Abstract
Objective
To evaluate if high‐volume lavage results in a negative bacterial culture of egress fluid, regardless of pathogen in an experimental model of equine septic arthritis.
Study design
Experimental study.
Animals
A total of 11 horses in three groups (lipopolysaccharide [LPS] Group 1, n = 4; Escherichia coli [E. coli] Group 2, n = 4; Staphylococcus aureus [S. aureus] Group 3, n = 3).
Methods
A middle carpal joint of each horse was injected with LPS (synovitis Group 1), E. coli (septic arthritis Group 2) or S. aureus (septic arthritis Group 3). Arthroscopic lavage of the middle carpal joint was performed 24‐h post‐injection. Egress fluid samples were collected every liter for bacterial culture. Liter samples 1, 5, 10, 15, and 20 were submitted for bacterial culture. Subsequent samples were submitted based on the results of the initial dataset.
Results
Predicted probability of a positive culture after 1, 5, 10, 15, and 20 L for E. coli and S. aureus, respectively were: 44.7% (p = .04) and 98.3% (p ≤ .001) after 1 L, 42.6% (p = .02) and 94.3% (p ≤ .001) after 5 L, 40.1% (p = .02) and 77.8% (p = .001) after 10 L, 37.6% (p = .04) and 42.5% (p = .08) after 15 L, and 35.2% (p = .09) and 13.5% (p = .32) after 20 L. LPS cultures (n = 4) were negative at every timepoint. Post‐lavage 50% (2/4) of E coli. and 100% (3/3) S. aureus horses had positive synovial fluid cultures.
Conclusion
A total of 20 L of lavage fluid was not sufficient to result in a sustained negative bacterial culture.
Clinical significance
High‐volume arthroscopic lavage alone is not sufficient as a treatment for septic arthritis.
1. INTRODUCTION
Septic arthritis is a serious, potentially life‐threatening or career ending condition in horses. Penetrating trauma and iatrogenic contamination are the most frequent causes in adults, and hematogenous spread is most common in foals. 1 , 2 , 3 , 4 , 5 Reported survival rates of horses vary widely from 42% to 98% 3 , 4 , 6 , 7 , 8 with adults having a better prognosis than foals. Return to function has been reported to be 26%–81%. 3 , 4 , 6 , 7 , 8 Diagnosis relies on cytological analysis and bacterial culture of synovial fluid samples. 9
An aggressive multimodal treatment approach is advocated and includes systemic and local antimicrobials, anti‐inflammatories, analgesia, and joint lavage. Thorough lavage is indicated to remove debris, inflammatory products, and microorganisms from within the joint. Many methods of lavage have been described including arthrotomy, drains, through and through needle lavage, and arthroscopy. 2 , 4 Arthroscopic lavage has been promoted over other methods as visualization of the joint allows for debridement of damaged cartilage and removal of hyperplastic synovium. 1 , 4 , 5 , 10 , 11 , 12 , 13
Large volumes of sterile crystalloid fluids for joint lavage have been recommended. 2 , 4 , 9 , 14 However, there is very little data available on what volume is adequate. One previous study looking at the retrieval of colored microspheres from normal cadaver tarsocrural joints found that most were retrieved within the first 1 L of the lavage. That study concluded that for particulate removal, as little as 1–2 L may be adequate, but in cases of joint sepsis, higher volumes may be required due to inflamed synovium and fibrin deposits. 15 To the author's knowledge, there have been no studies performed looking at the lavage volume required for a negative bacterial culture in horses with induced septic arthritis and synovitis. The objective of this study was to evaluate if high‐volume lavage results in a negative bacterial culture of egress fluid, regardless of pathogen in an experimental model of equine septic arthritis. The hypothesis of this study was that 20 L of fluid would result in a negative bacterial culture in all horses, regardless of the pathogen utilized.
2. MATERIALS AND METHODS
2.1. Animals
A total of 11 healthy adult Quarter Horses (6 mares and 5 geldings) with a mean age of 10 ± 3 years (range: 7–18 years) and mean bodyweight of 528 ± 29 kg (range: 483–580 kg) were included in the study. All procedures were approved by the University of Saskatchewan Animal Care and Use Committee and Animal Research Ethics Board (AUP#20180048). Horses were considered to be healthy and free of musculoskeletal disease based on thorough physical examination, lameness examination, and blood work (complete blood count, biochemistry profile and measurement of systemic blood serum amyloid A). Horses were evaluated at the walk and trot and no significant gait abnormalities were observed.
2.2. Experimental design
To determine the efficacy of eradication of synovial joint infection during arthroscopic lavage, three experimental models were used. A non‐septic synovitis model induced by lipopolysaccharide 16 served as a control group, a septic arthritis model using Escherichia coli, 16 and a septic arthritis model using Staphylococcus aureus 17 served as two treatment groups.
Horses were randomly assigned into one of the following three groups and had their left middle carpal joint injected: LPS group (synovitis, n = 4), E. coli group (septic arthritis, n = 4), and S. aureus group (septic arthritis, n = 3).
2.3. Experimental induction of non‐septic synovitis and septic arthritis
Synovitis was induced using a previously described lipopolysaccharide model. 16 , 18 Briefly, 50 ng of lipopolysaccharide (LPS) was diluted into 1 mL of sterile phosphate‐buffered saline solution, aliquoted, and then stored at −80°C until used. Aliquots of LPS (50 ng/mL) were then thawed prior to inducing synovitis and stored on ice until injection of the assigned middle carpal joint was performed.
For the E. coli group, septic arthritis was induced with an inoculation dose of 1.0 × 108 colony‐forming units (CFU) of E. coli using a previously described bacterial/septic arthritis model in horses. 18 Briefly, the strain used was isolated from a clinical case presented to the Veterinary Medical Center, diagnosed and treated for septic arthritis. The stock culture was divided into 50 μL aliquots of 1.0 × 1010 CFU/mL and thereafter stored at −80°C. Bacteria were cultured in blood culture medium at 37°C in aerobic conditions to the mid‐exponential phase of growth. Bacterial cells were then harvested by centrifugation, washed in sterile phosphate buffered saline (PBS, pH 7.4), and resuspended in fresh sterile PBS to 1.0 × 108 CFU/mL as previously described. 18
For the S. aureus group, a culture of S. aureus previously isolated from a clinical case of equine septic arthritis was inoculated (provided by Prairie Diagnostic Services, Saskatoon, Saskatchewan). The aliquots of 50 μL were stored at −80°C. Individual aliquots were used to prepare each injection. To prepare the inoculum, stored aliquoted bacteria were cultured in 10 mL of brain‐heart infusion (BHI) broth (BD Bacto Brain Heart Infusion; Becton Dickinson) at 37°C on a shaking platform at 180 rpm in aerobic conditions overnight. The next morning, 300 mL of the overnight culture was transferred into 30 mL of BHI broth and cultured for 3 h in the same conditions to reach the mid‐exponential phase of growth. The bacterial suspension was adjusted to optical density (OD) 0.05 (NanoDrop 2000c Spectrophotometer; Thermo Scientific, Waltham, Massachusetts), which was determined to correspond approximately to 107 CFU/mL based on a previously determined standard growth curve, and then diluted 1:10 to obtain final concentration of 106 CFU/mL. The exact CFU of each inoculum was subsequently determined by titration of serial dilutions and culture on Colombia blood agar. The mean ± SD CFU per 1 mL of inoculum determined by titration was 0.81 ± 0.17 × 106 CFU/mL. The standard growth curve was determined by inoculating 100 mL BHI broth with S. aureus stock suspension. After 2 h of aerobic incubation at 37°C, a sample of the broth culture was taken for optical density measurement and bacterial colony count by subculturing on blood agar plates in triplicates. This was repeated eight times every hour. Average bacterial count was plotted against optical density, and straight line was fitted in Excel (Microsoft) with good fit (R 2 = 0.98).
All horses were sedated with detomidine hydrochloride (0.015 mg/kg, IV) and the left carpus was aseptically prepared in a routine fashion. A 20‐gauge needle attached to a 6‐mL syringe was then inserted into the middle carpal joint using a dorsolateral approach and a synovial fluid sample was collected and used as an internal control for this study. Thereafter, the syringe was replaced with a 3‐mL luer‐lock syringe containing 1 mL of either 50 ng LPS, 1.0 × 108 CFU of E. coli, or 0.8 × 106 CFU of S. aureus and the solution was delivered into the middle carpal joint.
2.4. Surgical procedure and sample collection
All horses underwent arthroscopic lavage 24 h post induction of synovitis and septic arthritis. All horses received sodium penicillin (Penicillin G, sodium for injection, USP; Pharmaceutical Partners of Canada, Richmond Hill, Ontario), 22 000 IU/kg bodyweight (BW), IV, gentamicin sulfate (Gentocin; Intervet Canada, Kirkland, Quebec), 6.6 mg/kg BW, IV, and phenylbutazone (Phenylbutazone; Rafter 8 Products, Calgary, Alberta), 4.4 mg/kg BW, IV, 30 min before surgery. Horses were premedicated with xylazine hydrochloride (Rompun; Bayer Health Care, Toronto, Ontario), 1 mg/kg BW, IV. Anesthesia was induced with ketamine hydrochloride (Vetalar, Bioniche, Bellevillle, Ontario), 2.2 mg/kg BW, IV, and diazepam (Diazepam, Sandoz Canada, Quebec), 0.1 mg/kg BW, IV, and maintained with isoflurane in oxygen. Horses were positioned in dorsal recumbency with the front limbs suspended. The assigned carpus was clipped, aseptically prepared, and draped for a routine carpal arthroscopic surgery. A 20 gauge, 1.5 in needle was placed using the dorsolateral approach to the middle carpal joint and a 3 mL synovial fluid sample was collected. Arthroscopic surgery was performed using the previously described approach to the middle carpal joint. 19 Briefly, a 5.5 mm diameter arthroscopic cannula with blunt obturator (Karl Storz Veterinary Endoscopy, Goleta, California) was introduced into the dorsolateral aspect of the middle carpal joint in a routine manner, the obturator was replaced with a 30° 4 mm diameter arthroscope (Karl Storz Veterinary Endoscopy). A 4.5 mm diameter egress cannula (Karl Storz Veterinary Endoscopy) was placed in the dorsomedial pouch of the joint in a routine manner. 19 After the initial articular examination, the middle carpal joint was copiously lavaged using 20 L of lactated Ringer's solution. Sterile tubing was fitted to a 4.5 mm egress portal (Karl Storz GmbH & Co. KG, Tuttlingen, Germany) and used for fluid collection. Fluid samples were collected into 1.5 mL sterile microcentrifuge tubes after each liter of lavage fluid was administered during the arthroscopic procedure. All samples were stored at −80°C until bacterial culture analysis. Skin incisions were then closed using 2‐0 monofilament polybutester in a simple interrupted suture pattern. Thereafter, the middle carpal joint was injected with 500 mg of gentamicin sulfate, and a light sterile bandage was applied over the incisions before recovery.
In conjunction with this study, another study was carried out to investigate the response to treatment and the eradication of septic arthritis in experimental horses. As part of the concurrent study all horses received sodium penicillin (Penicillin G, sodium for injection, USP; Pharmaceutical Partners of Canada), 22 000 IU/kg BW, IV every 6 h, gentamicin sulfate (Gentocin; Intervet Canada), 6.6 mg/kg BW, IV, every 24 h and phenylbutazone (Phenylbutazone; Rafter 8 Products), 4.4 mg/kg BW, IV, every 24 h postoperatively for 6 days.
2.5. Synovial fluid sample collection
On day 1 postoperatively horses were sedated with detomidine hydrochloride (0.015 mg/kg, IV) and the left carpus was aseptically prepared in a routine fashion. A 20 gauge, 1.5 in needle was placed using the dorsolateral approach to the middle carpal joint and a 3 mL synovial fluid sample was collected for bacterial culture.
2.6. Cytological evaluation of synovial fluid
To confirm the models of synovitis and septic arthritis for this study, synovial fluid samples were collected into 0.5 mL ethylenediaminetetraacetic acid (EDTA) (BD Microtainer; Becton, Dickinson, Franklin Lakes, New Jersey) tubes and submitted for cytological evaluation to an independent laboratory (Prairie Diagnostic Services Inc., Saskatoon, Saskatchewan, Canada) for analysis. The following parameters were determined: gross fluid appearance, nucleated cell count (TNCC), total protein (TP), percentage of neutrophils (PN) and their morphology. Values of TNCC over 30.0 × 109/L, PN greater than 80% and TP greater than 40 g/L were considered as the clinical threshold for equine septic arthritis. 9
2.7. Bacterial culture of synovial fluid samples
Synovial fluid samples were collected in 1.5 mL sterile microcentrifuge tubes for bacterial culture. Bacterial culture and analysis were performed by a single pathologist blinded to the treatment (RVK). A total of 100 μL of synovial fluid was inoculated on a Columbia blood agar (CBA) with 5% sheep blood culture plate (Oxoid, Nepean, Ontario, Canada), and incubated aerobically at 37°C (VWR Incubator; Sheldon Manufacturing, Cornelius, Oregon) for 72 h. A total of 200 μL of synovial fluid was inoculated into blood culture medium (Oxoid SIGNAL Blood Culture System Medium, Basingstoke, Hampshire, UK) and into 100 mL of brain‐heart infusion (BHI) broth (BD Bacto Brain Heart Infusion; Becton, Dickinson) and both were incubated under aerobic conditions on an orbital shaker platform (C24 Incubator Shaker; New Brunswick Scientific) at 37°C and 180 rpm for 72 h. Results were recorded as either positive or negative for the bacteria the horse had been inoculated with. For this study, a negative culture was defined as if no bacterial growth was present on any of the culture media. A culture was considered positive if there was at least one colony of the inoculated bacteria.
2.8. Lavage fluid sample analysis
Synovial fluid samples in sterile 1.5 mL microcentrifuge tubes from liters 1, 5, 10, 15, and 20 were submitted to an independent laboratory (Prairie Diagnostic Services Inc) for bacterial culture. Results were recorded as either positive or negative for the bacteria the horse had been inoculated with. Subsequent samples were submitted for culture‐based results of the initial dataset. If results showed that horses had a positive bacterial culture in one sample and a negative culture on the next sample, then the fluid samples for the liters between the positive and negative cultures were submitted to determine that exact liter where the horse no longer had a positive culture.
2.9. Statistical analysis
Data was checked, cleaned and stored in excel (Microsoft 365). Data analysis employed descriptive analysis, and a generalized estimating equation (GEE) population averaged model completed in STATA 17 (StataCrop Inc). Data are described by percentage of positive cultures by day and by lavage volume. GEE, accounting for repeated sampling within horse, employed a logit function using an exchangeable correlation matrix to assess probability of positive culture across five lavage volumes by pathogen type (E. coli and S. aureus). Data are presented as percentage (predicted probability) and corresponding 95% CIs as calculated by the margins command.
3. RESULTS
3.1. Cytological evaluation of synovial fluid
Prior to inoculation of LPS, E. coli, or S. aureus all horses had normal TNCC of <1.0 × 109/L (median: 0.1 × 109/L range: 0.1–0.9 × 109/L) and few RBCs present (median: 0.1 × 1012/L range: 0.1–0.6 × 1012/L). TP was mildly elevated in two horses (Horse ID: E1 and S2) at 25 g/L prior to experimental induction. In three horses (Horse ID: E4, E5, and L3) there was not a sufficient sample quantity to measure TP. All other horses (n = 6/11) had TP values of <20 g/L (median: 11 g/L range: 8–25 g/L) and were considered normal. On day 1, all horses in all groups, including the non‐septic synovitis group had TNCC values greater than the septic threshold value of 30.0 × 109/L9 (median: 179.1 × 109/L range: 166.8–265.4 × 109/L). RBC numbers were not numerically different (median: 0.13 × 1012/L range: 0.11–0.55 × 1012/L). TP was elevated in all horses, in all groups, beyond the septic threshold of 40.0 g/L9 (median: 71 g/L range: 53–90 g/L). Values for each horse on days 0 and 1 are presented in Table 1.
TABLE 1.
Synovial fluid cytology parameters by individual horse.
| Day 0 | Day 1 | |||||
|---|---|---|---|---|---|---|
| Horse ID | TNCC (×109/L) | RBC (×1012/L) | TP (g/L) | TNCC (×109/L) | RBC (×1012/L) | TP (g/L) |
| E1 | 0.3 | 0.12 | 25 | 179.1 | 0.13 | 75 |
| E2 | 0.6 | 0.19 | 8 | 218 | 0.13 | 53 |
| E4 | 0.1 | 0.1 | 0 | 254.6 | 0.32 | 71 |
| E5 | 0.1 | 0.1 | 0 | 214.8 | 0.12 | 73 |
| L1 | 0.1 | 0.1 | 11 | 169.5 | 0.15 | 59 |
| L3 | 0.1 | 0.1 | 0 | 55.1 | 0.1 | 60 |
| L4 | 0.1 | 0.1 | 20 | 112.6 | 0.1 | 65 |
| L5 | 0.1 | 0.1 | 16 | 133.4 | 0.22 | 65 |
| S1 | 0.1 | 0.1 | 20 | 166.8 | 0.21 | 90 |
| S2 | 0.9 | 0.6 | 25 | 188.2 | 0.11 | 74 |
| S3 | 0.1 | 0.1 | 9 | 265.4 | 0.55 | 85 |
Note: Day 0 parameters are prior to induction of septic arthritis and synovitis. Day 1 parameters are 24 h following induction of septic arthritis and synovitis and prior to arthroscopic lavage.
Abbreviations: RBC, red blood cells; TNCC, total nucleated cell count; TP, total protein.
3.2. Bacterial culture of synovial fluid samples
All cultures from all horses were negative at day 0 (n = 11). The LPS non‐septic synovitis group all (n = 4) had negative cultures at day 1. All horses in the induced septic arthritis groups had positive bacterial cultures of either S. aureus (n = 3) or E. coli (n = 4) on day 1 prior to arthroscopic lavage. Bacterial cultures on all horses in the LPS group (n = 4) remained negative on day 2 post lavage. In the E. coli septic arthritis groups 50% (2/4) of horses had positive cultures on day 2. The S. aureus septic arthritis group horses all had positive cultures (n = 3) on day 2 (Table 2).
TABLE 2.
Bacterial culture results by horse, reported as positive (Y) or negative (N) for the inoculated pathogen pre‐ and post‐arthroscopic lavage.
| Horse ID | Pathogen | Day 1 (pre‐lavage) | Day 2 (post‐lavage) |
|---|---|---|---|
| E1 | E. coli | Y | Y |
| E2 | E. coli | Y | N |
| E4 | E. coli | Y | N |
| E5 | E. coli | Y | Y |
| S1 | S. aureus | Y | Y |
| S2 | S. aureus | Y | Y |
| S3 | S. aureus | Y | Y |
| L1 | LPS | N | N |
| L3 | LPS | N | N |
| L4 | LPS | N | N |
| L5 | LPS | N | N |
Abbreviations: LPS, lipopolysaccharide; N, negative; Y, positive.
3.3. Bacterial culture of arthroscopic lavage fluid
Cultures of LPS horses (n = 4) were negative at every timepoint during arthroscopic lavage. After 1 L of lavage 50% (2/4) of the E. coli septic arthritis horses and 100% (3/3) of the S. aureus septic arthritis horses had a positive culture. The predicted probability of a positive culture after 1 L was 44.7% (95% CI: 3.2–86.1%, p = .04) for E. coli and 98.3% (95% CI: 91.5–105.1%, p ≤ .001) for S. aureus. At the 5 L lavage timepoint 25% (1/4) of the E. coli group horses and 67% (2/3) of the S. aureus horses had positive cultures. This corresponded to a positive predicted probability of 42.6% (95% CI: 6.2–79%, p = .02) and 94.3% (95% CI: 77.2–111.4%, p ≤ .001), respectively for each group. At the 10 L lavage two horses, one from each of the septic groups (Horse E5 and Horse S3) had a positive culture after being negative at the 5 L timepoint. This resulted in 50% (2/4) of the E. coli and 100% (3/3) S. aureus horses having a positive culture. For E. coli the positive predicted probability at 10 L was 40.1% (95% CI: 6.4–73.7%, p = .02) and for S. aureus it was 77.8% (95% CI: 37.7–117.8%, p = .001). At the 15 L timepoint 50% (2/4) of E. coli horses and 33% (1/3) of the S. aureus horses had positive cultures. The positive predicted probability at 15 L was 37.6% (95% CI: 2.2–73.0%, p = .04) and 42.5% (95% CI: 0.0–89.6%, p = .08) for each group respectively. At the final timepoint of 20 L, 25% (1/4) of the E. coli horses and 0% (0/3) S. aureus horses had positive cultures. The positive predicted probability at 20 L was 35.2% (95% CI: 0.0–75.6%, p = .09) and 13.5% (95% CI: 0.0–40.3%, p = .32) for each group, respectively. Individual horse data is represented in Tables 3 and 4.
TABLE 3.
Bacterial culture by volume of lavage for individual horses with percentages of positive culture (Y) reported by group for each timepoint.
| Horse ID | Pathogen | 0 L (pre‐lavage) | 1 L | 5 L | 10 L | 15 L | 20 L |
|---|---|---|---|---|---|---|---|
| E1 | E. coli | Y | Y | Y | Y | Y | Y |
| E2 | E. coli | Y | N | N | N | N | N |
| E4 | E. coli | Y | N | N | N | N | N |
| E5 | E. coli | Y | Y | N | Y | Y | N |
| 100% | 50% | 25% | 50% | 50% | 25% | ||
| S1 | S. aureus | Y | Y | Y | Y | N | N |
| S2 | S. aureus | Y | Y | Y | Y | Y | N |
| S3 | S. aureus | Y | Y | N | Y | N | N |
| 100% | 100% | 67% | 100% | 33% | 0% | ||
| L1 | LPS | N | N | N | N | N | N |
| L3 | LPS | N | N | N | N | N | N |
| L4 | LPS | N | N | N | N | N | N |
| L5 | LPS | N | N | N | N | N | N |
| 0% | 0% | 0% | 0% | 0% | 0% |
Abbreviations: LPS, lipopolysaccharide; N, negative; Y, positive.
TABLE 4.
Predicted probability of a positive culture after various lavage volumes generated using a GEE model.
| Pathogen | Lavage (L) | Predicted probability | 95% CI | Interval | p‐value |
|---|---|---|---|---|---|
| E. coli | 1 | 44.7% | 3.2% | 86.1% | .04 |
| E. coli | 5 | 42.6% | 6.2% | 79.0% | .02 |
| E. coli | 10 | 40.1% | 6.4% | 73.7% | .02 |
| E. coli | 15 | 37.6% | 2.2% | 73.0% | .04 |
| E. coli | 20 | 35.2% | 0.0% | 75.6% | .09 |
| S. aureus | 1 | 98.3% | 91.5% | 105.1% | <.001 |
| S. aureus | 5 | 94.3% | 77.2% | 111.4% | <.001 |
| S. aureus | 10 | 77.8% | 37.7% | 117.8% | <.001 |
| S. aureus | 15 | 42.5% | 0.0% | 89.6% | .08 |
| S. aureus | 20 | 13.5% | 0.0% | 40.3% | .32 |
Abbreviation: GEE, generalized estimating equation.
For the E. coli group the median of last positive culture was 10 L (range: 0–20 L) for lavage volume. In the S. aureus group the median of last positive culture was 12 L (range: 12–15 L) for lavage volume. Data for individual horses is represented in Table 5.
TABLE 5.
Last positive bacterial culture volume by individual horse with calculated median for each species of inoculate bacteria.
| Horse ID | Pathogen | Lavage positive |
|---|---|---|
| E1 | E. coli | 20 L |
| E2 | E. coli | 0 L |
| E4 | E. coli | 0 L |
| E5 | E. coli | 18 L |
| S1 | S. aureus | 12 L |
| S2 | S. aureus | 15 L |
| S3 | S. aureus | 12 L |
Note: Median: E. coli 10 L; S. aureus 12 L.
4. DISCUSSION
In this study, 20 L of lavage fluid were not sufficient to result in a sustained negative bacterial culture. All horses in the S. aureus group and 25% (1/4) horses in the E. coli group had negative bacterial cultures of egress fluid by the end of 20 L. However, all S. aureus horses and 50% (2/4) of the E. coli horses had positive synovial fluid bacterial cultures the day following arthroscopic lavage. These results support the previously recommended use of postoperative systemic antimicrobial therapy, local antimicrobial therapy, and/or IV regional limb perfusion as part of an aggressive multimodal treatment approach for septic arthritis. 1 , 2 , 4 , 5 , 20 , 21
In combination with joint lavage, systemic and local antimicrobial therapy is recommended to provide a robust treatment protocol in horses with septic arthritis. Regardless of the suspected organism, broad spectrum systemic antimicrobial therapy should be initiated as soon as a diagnosis of septic arthritis is made. 1 , 2 , 4 , 5 , 21 To achieve higher intra‐articular concentrations of antimicrobials the use of local delivery systems such as intra‐articular administration, regional limb perfusion, or intra‐articular antibiotic impregnated polymethylmethacrylate (PMMA) beads is advocated. 1 , 2 , 4 , 5 , 21 The results of this study highlight the importance of continued use of antimicrobial therapy postoperatively to eradicate infection in synovial structures as 71% (5/7) horses included in this study had positive bacterial cultures of synovial fluid day 2 post‐lavage.
The goal of high‐volume lavage in cases of septic arthritis is to achieve the physical removal of inflammatory cells and mediators, bacteria, fibrin, foreign material, and other debris. 1 , 2 , 4 , 29 A balanced polyionic electrolyte solution is the fluid of choice for high‐volume lavage 4 , 10 , 13 , 29 , 30 and that lavage should continue until all areas of the synovial cavity are clear. 10 , 29 In this study, 20 L of balanced polyionic electrolyte solution was utilized based on a previously published study on horses with experimentally induced septic arthritis and synovitis. 16
One horse in the E. coli group (E5) and one horse in the S. aureus group (S3) had negative bacterial cultures following 5 L of lavage volume. However, both horses had positive bacterial cultures following 10 L of lavage volume. One possible explanation for this is that after 5 L of lavage volume, all free‐floating bacteria may have been cleared from the joint space; however, bacteria entrapped in hyperplastic synovium may have still been present 13 , 29 and required an increased volume of lavage to be cleared. Previous studies have shown that multiple bacterial species, including E. coli and S. aureus, produce pathogenicity factors such as adhesion molecules and biofilms, which allow them to persist in synovial space and elude culture. The exact mechanisms are not completely understood, and this study also demonstrates the importance of further research to understand these mechanisms. 34 , 35
Horses in this study did not have a synovectomy performed. Synovectomy of hyperplastic, infected, inflamed or hyperemic synovium has previously been advocated for in septic arthritis to decrease bacterial count and reduce the production of inflammatory mediators. 5 , 11 , 13 All horses in this study were documented to have hyperplastic synovium during surgery. Therefore, future studies could include synovectomy to determine if it has an impact on the volume of lavage required for a negative bacterial culture.
The type of bacteria, chronicity of infection, and individual horse variation may impact the volume required to achieve a negative culture at the end of arthroscopic lavage. This study only utilized S. aureus and E. coli isolated from clinical cases for the induction of septic arthritis. These are the most common bacterial species present in cases of septic arthritis in adult horses. 1 , 3 , 23 , 24 However, other species may be present 14 , 24 , 25 and may respond differently to lavage volume. All horses in this study received the same inoculation dose of bacteria based on previously reported models of induced synovitis and septic arthritis, 16 , 18 , 22 therefore we cannot predict how varying bacterial loads impact the volume of lavage required for a negative bacterial culture. All horses in this study were treated 24 h following induction of synovitis and septic arthritis while in the acute phase of sepsis. One study found that time to referral and time from admission to surgery had no association with survival. 26 Similarly, another study did not find any association between time from contamination and presentation and survival. 6 Based on these previous reports, chronicity of septic arthritis may not influence survival, but there are no reports on how chronicity impacts the volume of lavage required during arthroscopic lavage. The current study cannot be used to predict how much volume is needed for a negative bacterial culture in chronic cases of septic arthritis as the presence of fibrin, pannus, or increased synovial hyperplasia may influence the volume required to achieve a negative bacterial culture.
The findings in this study contrast with a report where most microspheres in a tarsocrural joint were retrieved following 1 L of lavage volume. 15 It also contrasts a similar report where most microspheres in the metacarpo−/metatarsophalangeal joint were recovered in the first 2 L of egress fluid. 31 Factors accounting for the differences may include the fact that the middle carpal joints in the current study were of live horses with septic arthritis and synovitis as opposed to being normal cadaver limbs with synovial structures unaffected by disease. It is also possible that the microspheres in that study did not display the same behavior as bacteria within the joint during arthroscopic lavage such as the ability to attach to hyperplastic synovium. The microspheres utilized in both of those studies were 15 μm, the previously reported size of S. aureus is between 0.5–1.0 μm in diameter and this bacterium grows in clusters, pairs, or short chains. 32 The previously reported size of E. coli is 1.0–2.0 μm long with a radius of 0.5 μm. 33 The size difference between the microspheres and the live bacterium could also account for the differences. It also contrasts previous recommendations which suggest 2–6 L of lavage volume may be adequate in cases of septic arthritis. 2 , 27 , 28 These previous reports all recommend joint lavage as part of a multimodal treatment plan, including antimicrobial therapy, as treatment for septic arthritis. Horses may improve in clinical cases due to this multimodal approach despite the fact that all bacteria are not cleared from the joint during a low volume lavage.
The results of this study highlight the importance of postoperative synovial fluid sampling to help guide treatment decisions in cases of septic arthritis. Despite negative intraoperative lavage fluid bacterial cultures, 100% (3/3) of the S. aureus horses and 50% (2/4) of the E. coli horses had positive synovial fluid bacterial cultures on day 1 postoperatively. A study on experimentally induced septic arthritis in horses determined that total nucleated cell count, bacterial culture, and polymerase chain reaction (PCR) can be used to accurately indicate the elimination of an infection in horses with septic arthritis. 18
The main limitation in this study was small group sizes. Confidence intervals for predicted probabilities were very wide because of small sample size; subsequently statistical significance should be interpreted cautiously. Limitations associated with small group numbers resulted in this study having lower power to detect a difference that might have been anticipated with larger groups. Due to the nature of this study being an invasive experimental protocol which the horses were not euthanized after there was a limitation regarding the number of horses that could be included due to the animal use protocols at the investigating institution. Future studies with larger groups would improve the statistical power and may result in narrower confidence intervals. Another limitation of the study was that during arthroscopic lavage only the dorsolateral approach to the middle carpal joint was utilized. The palmar compartment of the joint was not entered in this study; future studies could utilize both a dorsolateral approach and palmar approach to the middle carpal joint for lavage to determine if there is any effect on volume required for a negative bacterial culture.
In conclusion, this study highlights that although 20 L of lavage fluid may be adequate to achieve a short‐term negative bacterial culture of egress fluid in some cases of septic arthritis, it cannot be relied upon to consistently achieve these results. An aggressive multimodal approach is advocated for due to the finding that even horses with a negative culture at the conclusion of surgery had positive bacterial cultures the following day. Further research is needed to determine the influence of lavage volume on bacterial culture in cases of septic arthritis.
AUTHOR CONTRIBUTIONS
Friedlund AM, DVM: Contributed to study execution, data analysis and interpretation, and preparation of the manuscript. Thomas KL, DVM, MSc, DACVS (Large Animal): Contributed to study design, study execution, data analysis and interpretation, and preparation of the manuscript. Bracamonte JL, DVM, DVSc, DECVS, DACVS (Large Animal): Contributed to study design, study execution, data analysis and interpretation, and preparation of the manuscript. Koziy RV, DVM, PhD, DACVP: Contributed to study execution, data analysis and interpretation. Epp T, DVM, PhD: Contributed to data analysis and interpretation. All authors gave their final approval of the manuscript.
CONFLICT OF INTEREST STATEMENT
The authors declare no conflicts of interest.
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