What information can be gathered from a synovial fluid aspirate?

Synovial fluid aspiration is often used to gather information about a possible periprosthetic infection. 

When a sufficient volume is recovered, laboratory tests may help surgeons evaluate the likelihood of a periprosthetic infection; however the aspiration attempt may not yield fluid even if it is image guided. To be clinically useful, the aspiration needs to be carried out several weeks in advance of a potential surgical revision to allow sufficient time for laboratory tests and culture results to be finalized.

The authors of Synovial Fluid Cutibacterium acnes Antigen Is Detected Among Shoulder Samples with High Inflammation and Early Culture Growth presented a three pronged analysis of 1,365 de-identified synovial fluid samples, of which 1,150 were culture-negative and 215 were culture-positive.  94 of the culture positive samples were positive for Cutibacterium and 121 for other organisms.

The samples were analyzed by (1) time to culture positivity (known to be a reflection of the load of bacteria in the sample), (2) a validated  C. acnes antigen immunoassay test, and (3) a synovial fluid inflammation score calculated from 4 tests on synovial fluid: C reactive protein,  alpha-defensin, WBC count and percent polymorphonuclear cells.  

They found that 

(1) The samples tended to cluster into high inflammation and low inflammation groups for both all specimens

and for those in specimens that were culture positive for Cutibacterium

(2) C. acnes antigen levels demonstrated moderate-strong positive correlation with inflammation, with 166-fold higher levels of C. acnes antigen in high-inflammation samples compared with low-inflammation samples. 

(3) The days to C. acnes culture positivity demonstrated weak- inverse correlation with inflammation, with 1.5-fold earlier growth among the 67 high- inflammation samples compared to the low inflammation samples. Because a positive culture depends on discernible colonies on the culture plate and because the threshold for discernability relates to the number of bacteria present, low loads of bacteria in the sample are expected to have longer time to culture positivity. 

The relationship between C acnes antigen levels (left) and days to culture positivity (right) for samples with low inflammation and high inflammation is shown below.

(4) 19.0% of high-inflammation, culture-negative fluid samples demonstrated elevated C. acnes antigen.  Elevated C. acnes antigen was observed in only 0.38% of the low-inflammation culture-negative fluid samples and in only 4.9% of the high-inflammation non-C. acnes-positive cultures. 

Here it is of note that synovial fluid cultures are not uncommonly culture negative when tissue and explant cultures are positive. This may be because there may be Cutibacterium in biofilm form in tissue but not in planktonic (free floating) form in fluid. Thus these shoulders may not have been culture negative if tissue and explant cultures had been available. Absence of evidence is not evidence of absence.

Comment: One way to put these findings together is that low loads of Cutibacterium (as indicated by longer times before cultures become positive and by low antigen levels) may be insufficient to cause a tissue damaging inflammatory response on the part of the host. If one uses the definition of infection as "bacteria doing harm", these shoulders may not meet that definition, even though there are bacteria present. On the other hand, high loads of bacteria are likely to have shorter time to culture positivity, higher antigen levels and more inflammation.

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Here are some videos that are of shoulder interest
Shoulder arthritis - what you need to know (see this link).
How to x-ray the shoulder (see this link).
The ream and run procedure (see this link).
The total shoulder arthroplasty (see this link).
The cuff tear arthropathy arthroplasty (see this link).
The reverse total shoulder arthroplasty (see this link).
The smooth and move procedure for irreparable rotator cuff tears (see this link).
Shoulder rehabilitation exercises (see this link). 

Why do routine cultures miss detecting Cutibacterium?

Cultures of specimens of joint fluid, deep tissue samples, and prosthetic explants (see link) are the gold standard for identifying the microbes responsible for periprosthetic joint infections (PJI). As a result it is important to optimize the sampling and culturing methods so that organism-specific treatment can be implemented. Samples of sufficient size and number are to be obtained from the joint using sterile technique. Control sterile samples are to be obtained to assess the level of environmental and laboratory contamination. Appropriate media are to be selected for broth, anaerobic and aerobic cultures for bacteria and, when indicated, for fungus, yeast and micobacteria as well. Finally, cultures need to be observed for an appropriately long time in view of the time to positivity (TTP) of the bacteria. This latter factor is important in that a too-short period of observation may miss detecting the bacteria.

The time to positivity has been studied for Cutibacterium in shoulder periprosthetic infections (PJI). For example the authors of Optimization of periprosthetic culture for diagnosis of Propionibacterium acnes prosthetic joint infection concluded that a minimum culture incubation period of 13 days should be applied to both aerobic and anaerobic culture media for all periprosthetic specimens. The authors of
Origin of Propionibacterium in Surgical Wounds and Evidence-Based Approach for Culturing Propionibacterium from Surgical Sites found that obtaining at least four specimens, observing them for seventeen days, and using three types of culture media optimize the recovery of Propionibacterium (Cutibacterium) from samples obtained at revision surgery.

Recently, the authors of Time to Positivity of Cultures Obtained for Periprosthetic Joint Infection sought to determine the TTP for pathogens commonly encountered in PJI in 536 patients who met the 2018 International Consensus Meeting (ICM) criteria for PJI and had a positive intraoperative culture. 

Synovial fluid samples had the lowest yield on culture, compared with samples taken from soft tissue and bone: 66.2% (1,199) of the soft-tissue samples, 58.6% (782) of the samples obtained from bone, while only 31.3% (112) of the synovial fluid samples were positive. An explanation for the lower sensitivity of fluid cultures is that the majority of microbes present in prosthetic joints with PJI are in a biofilm requiring tissue sampling rather than in a planktonic form accessible in joint fluid.  

When evaluating the median TTP according to specimen type, synovial fluid (TTP, 1.97 [1.1 to 3.1]; n = 112) exhibited the shortest TTP, followed by soft tissue (TTP, 3.17 [1.4 to 5.3]; n = 1,199) and bone (TTP, 4.16 [2.3 to 5.9]; n = 782). An explanation for the shorter TTP for fluid cultures is that planktonic bacteria are likely to have a faster growth rate than the sessile bacteria found in a biofilm.

The median TTP for all positive cultures was 3.3 days (IQR, 1.9 to 5.4). In order of TTP speed

Methicillin-resistant Staphylococcus aureus (TTP, 1.42 [1.0 to 2.8]; n = 85) was fastest, followed by

Gram-negative rods (TTP, 1.92 [1.0 to 3.9]; n = 163), 

Methicillin-sensitive Staphylococcus aureus (TTP, 1.95 [1.1 to 3.3] n = 393), 

Streptococcus species (TTP, 2.92 [1.2 to 4.3]; n = 230), 

Staphylococcus epidermidis (TTP, 4.20 [2.4 to 5.5]; n = 555), 

Candida species (TTP, 5.30 [3.1 to 10]; n = 63); the slowest was the commonest cause of shoulder PJI:

Cutibacterium acnes (TTP, 6.97 [5.9 to 8.2]; n = 197). Note in the chart below that almost all of the positive cultures for Cutibacterium would have been missed with the three day observation period that is typical of many labs.

The authors recommended holding cultures for 14 days to capture most of the pathogens encountered in PJI.

Comment: A culture "turns positive" when there are sufficient numbers of bacteria on the plates or in the broth to be noticed by the laboratory technologist. 

The time to turn positive is faster for organisms having shorter doubling times (e.g. MRSA from a joint fluid sample). 

It is also faster when the original sample submitted for culture has a greater number of bacteria in it (see chart below).

This study indicates that attempts to identify the organisms causative of periprosthetic infection can fail because of too-short periods of culture observation - especially for the organism most commonly responsible for shoulder periprosthetic infections: Cutibacterium.

It also shows that with most bacteria, but especially Cutibacterium, the results of cultures come far too late to guide the initial treatment. Thus initial surgical and antibiotic must be based on the physician's knowledge of the most likely causative organism. In the case of shoulder PJI, that is Cutibacterium.

You can support cutting edge shoulder research that is leading to better care for patients with shoulder problems, click on this link.

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Here are some videos that are of shoulder interest
Shoulder arthritis - what you need to know (see this link).
How to x-ray the shoulder (see this link).
The ream and run procedure (see this link).
The total shoulder arthroplasty (see this link).
The cuff tear arthropathy arthroplasty (see this link).
The reverse total shoulder arthroplasty (see this link).
The smooth and move procedure for irreparable rotator cuff tears (see this link).
Shoulder rehabilitation exercises (see this link).

What is a "false positive" culture?

The Incidence and Incubation Period of False Positive Cultures in Shoulder Surgery

These authors studied 95 patients having either an open and arthroscopic procedure group (excluding those with abnormal inflammatory labs, history of previous shoulder surgery, or corticosteroid injection within six months of surgery). There were 47 patients in the open group and 48 in the arthroscopic group. In the open group, surgeries included 22 replacement arthroplasties, 22 rotator cuff repairs, and 3 instability repairs. In the arthroscopy group, surgeries included 24 acromioplasties, 17 rotator cuff repairs, 4 distal  clavicle resections, and 3 instability repairs.

Three cultures were obtained for each patient: superficial tissue culture, tissue culture, and  “sterile” control swab. Cultures were held for 28 days and checked on regular intervals.

In the open group, positive culture results were reported for eight of the forty-seven patients (17.02%) with one patient yielding positive superficial and deep culture specimens, each growing a different organism. Nine of the 141 cultures (6.3%) obtained were positive for bacterial growth. Five positive cultures were from superficial tissue, two positive cultures were isolated from deep tissue, and two positive cultures were isolated from the control “sterile” swab  group. All positive bacterial cultures were reported within seven days.  C. acnes was isolated in three of the forty-seven patients (6.4%), twice from a superficial tissue specimen and once from a deep tissue specimen. C. acnes was the most common isolated organism (33% of positive cultures) in the open group followed by Coagulase-negative Staphylococcus aureus (CoNS, 22% of positive cultures). All three patients yielding positive C.  acnes growth were men.

In the arthroscopic group, positive culture results were reported five of forty-eight patients (10.4%). Eight of the 144 cultures (5.5%) were positive for bacterial growth and one of the 144 cultures (0.7%) was positive for “mold”. Of the nine positive cultures, three were  obtained from superficial tissue, three were obtained from deep tissue, and three were obtained from the “control” swab. Two patients had more than one positive culture with one of the patients growing Methicillin Resistant Staphylococcus aureus (MRSA) from the superficial, deep, and “sterile” control cultures. All positive bacterial culture results were reported within seven 158 days. Only one specimen was isolated beyond one week, which was positive with “mold” at twenty-six days.  No specimens from the arthroscopic group were positive for C. acnes. The most common isolated organism was MRSA (50% of positive cultures) followed by CoNS (25% of positive cultures). 

They reported a "false-positive rate in open shoulder surgery of 17.02% and 10.4% in arthroscopic shoulder surgery.

The incidence of positive C. acnes cultures was 6.4% in the open group while C. acnes was not isolated in the arthroscopic group. All positive bacterial cultures were reported within seven days  of collection. 

Holding cultures longer than 14 days did not lead to an increased rate of false positive culture results.

In their Discussion, the authors pointed to " disturbing findings when reviewing the results of our study. One concern, was the high rate of lab errors. We had to exclude five patients due to blatant errors, including lost samples and removing cultures prior to 28 days. Additionally, five of the eighteen positive cultures were from the “sterile” control swabs, which suggest a high rate of contamination during sampling, transport, or laboratory handling."

Comment: The authors state in their discussion, "We are not able to prove the positive cultures are truly false positives." This begs the question, "what does it mean to state that a culture is "falsely positive"?" It seems more rational to state that no positive culture is "falsely" positive: if bacteria grew, the culture was positive; end of story. Positive cultures from "control" swab specimens are not "falsely positive": somewhere along the line the swab or the medium in which it was placed or the plates on which it was cultured acquired bacteria - truly.  Surgeons should periodically submit control swabs as quality assurance of their culturing system; see this link. If there is a substantially positive control swab it's time to check each step of the process to see where the bacteria are coming from (fingers, gloves, swabs, air) and when. Positive control swabs confound the interpretation of culture swabs from patients - patient cultures cannot be properly evaluated without knowing the results of local control swabs.

Similarly, positive cultures from specimens obtained from asymptomatic patients are not "false positives", although they may be unexpected. The bacteria cultured must have come from somewhere. If concurrent control swabs are negative, it is likely that the bacteria came from the patient - truly. That doesn't mean that the bacteria were doing harm, just they were there.

Finally, assessing the load or degree of positivity of bacteria in a cultured specimen can be helpful in interpreting the result. See this link and see this link.

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The challenge of diagnosing a propionibacterium "infection"

Optimal cultivation length to isolate Propionibacterium acnes in suspected bone and joint infections is > 7 days

JCM Accepted Manuscript Posted Online 12 October 2016

J. Clin. Microbiol. doi:10.1128/JCM.01435-16

These authors retrospectively studied whether reducing cultivation time to 7 days allowed accurate diagnosis without losing sensitivity. They identified patients with at least one positive P. acnes sample between 2005 and 2015 and grouped into ‘infection’ and ‘no infection’. They defined an ‘infection’ when at least two samples from the same case were positive. Clinical and microbiological data including time to positivity for different cultivation methods were recorded.

They identified 70 cases meeting their definition of  a P. acnes ‘infection’, in which 262 out of 379 samples were positive (69.1%). 47 cases did not fulfill our criteria for a proven ‘infection’ with only one positive sample (47 out of 215 samples, 21.9%). The most common sample site was shoulder (total n = 77), followed by hip (n=26). In the ‘infection’ group, they diagnosed PJI in 35 (50%) cases, and implant-associated infections with large (plates, intramedullary nail) or small implants (screws, anchors) in 10 (14.3%) and 17 (24.3%), respectively. Five cases presented with septic arthritis (arthroscopy-associated), and three cases with osteomyelitis (two in the shoulder after infiltration or trauma and one in the clavicula associated with b-cell lymphoma). In the ‘no infection’ group there were pain due to mechanical reasons in 22 (46.8%), aseptic loosening of an implant in 9 (19.2%), and
 other reasons in 16 (34%) cases diagnosed at revision surgery.

The cases of P. acnes "infection" had a significant faster median time to positivity of 6 days (range 2- 11 days) compared to 9 days in 47 cases with P. acnes identified as a contamination (p<0.0001).

 In 15 of 70 (21.4%) patients with an "infection", tissue samples were positive after day 7 and in 6 patients (8.6%) after day 10. Highest sensitivity was detected for thioglycolate broth (66.3%) and best positive predictive values for anaerobic agar plates (96.5%). A prolonged transportation time from the operating theatre to the microbiological laboratory did not influence time to positivity of P. acnes growth. By reducing the cultivation time to 7 days, false negative diagnosis would increase by 21.4%, thus they recommend culturing biopsies in suspected bone and joint infections to detect P. acnes for at least 10 days . They would have missed 6 cases (8.6%) if they had stopped cultures at day 10.

Their yield with sonication was lower than expected and they suggest the possibility that P. acnes might be inhibited or killed using ultrasound baths.

They conclude that a prolonged cultivation time is necessary for P. acnes  identification. They would have missed 21.4% of the P. acnes infections if cultivation time had been reduced to 7 days. Thioglycolate broth as an enrichment method for tissue samples showed a high sensitivity. The best positive predictive value was seen with direct incubation on anaerobic agar plates. Time to positivity of P. acnes growth  did not seem to be affected by a prolonged transportation time, which showed that P.  acnes in the biofilm of musculoskeletal infections can survive for hours even in a fastidious environment.

Comment:  This study effectively points out that longer incubation times result in a higher rate of culture positivity for Propionibacterium and the importance of anaerobic and broth cultures.

The definition of 'infection' as two or more positive cultures is, however, arbitrary due to the fact that Propionibacterium are not evenly distributed throughout the wound (see this link); whether or not a given specimen is culture positive depends on the 'luck of the draw'.

Rather than applying an arbitrary definition of 'infection' (and there are many), it would be clearer to report the number of specimens submitted from (a) tissue, (b) explant, and (c) fluid and the percent of each that was culture positive.

The bacterial load matters. Higher total numbers of bacteria in the wound will increase the percent of specimens that are culture positive, but a low percent of positive cultures does not mean the wound is aseptic.

We could take a number of samples of this lawn and not find two that show a mushroom, but that doesn't mean mushrooms are not really there.

Higher numbers of bacteria in a specimen will result in a shorter time to the recognition of a culture being positive, but a long time to positivity does not mean that the result is a 'contaminant'.

Consider the example below in which 1000 bacteria need to be present in order for the culture is recognized as positive. The red specimen contained 4 bacteria while the blue specimen contained one. The red specimen was 'culture positive' at nine days, while the blue specimen was interpreted as showing 'no growth' at 10 days.

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Failed shoulder arthroplasty - how badly is it infected?

Early Versus Late Culture Growth of Propionibacterium acnes in Revision Shoulder Arthroplasty.

Theses authors retrospectively reviewed 208 revision arthroplasty cases. For all patients included in the study, at least two culture specimens (synovial fluid and/or tissue) were obtained (mean, 4.2 ± 1.4 samples; range, two to seven samples).  Tissue and fluid were cultured anaerobically for a mean (and standard deviation) of 13.1 ± 3 days. A positive culture was found for ninety-two (44%).  P. acnes was identified in culture in sixty-one (29%) of the cases; in seven of those cases, coagulase-negative Staphylococcus  (CONS) was also identified. Organisms identified in the other culture-positive cases included isolated CONS (twenty-two cases), Enterobacter cloacae (two cases), diphtheroid bacilli (two cases), and one case each of methicillin-sensitive S. aureus  (MSSA), Staphylococcus epidermidis, Staphylococcus lugdunensis, Peptostreptococcus  magnus, and Enterococcus gallinarum.

This study concerned 46 cases that had P. acnes only positive cultures. Cases were categorized into one of two groups for analysis: probable true positive or probable contaminant (false-positive) on the basis of culture results and perioperative findings.

Among the cases that were positive the time to P. acnes culture growth was significantly shorter (p = 0.002) in the probable true-positive culture group compared with the probable contaminant group (median of five days compared with nine days). Among the thirty-seven cases in the probable true-positive group, no culture result turned positive after eleven days, whereas in the probable contaminant group, cultures turned positive after this time point in 44% (four of nine) of the cases. There were also significantly fewer days to P. acnes culture growth among cases with a higher number of positive cultures (p = 0.001) and a higher proportion of positive cultures (p < 0.001), regardless of group classification.

Comment: 

There is no such thing as a false positive culture - if bugs grow the culture is positive. There is no practical way of determining whether the bacteria cultured existed in the surgical wound or whether the specimen was contaminated by the operating room or laboratory environment. 

Both the percent of specimens that are culture positive and the time for cultures to become positive depend on the number of bacteria present in the shoulder, so it is not surprising that the two are related as shown by this plot of the data from this study.

The clinical manifestations of a shoulder infection are also expected to be related to the number of bacteria in the shoulder, so those shoulders in the upper left corner are more likely to have clinical findings that those in the lower right corner.

Our interpretation of the data in this study is that the percent of cultures that are positive, the time it takes for the cultures to become positive, and the degree of clinical manifestation of the infection are all eflections of the severity of the infection. There is no additional benefit in trying to assign some culture results to a 'contaminant' or 'false positive' category. This information on the severity of the infection may influence the decision about how the infection should be managed. 

We need to remember:

(1) Shoulders containing greater numbers of bacteria are more likely to have clinical manifestations.

(2) Shoulders containing greater numbers of bacteria are more likely to have a a high percentage of cultures that are positive.

(3) Shoulders containing greater numbers of bacteria are more likely to have shorter times for cultures to become positive. The reason is that a culture becomes positive when the laboratory technologist can see growth on the culture plate. This growth is visible when a threshold number of bacteria have grown. The time for this threshold to be reached depends  on the amount of bacteria streaked out on the culture plate - the size of the innoculum. The size of the innoculum, in turn, depends on the number of bacteria in the shoulder.

Thus the authors' conclusion"There were also significantly fewer days to P. acnes culture growth among cases with a higher number of positive cultures (p = 0.001) and a higher proportion of positive cultures (p < 0.001), regardless of group classification."

We all continue to struggle with the best way to manage patients with these different types of culture results. Progress comes in small steps, but one of the most important ones is recognition of the need, as explained here, to obtain at least 5 specimens from tissue or explants, to culture these specimens on aerobic and anaerobic media, to hold the cultures for 17 days, and to report the results in a way that indicates the source, the number of specimens and the number of specimens that were culture positive, as in this example:  Propi: Tissue: 2+/3, Explants: 3+/3, Fluid 0+/1

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