Abstract
Aims
Preoperative diagnosis is important for revision surgery after prosthetic joint infection (PJI). The purpose of our study was to determine whether reverse transcription-quantitative polymerase chain reaction (RT-qPCR), which is used to detect bacterial ribosomal RNA (rRNA) preoperatively, can reveal PJI in low volumes of aspirated fluid.
Methods
We acquired joint fluid samples (JFSs) by preoperative aspiration from patients who were suspected of having a PJI and failed arthroplasty; patients with preoperative JFS volumes less than 5 ml were enrolled. RNA-based polymerase chain reaction (PCR) and bacterial culture were performed, and diagnostic efficiency was compared between the two methods.According to established Musculoskeletal Infection Society (MSIS) criteria, 21 of the 33 included patients were diagnosed with PJI.
Results
RNA-based PCR exhibited 57.1% sensitivity, 91.7% specificity, 69.7% accuracy, 92.3% positive predictive value, and 55.0% negative predictive value. The corresponding values for culture were 28.6%, 83.3%, 48.5%, 75.0%, and 40.0%, respectively. A significantly higher sensitivity was thus obtained with the PCR method versus the culture method.
Conclusion
In situations in which only a small JFS volume can be acquired, RNA-based PCR analysis increases the utility of preoperative puncture for patients who require revision surgery due to suspected PJI.
Cite this article: Bone Joint Res. 2020;9(5):219–224.
Article focus
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This study surveyed the value of reverse transcription-quantitative polymerase chain reaction (RT-qPCR) in revealing PJI with low volumes of aspirated fluid preoperatively.
Key messages
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The diagnostic efficiency between RNA-based polymerase chain reaction (PCR) and bacterial culture was compared, and a significantly higher sensitivity was obtained with the PCR method versus the culture method.
Strengths and limitations
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In the present study, we analyzed cases in which less than 5 ml of a joint fluid sample (JFS) was obtained preoperatively and evaluated a PCR-based method for preoperative detection that is less dependent on the sample volume and concentration of bacteria.
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The small number of samples (21) used in this study is a limitation.
Introduction
Prosthetic joint infection (PJI) is a serious complication of arthroplasty. The incidence rate of PJI for primary arthroplasty is 0.5% to 3.0%, whereas that following revision surgery is between 4.0% and 6.0%.1,2
Because different treatment strategies are used for patients with suspected PJI (including PJI and aseptic loosening) who require revision surgery, a clear preoperative diagnosis of suspected PJI is ideal. However, imaging methods of preoperative diagnosis have weak sensitivity and low specificity;3 serological inflammatory tests are highly sensitive4 but have low specificity.5,6 Preoperative joint fluid aspiration and culture are indicated if an infection is suspected due to the results of the above tests.7 Therefore, the utility of preoperative aspiration culture has been frequently questioned because of its high false-negative rate, which can be caused by microbe-related factors, e.g. a paucity of bacteria in the joint fluid, highly fastidious bacterial growth, the biofilm nature of the PJI, or the effects of primary antibiotic therapy.8,9 Furthermore, as conventional culture usually requires 3 ml to 5 ml of sample, joint fluid sample (JFS) volumes from preoperative punctures can be insufficient,10 resulting in an even lower positive rate. Accordingly, it is necessary to improve diagnostic performance when using low-volume JFSs from preoperative aspiration.
Detection of 16S ribosomal RNA (rRNA) by polymerase chain reaction (PCR) might increase the detection rate of microorganisms in PJIs with small amounts of sample.11,12 Most methods rely on bacterial DNA detection, which leads to higher sensitivity compared to culture methods.13–15 However, the false-positive rate of DNA-based PCR is higher than that of culture,16 and exogenous bacterial DNA that originates from percutaneous aspiration may account for this issue. Studies have shown that the instability of RNA under natural conditions can reduce the false-positive rate caused by exogenous contamination in simulated joint infections.17 Moreover, clinical studies have reported that the sensitivity of RNA-based PCR is the same as that of bacterial culture while exhibiting a lower false-positive rate.18–20 Regardless, the samples used in these studies were acquired intraoperatively and were greater in volume than those collected preoperatively, which may have enhanced sensitivity. Thus, it remains to be determined whether diagnostic efficacy can be improved by RNA-based PCR detection of bacteria in low volumes of JFSs acquired from preoperative aspiration.
As a volume of 4 ml to 5 ml JFS is usually needed for routine culture,21,22 suspected PJI patients with preoperative aspiration JFS volumes of less than 5 ml were enrolled in this study. The diagnostic efficiencies of RNA-based PCR and bacterial culture using preoperative joint fluid were compared to investigate whether RNA-based real-time quantitative PCR can improve the accuracy of PJI diagnosis relative to bacterial culture when using low volumes of preoperatively aspirated synovial fluid from suspected PJI cases.
Methods
Patient selection and sample collection
The patients included in this study were chosen from those who underwent revision surgery in our hospital due to suspected PJI between January 2014 and December 2016. The inclusion standards were as follows: based on symptoms, medical history, and clinical data, patients suspected of having PJI and who finally received revision surgery. A diagnosis of PJI was made according to Musculoskeletal Infection Society (MSIS) criteria for PJI. One of the following must be met for diagnosis of PJI: a sinus tract communicating with the prosthesis; a pathogen is isolated by culture from two separate tissue or fluid samples obtained from the affected prosthetic joint; four of the following six criteria exist: 1) elevated ESR and CRP (ESR > 30 mm/hour; CRP > 10 mg/l); 2) elevated synovial fluid white blood cell (WBC) count (> 3,000 cells/μl); 3) elevated synovial fluid neutrophil percentage (> 65%); 4) presence of purulence in the affected joint; 5) isolation of a microorganism in one periprosthetic tissue or fluid culture; 6) more than five neutrophils per high-power field in five high-power fields observed from histological analysis of periprosthetic tissue at ×400 magnification. JFS obtained by puncture before revision, with a total sample volume of less than 5 ml and more than 2 ml (the minimum volume for both culture and PCR methods). The exclusion standards were as follows: incomplete laboratory and clinical information available; contaminated specimens or specimens suspected of being contaminated; specimens obtained from patients with infections in other body parts that might influence the accuracy of detection. For each included patient, medical history, demographic characteristics, physical signs, serological inflammatory markers, WBC count and polymorphonuclear (PMN)% of JFS, intraoperative findings, and culture results were recorded. Patients who met the standard described by the MSIS were designated as having PJI.23 All preoperative aspirations and revision surgeries were performed under standard conditions with written informed patient consent. The study was approved by the Ethics Committee of the First Affiliated Hospital of Fujian Medical University, Fuzhou, China (Ethics Number: [2014] 047). According to described guidelines, the preoperative aspiration procedures were conducted in a sterile operating room.24 After draping with a sterile towel, a sterile needle was used to aspirate the joint cavity, without local anaesthesia, to acquire a JFS. The extracted joint fluid from each subject was divided into two parts: 1 ml to 3 ml was used for culture and cell counting; and the residual 0.5 ml to 1 ml was reserved for RNA extraction and detection.
Preoperative culture
Since only the cases with a preoperative sample volume of less than 5 ml were involved in this study, the sample was preferentially injected into a BACTEC Peds Plus bottle (Becton-Dickinson GmbH, Heidelberg, Germany), which requires a smaller volume of joint fluid (1 ml to 3 ml) for culture.25,26 Except for two cases, whose infection was secondary to periodontal disease, anaerobic infection was suspected and the BACTEC Anaerobic/F bottle (Becton-Dickinson GmbH) was used (coincidentally, the amount of joint fluid obtained in these two cases was 5 ml; after PCR, 4 ml was still used for culture). Furthermore, to allow growth of fastidious bacteria, the time for culture was extended from the traditional length of one week to two weeks.27
Preoperative RNA-based PCR
Preoperatively obtained JFS was added to an RNA protection reagent (76163; Qiagen, Valencia, California, USA) immediately after collection and then centrifuged and lysed according to the same method used to obtain DNA. RNeasy Mini Kit (74102; Qiagen) was used to extract total RNA. Prior to RNA elution, the samples were treated with DNase to remove DNA contamination.
In line with the manufacturer’s instructions, 1 µg of total RNA was reverse-transcribed using random primers. After obtaining complementary DNA (cDNA) samples, quantitative polymerase chain reaction (qPCR) was performed in line with standard protocols using a SYBR Green Kit (RR820A; Takara, Dalian, China) in an Applied Biosystems (ABI) 7500 PCR instrument. Briefly, 1 µl of cDNA was combined with 19 µl of reaction mixture containing primers at 0.5 µM and 0.5X SYBR Green mixture. We used the consensus primer pair to amplify the bacterial 16S rRNA gene (primers are listed in Supplementary Table i). The PCR cycling conditions were as follows: 50°C for ten minutes, 95°C for five minutes, and 40 cycles of 95°C for ten seconds and 60°C for 30 seconds. Sterile synovial fluid was used as a negative control. As a positive control, a standard strain of Escherichia coli was added to sterile synovial fluid at a concentration of 1010 CFU/ml. A two-cycle difference from the sterile baseline was considered a detectable positive result. Amplification products were collected into 1.5 ml Eppendorf tubes and prepared for sequencing using the ExoSAP-IT reagent (Affymetrix, Santa Clara, California, USA). Sequencing was performed using the BigDye Terminator method with a 3730xl Genetic analyzer (Applied Biosystems, Foster City, California, USA). The sequences generated were compared against those in the National Center for Biotechnology Information (NCBI) GenBank database.28
Intraoperative culture
Samples were taken at multiple sites of suspected infection, including necrotic and purulent tissues and sites of bone erosion (five samples from at least three sites for each patient). After collection, tissues were ground for one minute in brain-heart infusion broth with a mortar and pestle, and the homogenate was cultured on aerobic and anaerobic sheep-blood agar plates (BD Diagnostic Systems, Heidelberg, Germany). Each intraoperatively removed prosthesis was placed into a sterile box with 200 ml to 400 ml of Ringer’s solution (Caiyou Industry, Shanghai, China), to make sure the prosthesis was totally immersed in liquid. The box was vortex-mixed for 30 seconds, subjected to sonication (40 kHz, 25°C to 37°C) for ten minutes, and then vortex-mixed again for 30 seconds. After that, the sonicate fluid was centrifuged at 3,000 g for ten minutes, the supernatant was discarded, and 10 ml of the sediment was injected into a BACTEC Plus culture bottle (Becton-Dickinson GmbH, Heidelberg, Germany). Joint fluid samples obtained during operation were cultured using the same method for the same incubation time used for preoperative samples. We combined the results of synovial fluid, tissue, and prothesis sonication fluid culture to get a comprehensive positive bacterial culture result.
Statistical analysis
All statistical calculations (sensitivity, specificity, accuracy, positive and negative predictive values (PPV and NPV), positive likelihood ratio (LR+), and confidence intervals (CI)) were performed using SPSS v.23.0 (IBM, Armonk, New York, USA). These values were further evaluated with the McNemar test (sensitivity and specificity) and chi-squared test (accuracy, PPV, and NPV). The statistical significance threshold was set at p = 0.05.
Results
Analysis of included cases
In line with the inclusion standard, 42 patients qualified for our study. Among these, six with incomplete clinical data and three with suspected cases of specimen contamination during transfer were excluded. Therefore, 33 patients were ultimately included in our study. In line with MSIS PJI diagnostic criteria, 21 cases were diagnosed with PJI (72.3% of the included cases), including six males and 15 females aged 49 to 88 years (mean 63.9 years (SD 6.5)); 16 patients underwent hip arthroplasty and five underwent knee arthroplasty. A total of 12 cases were diagnosed as non-PJI, including three males and nine females aged 53 to 77 years (mean 67.6 years (SD 9.3)); nine patients underwent hip arthroplasty and three underwent knee arthroplasty. The causes for the previous arthroplasties are provided in Supplementary Table ii.
Comparison of preoperative aspiration culture and PCR
Analyses of preoperatively aspirated JFSs via culture and PCR showed a sensitivity, specificity, PPV, NPV, accuracy, and LR+ of 28.6%, 83.3%, 75.0%, 40.0%, 48.5%, and 1.71, respectively, for the preoperative culture method and 57.1%, 91.7%, 92.3%, 55.0%, 69.7%, and 6.86, respectively, for the RNA-based PCR method. The sensitivity of the PCR technique was notably superior to that of the culture technique for detecting bacteria from preoperatively aspirated JFS (p < 0.05). However, no significant difference between the two techniques was found for specificity, NPV, PPV, accuracy, or LR+ value (Supplementary Table iii).
Microorganisms detected by culture and PCR
Of the 21 cases diagnosed as PJI, preoperative low-volume JFS culture (all samples < 5 ml) identified six positive cases (28.6% sensitivity) and two false positives (83.3% specificity), whereas intraoperative sample culture identified 17 positive cases (81.0% sensitivity) and no false positives (100% specificity). The sensitivities of both preoperative culture and PCR were significantly lower than that of intraoperative culture (p < 0.05). The type and frequency of infecting organisms in culture and PCR were analyzed, and the results are shown in Supplementary Table iv. Staphylococcus epidermidis was found to be the most common microorganism (Supplementary Table iv). PCR amplicon sequencing provided poor resolution for species-level identification of coagulase-negative staphylococci (CoNS). Five Staphylococcus species, three Staphylococcus aureus species, and two CoNS species were identified by sequencing, confirming that Staphylococcus was the predominate infecting organism. Interestingly, although intraoperative culture showed better diagnostic efficiency than did RNA-based PCR, the latter method did detect bacteria in two false-negative cultures (Supplementary Tables v and vi, cases 3 and 4).
Utility of RNA-based PCR in cases of false diagnosis by culture
We observed several instances of missed diagnosis or misdiagnosis among the preoperative sample cultures, as presented in Supplementary Tables v and vi. For cases 1 and 2, the results of preoperative serological inflammatory tests were above the threshold, the puncture fluid was not purulent, and the total sample volume was approximately 5 ml (4 ml was used for culture and WBC counting; 1 ml was used for RNA-based PCR). The WBC count and PMN% were below the cutoff values in case 1 but above the values in case 2. The results of preoperative culture were negative in both cases. Definite PJI diagnosis was made for cases 1 and 2 by intraoperative culture, which revealed infections with Bacteroides fragilis and E. coli, respectively, and preoperative RNA-based PCR was positive in both cases. For cases 3, 4, and 5, the patients experienced joint swelling and pain after the primary arthroplasty and were treated with antibiotics for three days, one week, and four weeks, respectively, before synovial fluid was obtained. Therefore, although the results for serological inflammatory markers, WBC counts, and PMN% were above threshold values, the results of pre- and intraoperative culture were negative. During each operation, a large amount of pus was found in the joint cavity, the results of intraoperative pathology were positive, and PJI diagnosis was confirmed according to MSIS minor criteria. Preoperative RNA-based PCR results were positive for cases 3 and 4 and negative for case 5. All five patients described above underwent standard two-stage revision, with cement spacers inserted at the first stage. In case 6, PJI was suspected one year after primary arthroplasty due to joint pain and high skin temperature. The results of synovial puncture fluid culture suggested an infection with Bacillus subtilis, but other indicators did not support PJI, including RNA-based PCR. Ultimately, infection was excluded by intraoperative findings, and a one-stage revision was performed for aseptic loosening.
Discussion
A previous meta-analysis of PJI diagnosis in 34 studies indicated that preoperative hip and knee joint fluid culture has 65% to 78% sensitivity and 93% to 97% specificity.9 Unlike these previous studies, we only analyzed cases in which less than 5 ml of a JFS was obtained preoperatively. According to our results, among 21 cases of definite PJI eight positive cases were identified by preoperative joint fluid culture; the cultures revealed a spectrum of bacteria commonly found in musculoskeletal infections, but the sensitivity of this method was not as high as that described in previous studies.29,30 Interestingly, the sensitivity of intraoperative culture (including analysis of tissue, joint fluid, and sonicated fluid) was greatly increased. This discrepancy is likely related to the specimen volume. Positive culture results usually require multiple samples, thus requiring greater volume, especially when diagnosing infections caused by fastidious bacteria.31 Therefore, we evaluated a PCR-based method for preoperative detection that is less dependent on the sample volume and concentration of bacteria. Because the puncture approach has a great risk of introducing DNA from exogenous bacteria, a higher false-positive rate is obtained when employing DNA-based PCR.32 Furthermore, we have compared these two methods in a previous study, and demonstrated that the specificity of RNA-based PCR was superior to that of DNA-based PCR, while the sensitivity was similar.20 In this study, the joint fluid was limited; if we performed a DNA-based PCR additionally, the bacterial cultures would be affected due to low volumes of samples. Therefore, we utilized only RNA-based PCR to detect bacteria in joint fluid obtained before surgery.20 The results revealed higher specificity and sensitivity of RNA-based PCR than those of culture for low-volume preoperatively aspirated samples, although the sensitivity of RNA-based PCR was not as high as that of intraoperative sample culture.
We identified several false-negative and false-positive results based on preoperative joint fluid culture (Supplementary Tables v and vi). One explanation for the negative culture results for cases 1 and 2 is that an insufficient amount of joint fluid was obtained before surgery; because additional specimens were obtained during surgery, a definitive PJI diagnosis was established by intraoperative culture. RNA-based PCR was accurate in cases 1 and 2, despite their low sample volumes. Antibiotics were applied preoperatively in cases 3, 4, and 5, with normal results in serological inflammation tests and negative culture results for both pre- and intraoperative samples. Nonetheless, PJI was confirmed based on typical patient symptoms, observation of pus-filled joint cavities, and examination of frozen intraoperative samples. RNA-based PCR showed positive results for cases 3 and 4 and a negative result for case 5, which may be related to the duration of preoperative antibiotic use. In cases 3 and 4, antibiotics were used for less than one week; in case 5, antibiotics were used for more than four weeks. Studies have indicated that 16S rRNA can play a role as an indicator of cell activity,33 and that it cannot be detected one week after antibiotic treatment has blocked bacterial growth.34 Therefore, RNA-based PCR detection is more sensitive than bacterial culture for patients who have used antibiotics before surgery; regardless, there are time windows in which false-negative diagnoses are possible. In case 6, a false-positive result was obtained with preoperative bacterial culture, although RNA-based PCR did not. One possible explanation is that exogenous RNA introduced by puncture was degraded during the extraction process, highlighting another possible advantage of RNA-based PCR compared to DNA-based PCR and culture.
In our study, we used universal primers to identify the presence of bacteria, although these primers do not permit species discrimination. Consequently, we sequenced the amplified PCR products to identify the organisms, but consistent with a previous report this method was limited to recognizing CoNS at the genus level.30 Although the 16S rRNA sequence is highly conserved, it also harbours variable regions. As studies using rRNA-based reverse transcription-quantitative polymerase chain reaction (RT-qPCR) methods to discover hypervariable regions of 16S rRNA have been conducted to distinguish among different Staphylococcus species,19,35 designing species-specific primers for bacterial identification at the level of species may be possible in following studies.36
Because our study was a single-centre study and the observed PJI occurrence was low, the number of specimens acquired was relatively small, which may lead to bias. In addition, for two patients different culture bottles from the same company were used, which may also cause bias. Future multicentre trials with larger numbers of cases are warranted to confirm our findings.
In this study, the majority of the limited volume of joint fluid obtained before revision surgery was used for inoculating bacterial culture, whereas only 1 ml of each specimen was used for PCR analysis. Therefore, RNA-based PCR is compatible with bacterial culture methods. It is thus recommended that RNA-based PCR be performed when preoperatively aspirated joint fluid is limited, and when patients have not been using antibiotics for an extended period (less than one week). Further work is warranted to improve the efficiency of PCR for preoperative diagnosis of PJI. In future work, we will use a more stable RNA extraction system, and stricter operation in specimen transportation and PCR process to reduce the risk of contamination. Additionally, we will use primers for a broader bacterial spectrum to increase the diagnostic efficiency of PCR.
Acknowledgements
None declared
Open access
This is an open-access article distributed under the terms of the Creative Commons Attribution Non-Commercial No Derivatives (CC BY-NC-ND 4.0) licence, which permits the copying and redistribution of the work only, and provided the original author and source are credted. See https://creativecommons.org/licenses/by-nc-nd/4.0/.
Supplementary Material
Tables showing the primers used in this study, clinical data of patients with suspected infections, a comparison of diagnostic efficiency between culture and polymerase chain reaction, the organisms of prosthetic joint infection detected by culture and polymerase chain reaction, and typical cases misdiagnosed by preoperative culture.
Funding statement
This work was supported by the National Natural Science Foundation of China (81772251) and Fujian Province natural science fund (2018I0006, 2018Y4003).
No benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article.
Conflict of interest statement
None declared
Ethical review statement
The study was approved by the Ethics Committee of the First Affiliated Hospital of Fujian Medical University, Fuzhou, China (Ethics Number: [2014] 047).
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