (%))	Left	44 (64.71%)	41 (56.94%)	0.883	0.347
	Right	24 (35.29%)	31 (43.06%)

Laboratory data

The results of the study revealed that microbial cultures were negative in patients clinically diagnosed with aseptic loosening during prosthetic revision (Table 2). However, DNA testing indicated that 54.41% (n = 37) of these patients with aseptic loosening still had microbial infections, including both Gram-positive and Gram-negative species. Among them, 83.78% (n = 31) had Gram-negative infections, while 16.22% (n = 6) had Gram-positive infections. It is noteworthy that all of these patients had multiple microbial infections, and the types and percentages of these infections are presented in Table 3. Our focus is on the six microbial strains with the highest levels, namely Escherichia coli, Streptococcus sanguinis, Sphingomonas melonis, Comamonas aquatica, Mitsuaria chitosanitabida, and Acidovorax delafieldii, all of which are considered low virulence strains.

Table 2.

The results of microbial testing of arthroplasty implants in two groups of patients

Microbial testing		Group A(n = 68)	Group B(n = 72)
DNA detecting results	Gram-positive microbes (G+)	6	66
	Gram-positive microbes (G+)	31	6
	Negative results	31	0
Microbes culture results	Gram-positive microbes (G+)	0	60
	Gram-negative microbes (G-)	0	6
	Negative results	68	6

Table 3.

Positive DNA test results for group A [n (%)]

Microbes	PCR Positivity Count
Escherichia coli	25 (67.57%)
Sphingomonas melonis	12 (32.43%)
Mitsuaria chitosanitabida	12 (32.43%)
Streptococcus sanguinis	6 (16.22%)
Comamonas aquatica	6 (16.22%)
Acidovorax delafieldii	6 (16.22%)

In the investigation of patients with PJI, only 6 out of 72 patients had negative microbial culture results (Table 2). Among the remaining patients, 90.9% of the infections were caused by Gram-positive cocci, while only 9.1% were caused by Gram-negative bacteria. Staphylococcus aureus, MRSA, Serratia marcescens and Streptococcus mutans accounted for 45.45%, 36.36%, 9.09% and 9.09% of the infections, respectively. The strains detected in the DNA test were consistent with the dominant strains identified through microbial cultures. Table 4 lists the types and frequencies of microbial infections identified from positive results of microbial culture and DNA testing.

Table 4.

DNA testing and microbial culture results for group B (Positive results) [n (%)]

Microbes	Culture Positivity Count	PCR Positivity Count
Staphylococcus aureus (G +)	30 (45.45%)	36 (50.00%)
MRSA (G +)	24 (36.36%)	24 (33.33%)
Serratia marcescens (G -)	6 (9.09%)	6 (8.33%)
Streptococcus mutans (G +)	6 (9.09%)	6 (8.33%)

Surgical details

In the comparison of surgical details between Group A and Group B, no significant differences were observed in any of the measured parameters (Table 5). Surgical time was similar between the two groups (P = 0.466). Blood loss during surgery and postoperative drainage volume showed no significant difference either (all P > 0.05).

Table 5.

Comparison of surgical details among the two groups of patients

Classification	Group A(n = 68)	Group B(n = 72)	t	P
Surgical Time (min)	74.25 ± 8.54	73.26 ± 7.37	0.732	0.466
Blood Loss (ml)	71.33 ± 7.49	70.08 ± 8.67	0.917	0.361
Postoperative Drainage Volume (ml)	335.87 ± 42.33	332.58 ± 40.68	0.470	0.639

Western Ontario and McMaster universities osteoarthritis index

In the comparison of the WOMAC scores at baseline between Group A and Group B, no significant differences were observed in any of the subscales (Fig. 2). The Pain Subscale scores were similar between the two groups (Group A: 15.35 ± 1.26, Group B: 14.85 ± 2.18; t = 1.699, P = 0.092). The Stiffness Subscale scores also did not differ significantly (Group A: 6.64 ± 2.18, Group B: 7.05 ± 1.76; t = 1.238, P = 0.218). Similarly, the Function Subscale scores showed no significant difference (Group A: 37.96 ± 3.52, Group B: 38.67 ± 4.87; t = 0.992, P = 0.323).

Fig. 2.

Comparison of WOMAC score among the two groups of patients (Baseline). (A) Pain Subscale; (B) Stiffness Subscale; (C) Function Subscale. WOMAC: Western Ontario and McMaster Universities Osteoarthritis Index

ns: no statistically significant difference

After TKA, we still observed no significant differences in the WOMAC subscales (Fig. 3). The Pain Subscale scores were (Group A: 6.22 ± 1.54, Group B: 6.45 ± 1.53; t = 0.864, P = 0.389), the Stiffness Subscale scores did not differ significantly (Group A: 3.25 ± 1.04, Group B: 2.98 ± 0.57; t = 1.861, P = 0.066), and the Function Subscale scores showed no significant difference (Group A: 7.56 ± 1.57, Group B: 7.45 ± 1.56; t = 0.440, P = 0.660). These results suggest that, in terms of knee symptoms evaluated by the WOMAC index, there were no remarkable differences between patients with aseptic loosening and those with PJI at both the preoperative and postoperative stages.

Fig. 3.

Comparison of WOMAC among the two groups of patients (After TKA). (A) Pain Subscale; (B) Stiffness Subscale; (C) Function Subscale

ns: no statistically significant difference

Quality of life

In the comparison of the SF-36 scores between Group A and Group B, no significant differences were observed in any of the eight subscales (Table 6). The PF scores were similar between the groups (Group A: 51.24 ± 22.17, Group B: 51.87 ± 22.12; t = 0.171, P = 0.865). The RP scores also did not differ significantly (Group A: 19.67 ± 6.47, Group B: 19.87 ± 7.98; t = 0.167, P = 0.868). The BP scores showed no significant difference (Group A: 60.07 ± 23.96, Group B: 62.08 ± 23.56; t = 0.501, P = 0.617). GH scores were also comparable (Group A: 53.29 ± 19.54, Group B: 53.25 ± 19.87; t = 0.012, P = 0.991). VT scores did not differ significantly (Group A: 45.77 ± 11.75, Group B: 45.75 ± 14.37; t = 0.012, P = 0.990). SF scores were similar (Group A: 60.87 ± 23.55, Group B: 61.88 ± 26.45; t = 0.238, P = 0.812). RE scores showed no significant difference (Group A: 66.23 ± 20.78, Group B: 66.34 ± 17.65; t = 0.033, P = 0.973). Finally, MH scores were also comparable (Group A: 67.96 ± 15.36, Group B: 67.93 ± 16.44; t = 0.009, P = 0.993).

Table 6.

Comparison of SF-36 score among the two groups of patients

Classification	Group A(n = 68)	Group B(n = 72)	t	P
PF	51.24 ± 22.17	51.87 ± 22.12	0.171	0.865
RP	19.67 ± 6.47	19.87 ± 7.98	0.167	0.868
BP	60.07 ± 23.96	62.08 ± 23.56	0.501	0.617
GH	53.29 ± 19.54	53.25 ± 19.87	0.012	0.991
VT	45.77 ± 11.75	45.75 ± 14.37	0.012	0.990
SF	60.87 ± 23.55	61.88 ± 26.45	0.238	0.812
RE	66.23 ± 20.78	66.34 ± 17.65	0.033	0.973
MH	67.96 ± 15.36	67.93 ± 16.44	0.009	0.993

SF-36: the MOS item short form health survey; PF: Physical Functioning; RP: Role-Physical; BP: Bodily Pain; GH: General Health; VT: Vitality; SF: Social Functioning; RE: Role-Emotional; MH: Mental Health

Discussion

Osteoarthritis of knee is extremely common in the growing elderly population [32]. The use of TKA is increasing significantly, and with it, the number of patients with postoperative implant failure requiring arthroplasty revision, with PJI and aseptic loosening of the prosthesis being the most common causes of arthroplasty revision [32].

In this study, we aimed to investigate the microbiological causes of PJI and aseptic loosening of prostheses. To do this, we processed the prostheses of patients with these conditions using ultrasonic shock treatment. The fluid obtained after this treatment was then subjected to DNA testing and microbial culture. By analyzing the results of both tests microbiologically, we aimed to identify the specific microbes responsible for these conditions [33–35].

Using microbial species detected by sonication fluid cultures from knee prostheses, we found that the distribution of most common pathogens was similar regardless of the type of arthroplasty. The most common genera of PJI in patients were Gram-positive microbes, with the most common group being coagulase-negative staphylococci, with Staphylococcus aureus and MRSA predominating. The percentage of Staphylococcus aureus infections and MRSA was 45.45% and 36.36%, respectively. This finding is similar to data from others, and our results also support other Gram-positive microbe’s view [32, 36]. In our study, the probability of detecting microbes in microbial culture in patients with PJI was as high as 91.67%, which may be related to our joint prosthesis ultrasonication solution application. Previous studies have shown that the sensitivity and accuracy of both DNA detection and microbial cultures increase after implementing high intensity and low frequency ultrasound waves to articular prostheses [37]. Of course, this could also be related to other influences that we overlook, such as previous antibiotic use [38]. In addition, geographic location may also play a role in microbial differences. A study from six countries on three continents showed that the proportion of the top 10 disease-causing microorganisms varied in each country. These geographic disparities may reflect differences in antibiotic stewardship practices, local pathogen prevalence, or diagnostic protocols, emphasizing the need for region-specific diagnostic algorithms.​​ However, there were no differences in the most common microorganisms in PJI [36].

The presence of low-virulence microbial infections in patients diagnosed with aseptic prosthesis loosening after TKA has not been discussed in much of the literature. Most of the literature suggests that aseptic loosening of joint prostheses is related to the presence of particulate wear debris generated by mechanical interaction between implant components and is caused by chronic inflammation resulting from the activation of resident immune cells in contact with the implant wear debris [39]. However, we performed DNA testing and microbial cultures of ultrasonically processed fluids from articular prostheses of revision patients after TKA who were clinically diagnosed with aseptic loosening of their prostheses. We have come up with new insights by analyzing their results. In our study, 54.41% of patients with aseptic loosening had DNA test results that showed the presence of Gram-positive and Gram-negative microbial infections, of which 83.78% were Gram-negative and 16.22% were Gram-positive. Previous authors have demonstrated that microbial biofilms are recognized and attacked by neutrophils [40]. In addition, in some cases, the biofilm cannot be effectively removed, which may lead to a persistent inflammatory response, resulting in osteoclastogenesis, osteolysis, and consequently implant loosening, which is often thought to be associated with delayed low-grade infections caused primarily by microbes [26, 27]. Since the infected microbes were of low virulence and the microbial culture results showed that the microbe infecting the patients with aseptic loosening were inactivated, the patients did not show any signs of infection. They were, therefore, clinically diagnosed with aseptic loosening of the prosthesis. But it did have a microbial infection, and that microbial infection was probably the cause of its development [41].

Through DNA testing, we found that the majority of patients with PJI had multiple microbial infections, with only a small number of patients having a single strain of infection, which is the same data as others [32]. It may be that many of the microbes detected by DNA are small in proportion or even not at all at pathogenic levels, but there is still some guidance for postoperative coadministration of antibiotics in revision patients with PJI. At the same time, we found that patients with aseptic loosening of articular prostheses in the presence of low-virulence microbial infections had multiple low-virulence microbial infections and did not have a single microbial infection. This may be because a single low-virulence microbe is unable to affect the aseptic loosening of the prosthesis too much, and it is only in the presence of multiple low-virulence microbes that the aseptic loosening of the joint prosthesis is affected [42–44]. By integrating ddPCR into our analysis, we aim to complement the existing microbial culture and DNA testing methods. This combined approach ensures a more comprehensive evaluation of microbial presence, enhancing diagnostic reliability and providing deeper insights into the etiology of both PJI and aseptic loosening [45]. The use of ddPCR thus represents a significant advancement in the microbiological diagnosis of joint prosthesis-related infections, supporting more informed clinical decision-making.

This study contributes to the existing body of literature by employing a novel combination of ddPCR and conventional microbial culture techniques to investigate the microbiological causes of PJI and aseptic loosening in patients undergoing revision TKA. Unlike previous studies that have primarily focused on high-virulence microbe in PJI, our research highlights the potential role of low-virulence microbe in both PJI and aseptic loosening. Specifically, we identified multiple low-virulence microbial infections in patients diagnosed with aseptic loosening, which were previously underexplored in the literature. By demonstrating that these low-virulence microbes, particularly Gram-negative species, may contribute to the development of aseptic loosening, our findings suggest a more nuanced understanding of the etiology of implant failure. Furthermore, the use of ddPCR allowed for a more sensitive and comprehensive detection of microbial presence, thereby enhancing diagnostic reliability and providing deeper insights into the complex interplay between microbial infections and implant integrity. These results not only expand the current knowledge base but also pave the way for future research aimed at optimizing treatment strategies for patients with TKA revisions.

However, this study has several limitations. As a retrospective study, it is prone to selection bias. The clinical characteristics and treatment protocols of the included patients may limit the generalizability of the findings. Additionally, due to the small sample size, many factors were difficult to control. Some low - prevalence pathogens might not have been adequately represented. Thirdly, ​​the inability of ddPCR to distinguish live from dead microbe raises uncertainty about the clinical relevance of detected low-virulence species, particularly in aseptic loosening cases where microbial inactivity was culture-confirmed.​​ Furthermore, not all low-virulence organisms identified by ddPCR may represent true infections, as environmental contamination during prosthesis handling or laboratory processing could contribute to false-positive signals.​ Therefore, future large - scale clinical trials are essential to validate these results and further explore the complex relationship between microbial infections and TKA failure.

Recent advances in the field of PJI diagnostics have introduced novel biomarkers that may enhance diagnostic accuracy and specificity. Traditional serum parameters such as CRP, erythrocyte sedimentation rate (ESR), and white blood cell count are widely used but lack specificity, especially in cases of chronic PJI caused by low-virulence microorganisms [46]. Novel biomarkers, including fibrinogen, D-dimer, interleukin-6 (IL-6), procalcitonin, neutrophil-to-lymphocyte ratio (NLR), and platelet count to mean platelet volume ratio (PC/mPV), have been investigated to improve diagnostic accuracy [18, 47]. Schindler et al. [48] conducted a systematic review identifying fourteen different novel biomarkers, with proteins being the most commonly studied category. Notably, calprotectin demonstrated high sensitivity (98.1%) and specificity (95.7%) when used as a point-of-care (POC) test at a threshold of ≥ 50 mg/mL. Although none of these novel biomarkers outperformed established parameters, they provide promising avenues for further research. Our study supports the integration of these biomarkers, particularly in combination with advanced molecular techniques like ddPCR, to refine diagnostic approaches and improve patient outcomes. Incorporating these novel biomarkers could lead to earlier and more accurate diagnosis, enabling timely and effective treatment strategies for patients undergoing TKA revisions.

Conclusion

This study innovatively employed ddPCR for DNA detection and microbiologic analysis in 140 patients undergoing revision total knee arthroplasty due to periprosthetic joint infection and aseptic loosening,, demonstrating that PJI cases predominantly involved high-virulence Gram-positive pathogens, while aseptic loosening correlated with occult polymicrobial infections dominated by low-virulence Gram-negative species. Based on these findings, surgeons should implement more sensitive diagnostic tools like ddPCR for preoperative screening to detect low-virulence infections, tailor antibiotic prophylaxis to target high-virulence pathogens such as Staphylococcus aureus and MRSA, closely monitor patients with a history of low-virulence infections postoperatively for signs of complications, and incorporate novel biomarkers such as calprotectin into routine diagnostics to improve PJI diagnosis accuracy. These adjustments can enhance diagnostic precision, reduce revision surgeries, and improve patient outcomes.

Author contributions

Xiao-Kai Liu: Conceptualization, Formal analysis, Writing-Original Draft. Heng-Xin Zhao: Conceptualization, Methodology, Investigation. Lan-Feng Ding: Methodology, Formal analysis, Data Curation. Yuan-He Wang: Conceptualization, Formal analysis, Data Curation. Shao-Qi Tian: Investigation, Methodology, Writing-Original Draft.

Funding

None.

Data availability

The datasets used during the present study are available from the corresponding author upon reasonable request.

Declarations

Ethics approval and consent to participate

The study protocol was approved by the Ethical Review Committee (Approval No. QYFY WZLL 28857) and was in accordance with the Declaration of Helsinki. Informed consent was waived because the research exclusively utilized de-identified patient data, which presents no risk of harm or impact on patient care. This waiver was granted in compliance with the regulatory and ethical guidelines relevant to retrospective research studies. The clinical trial registration number of the study was NCT06716883.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.