Abstract
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Culture-negative periprosthetic joint infections (PJI) are commonly described in the literature.
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By implementing a routine diagnostic workup and by optimizing tissue sampling and processing, the culture-negative rate can easily be reduced.
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When faced with a culture-negative PJI, several serological and molecular techniques are available that may aid in finding the causative microorganism. Clinical clues may guide the treating physician towards more atypical and rare microorganisms.
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A multidisciplinary team consisting of orthopaedic surgeons, microbiologists and infectious disease specialist are warranted in tailoring diagnostic testing and deciding on the surgical and antibiotic treatment approach.
Introduction
Historically, cultures are considered as the gold standard in diagnosing any type of infection. However, cultures can be negative in many infectious diseases, including periprosthetic joint infections (PJI), in particular when a patient is on antibiotic treatment or when microorganisms are present that are difficult to cultivate. For this reason, a PJI can be diagnosed in the absence of any positive (intraoperative) cultures. Apart from microbiological criteria, the recently introduced diagnostic criteria from the European Bone and Joint Infection Society (EBJIS) considers a PJI as confirmed solely based on the following criteria: (i) a sinus tract communicating with the joint and/or (ii) more than 3000 white blood cells per microlitre synovial fluid or a percentage of polymorphonuclear leukocytes above 80% and/or (iii) positive histology with five or more neutrophils seen in at least five high-power fields (1). It must be noted that it is important that alternative explanations for these positive criteria are ruled out prior to infection diagnosis. For example, comorbidities like metallosis, active gout and inflammatory arthritis may also result in high leucocyte counts in the synovial fluid (2, 3, 4). In addition, the cut-off value for leucocytes in the synovial fluid is less reliable when the patient is less than 6 weeks after joint arthroplasty (5). In these circumstances, non-microbiological positive criteria should be interpreted with caution, but an infection should be considered in these cases (1).
How big is the problem of culture-negative PJI?
According to the available literature, depending on the definition used and the cohorts analysed, the incidence of culture-negative PJIs appears to be between 5 and 40% (6). This high percentage is worrisome. The main reason for a high culture-negative rate is prior antibiotic treatment. In a case–control study performed by Malekzadeh et al., PJI patients who were on (prior) antibiotic treatment had a culture-negative rate of 64% and a four times higher odds of culture negativity compared to controls without antibiotic treatment (7). Physicians should be aware that, in most cases, this high culture-negative rate can be avoided. For instance, most patients with a late chronic PJI scheduled for revision surgery do not require any antibiotic treatment prior to surgery. The causative microorganism(s) are likely to be present from the time of prosthesis implantation, and it is very uncommon that patients with a late chronic PJI develop a bacteraemia or other complications that justify the use of antibiotics. Withholding antibiotic treatment in these patients is safe. In order not to miss the use of antibiotics, physicians should always actively ask the patient if the general practitioner started any antibiotic treatment. If the patient is on antibiotic treatment, this should be discontinued at least 2 weeks prior to revision surgery in order to have reliable intraoperative cultures. The only exception to start antibiotic treatment prior to infection surgery is when the patient is critically ill or has signs of sepsis. This might be the case in patients with an early acute postoperative PJI or a late acute hematogenous PJI. Nevertheless, even in these cases, isolating the microrganism is relatively easy when a sufficient amount of blood cultures is obtained (i.e. two sets) and when a synovial fluid aspiration is performed prior to the start of antibiotics. Unlike chronic infections, the likelihood of having positive synovial fluid and/or blood cultures is high in acute infections, in particular in hematogenous ones. In contrary to antibiotic treatment, antibiotic prophylaxis prior to surgical incision should always be administered as this hardly affects culture yield (8), and timely antibiotic prophylaxis is critical to protect the implant from (secondary) infections (9, 10).
Other ways to reduce culture negativity
Culture yield can be improved by implementing a routine clinical and microbiological diagnostic workup for patients with a suspected PJI. Apart from withholding antibiotic treatment prior to infection surgery, it is important that a sufficient amount of intraoperative tissue samples is obtained during infection surgery. To achieve the most optimal sensitivity and specificity, at least four to five samples should be send in for culture at the most suspected sites of infection (11, 12). In addition, the samples need to be transferred to the microbiology laboratory as soon as possible, and if the samples cannot be incubated directly, they should be stored at 4°C to ensure bacterial viability (13). Next to adequate sampling, samples should be optimally processed in the microbiology laboratory. During the last decades, many papers have been published on how culture yield can be increased. For example, the incubation time should be around 14 days when using conventional culture techniques (14, 15). In particular, Cutibacterium acnes needs time to grow. Routinely prolonging the incubation time beyond this point will increase the risk for contamination and is not recommended (15). Incubation time can be reduced when inoculating tissue samples, synovial fluid and/or sonication fluid in blood culture bottles (16, 17). Minassian et al. demonstrated that when tissue samples are inoculated in blood culture bottles, 9 days of incubation is sufficient (17). The time to positivity is even further reduced when sonication fluid is inoculated in blood culture bottles (16). The use of sonication is particularly of added value when the patient was on prior antibiotic treatment (18). In these cases, culture sensitivity can be increased by around 20–30% compared to tissue cultures alone (18).
When applying the above-mentioned criteria, the culture-negative PJI rate can be significantly reduced. Indeed, a recent study evaluated the culture-negative PJI rate when excluding patients who were on prior antibiotic treatment, in whom less than four intraoperative samples were obtained, and when samples were incubated for less than 14 days unless inoculated in blood culture bottles. When fulfilling these requirements, no culture-negative PJIs were diagnosed in acute infections, and only 5% of culture-negative PJIs were diagnosed in chronic infections (19). These numbers are encouraging and indicate that isolating the causative microorganism in biofilm-related infections is not as difficult as previously assumed.
What to do if cultures remain negative
What to do when, despite optimal tissue sampling and processing, intraoperative cultures remain negative and no other explanation for false-positive minor diagnostic criteria can be found? The pathogens to expect depend on clinical presentation, timing after surgery and whether the patient was on antibiotic treatment at the time of sampling. Figure 1 shows a diagnostic flow chart that can be used for culture-negative PJIs and depicts serological and molecular methods that are available for finding the causative microorganism(s).
Common molecular and serological tests
Several molecular techniques can be applied in culture-negative cases. 16S PCR is a technique that is routinely available in most microbiological laboratories. This PCR technique targets the 16S ribosomal region that is specific for bacteria, and thus all bacteria can – theoretically – be detected by using 16S PCR. A disadvantage of this technique is that it is only able to detect the microorganism that is most abundantly present, and thus other microorganisms that may be present in polymicrobial cases will be missed. 18S PCR uses the same technique but is specific for fungi.
Another molecular technique is targeted PCR, a PCR that is specifically designed to detect a specific bacterial species. This technique is more sensitive than the 16S technique and is available to target either one single microorganism (e.g. specifically targeting Coxiellaburnetii) or multiple microorganisms (e.g. a film array multiplex panel specifically targeting the most common microorganisms associated with PJI). Last, but not least, a molecular technique that has gained a lot of interest in recent years is next-generation sequencing (NGS). With NGS, it is possible to detect all potential microorganisms present in a sample, and for this reason it is ideal for polymicrobial PJIs or for cases in which rare microorganisms are suspected but no clinical clues are available to choose a specific targeted PCR. A disadvantage of the technique is that it is not available in all laboratories, and the clinical significance of the microorganisms detected is not always clear.
Serological tests in serum may be helpful to diagnose or rule out specific infectious diseases, specifically zoonotic diseases.
Clinical presentation
Dependent on the clinical presentation and timing after surgery, certain microorganisms can be expected in culture-negative PJIs.
In case of early postoperative PJIs (i.e. less than 3 months after the index surgery), the causative microorganism is likely to be introduced to the surgical wound during or right after surgery. For this reason, the usual (skin) commensal pathogens should be suspected, in particular when the patient was on prior antibiotic treatment as potential cause of culture negativity. Since early postoperative PJIs are polymicrobial in a significant proportion of cases (20), multiplex PCR panels or NGS is the preferred test to detect all causative pathogens. The same holds for late chronic infections presenting with a sinus tract, which is a well-known risk factor for a polymicrobial PJI (21).
For cases in which a monomicrobial PJI is expected, like a late chronic PJI without a sinus tract or a late acute hematogenous PJI, a diagnostic test like 16S PCR can be performed on intraoperative tissue samples in the absence of any diagnostic clues. Although a negative 16S PCR does not rule out an infection, a positive 16S PCR may aid in the diagnosis. Multiplex PCRs specifically designed for PJI or NGS can also be performed when available or when specific (atypical) microorganism(s) are clinically expected. Zoonotic diseases like Lyme disease, Q-fever, brucellosis and cat scratch disease typically present with an acute onset of symptoms (22, 23, 24). Serology performed on serum can easily rule out these diseases. In addition, positive serology may aid in diagnosis, but targeted PCRs at the site of infection should ideally be used for definitive confirmation. Rat bite fever, caused by Streptobacillusmoniliformis, can be detected by several molecular methods, including NGS and 16S PCRs (25). Apart from zoonotic disease, specific targeted PCRs are also available for Mycoplasma species and Tropherymawhipplei, microorganisms that are difficult to cultivate by culture and able to cause PJIs (26, 27). Targeted PCRs for these microorganisms are probably more sensitive but may be detected by other molecular methods also, like 16S PCRs and NGS.
Rare fungi should be suspected in immunocompromised patients, for example in organ transplant recipients or in patients who received a stem cell transplantation (28, 29). For histoplasmosis, the patient should have had exposure in an endemic area. Fungal infections may present with acute as well as with chronic like symptoms. In addition, tuberculosis (TB) of a prosthetic joint may be present throughout the entire spectrum of presentation. In rare cases, latent TB may become manifest after invasive surgery and/or during an acute PJI caused by another microorganism. However, in most cases, TB or other mycobacteria, often present in a more chronic fashion (30). Granulomas found in histology sections might provide a clinical clue for mycobacterial infections but are not always present. Depending on the local epidemiology and risk factors for TB exposure, targeted PCRs are available, and tissue samples should be incubated for atleast 6 weeks in which special media may be used.
Conclusion
The incidence of culture-negative PJIs is low when using a routine diagnostic workup and when adequate culture techniques are applied. When a culture-negative PJIs is diagnosed, several serological and molecular tests are available to find the causative microorganism. Timing after surgery and the clinical presentation need to be taken into account for choosing the most appropriate diagnostic test.
ICMJE conflict of interest statement
The author has no conflict of interest.
Funding
No funding was obtained for this study.
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