The influence of perfusate volume on antimicrobial concentration in synovial fluid following intravenous regional limb perfusion in the standing horse

Abstract

This study investigated the influence of perfusate volume on antimicrobial concentration in synovial fluid following intravenous regional limb perfusion (IVRLP) and assessed the efficacy of low volume IVRLP. The front limbs of 9 horses were randomly assigned to 1 of 3 volume groups: 10 mL (Group 1), 30 mL (Group 2), or 60 mL (Group 3). A tourniquet was applied distal to the carpus and the limbs were perfused with 500 mg genta-micin diluted to the assigned volume via a catheter placed in the lateral palmar digital vein at the level of the proximal sesamoid bones. Synovial fluid samples were collected from the metacarpophalangeal joint at 30 minutes, followed by removal of the tourniquet. Gentamicin concentration in synovial fluid was detected using a fluorescence polarization immunoassay. There were no statistically significant differences among gentamicin concentrations in synovial fluid among perfusate volume groups. Mean gentamicin concentration in Group 1 (125.9 μg/mL) was higher than Group 2 (82.7 μg/mL) and Group 3 (56.1 μg/mL).

Introduction

Septic synovitis and osteomyelitis are serious, debilitating diseases that are difficult and costly to treat (1–4). Effective therapeutic resolution depends on the use of an anti-microbial, to which the offending pathogens are susceptible, at a concentration greater than the minimum inhibitory concentration (MIC) at the site of infection (5,6). Intravenous regional limb perfusion (IVRLP) in the horse was first described in 1990 as a method of delivering high antimicrobial concentrations to infected tissues and synovial structures (7). Since then the technique has become a popular adjunctive therapeutic modality in dealing with these diseases and has been associated with positive outcomes (1,8,9). The advantages of IVRLP over systemic administration of antibiotics include administration of a lower drug dose while potentially achieving higher local concentrations and avoidance of systemic side effects (10,11).

In the basic form, IVRLP involves the placement of a tourniquet on the limb to achieve occlusion of the superficial vasculature and injection of an antimicrobial solution into a peripheral vein distal to the tourniquet (12). Several published studies in human and equine veterinary literature have examined the effectiveness of IVRLP (2,6,10,13) and compared the technique to other forms of local antimicrobial therapy (3,11,14–16). Variables have been identified that influence the success of IVRLP, including tourniquet properties (2,6), patient characteristics (10), and perfusate features, including antimicrobial selection (8,13) and volume (17). Intravenous regional limb perfusion of the distal limb has been performed with perfusate volumes of 20 mL (8,9) to 250 mL (18), with 60 mL being the choice of many authors (1,2,8,11,13–15). The ideal perfusate volume is unknown (12). One theory suggests that the greater the volume used, the higher the intravascular pressure achieved and subsequently better drug diffusion to the surrounding tissues (12). However, it is also possible that too great a volume may result in intravascular pressure that exceeds effective tourniquet pressure, resulting in perfusate leakage under the tourniquet (19) or extravasation at the site of injection (13).

The objective of this study was to determine if perfusate volume affects the concentration of gentamicin in synovial fluid following IVRLP and to investigate if clinically useful concentrations can be obtained with low volume IVRLP. It was hypothesized that low perfusate volume would be effective in achieving gentamicin synovial fluid concentration greater than MIC but low perfusate volume would not achieve as high gentamicin concentration in synovial fluid as higher perfusate volumes.

Materials and methods

Nine healthy adult mares of various breeds (mean age, 9 y; range: 4 to 15 y; mean weight, 485 kg; range: 417 to 553 kg) were included in the study. All horses were normal on physical examination and had no clinical signs of forelimb lameness. The project was conducted in a manner consistent with the US National Institutes of Health “Guide to Care and Use of Laboratory Animals” and the Animal Welfare Acts (United States Public Laws 89–544, 91–579, 94–279).

Intravenous regional limb perfusions were performed bilaterally at the same time in the 9 subjects (18 forelimbs). Each fore-limb was randomly assigned to 1 of 3 groups (6 limbs/group). Five hundred milligrams of gentamicin sulfate (GentaFuse, Butler Schein Animal Health, Dublin, Ohio, USA) were added to sterile saline solution to a total perfusate volume of 10 mL for Group 1, 30 mL for Group 2 and 60 mL for Group 3. Horses were sedated with detomidine hydrochloride (Dormosedan; Orion Corporation, Espoo, Finland), 0.01 mg/kg body weight (BW), intravenously (IV) and butorphanol tartrate (Torbugesic; Fort Dodge Animal Health, Fort Dodge, Iowa, USA), 0.01 mg/kg BW, IV. The area over the site of injection was clipped and aseptically prepared. A 15.2 cm × 2.7 m esmarch bandage (Medline Industries, Mundelein, Illinois, USA) was applied 10 cm distal to the accessory carpal bone. To ensure consistent tourniquet placement among patients, tourniquet pressure was measured with a custom-made device consisting of an aneroid gauge attached to a cuff (23 cm × 10 cm) filled with 50 mL of air (Figure 1) placed beneath the tourniquet. Prior to commencing the study, the tourniquet pressure measuring device was placed on the limbs of 3 horses and the esmarch tourniquet applied. Average limb occlusion pressure was determined by observing the cessation of arterial blood flow on Doppler ultrasound (180 mmHg for the device used). Tourniquets were applied to a pressure of 280 mmHg on all limbs during the study to allow for increased intravenous blood pressure during injection. Limbs were not exsanguinated prior to injection. A 22G, 32 mm IV catheter (Abbocath-T; Hospira, Sligo, Ireland) was placed in the lateral palmar digital vein at the level of the proximal sesamoid bones. Following removal of the stylet, a 76-cm, 4-mL volume, perfusate-primed extension set (Extension Set; Hospira, Lake Forest, Illinois, USA) attached to a syringe with the remaining perfusate was connected to the catheter and the perfusate was injected over 1 to 3 min. The extension set was flushed with 4 mL of air at the end of the injection and the sliding clamp of the extension set was applied. Catheters and extension sets were secured to the limb with a conforming bandage (Vetrap; 3M, St. Paul, Minnesota, USA) and remained in place for the duration of the perfusion. Tourniquets were removed 30 min after injection followed by removal of the catheter. To reduce post-procedure swelling, a 5-cm long ribbon of 1% diclofenac sodium cream (Surpass; Boehringer Ingelheim Vetmedica, St. Joseph, Missouri, USA) was applied topically to the site of catheter placement following removal of the catheter and a temporary pressure bandage was applied. Horses that showed signs of movement or arousal during the procedure were administered additional sedation.

Figure 1.

Device for measurement of sub-tourniquet pressure.

A single synovial fluid sample was obtained immediately before removal of the tourniquet for all limbs. The metacarpophalangeal joint was aseptically prepared and a 20G needle was inserted into the dorsal metacarpophalangeal joint pouch. The needle was introduced under the lateral edge of the common digital extensor tendon and directed medially and parallel to the frontal plane of the joint so synovial fluid could be gently aspirated.

All synovial fluid samples were frozen at −20°C for 48 to 52 h until analysis. Gentamicin concentrations were detected using a validated fluorescence polarization immunoassay (Dimension Xpand; Siemens, Illinois, USA). The lowest limit of detection using the fluorescence polarization immunoassay was 0.27 μg/mL. Prior to sample analysis, accuracy of the immunoassay was determined by testing 6 blank synovial fluid samples that had been spiked with a known amount of gentamicin sulfate. The coefficient of variation ranged from 2.1% to 9.4%. Samples to be analyzed were diluted with blank synovial fluid if outside the limit of the analyzer.

Gentamicin concentrations in synovial fluid samples were compared among groups using software for statistical analysis (STATA; StataCorp, College Station, Texas, USA). Data were assessed for normality by use of a Shapiro-Wilk test and then analyzed by use of Mann-Whitney rank-sum analysis. Bonferroni correction was performed for multiple comparisons. Values of P < 0.05 were considered significant.

Results

No complications were observed in association with the procedure. Four of the 9 horses required additional sedation during IVRLP due to arousal and movement. The remaining 5 horses displayed only occasional weight-shifting or resting of a limb during the procedure. All horses were examined the day after the procedure, swelling was not detected at the site of catheter placement and lameness was not observed.

Mean synovial fluid concentration of gentamicin was highest in Group 1 (mean 125.9 μg/mL; range: 6.3 to 433.3 μg/mL), followed by Group 2 (mean 82.7 μg/mL; range: 0.6 to 140.4 μg/mL), and Group 3 (mean 56.1 μg/mL; range: 0.4 to 230.3 μg/mL). The differences were not statistically significant (Group 1 versus Group 2: P = 0.75, Group 1 versus Group 3: P = 0.63 and Group 2 versus Group 3: P = 0.42).

Discussion

The perfusate volumes used in this study did not significantly affect gentamicin concentration in synovial fluid following IVRLP. The lowest perfusate volume of 10 mL achieved the highest mean gentamicin concentration in synovial fluid and although statistical significance was not found, a trend was seen for higher mean synovial fluid antimicrobial concentration with lower perfusate volumes. This finding suggests that diffusion down a concentration gradient (12) may be equally if not more important than venous distention (12) in the dissemination of an antimicrobial during IVRLP. Disadvantages to the use of large perfusate volumes include the considerable force required to administer the solution in the face of high intravascular pressure, the risk of perivascular leakage and longer administration time, as a steady rate of injection during IVRLP is recommended (13,19).

The antimicrobial solution becomes more concentrated with decreasing total perfusate volume, this may increase the risk of vasculitis occurring. Clinical signs of vasculitis were not seen in this study and have not been reported in IVRLP studies with aminoglycoside antibiotics. Parra-Sanchez et al (13) described the occurrence of perivascular edema and cellulitis in 3/7 horses undergoing IVRLP with enrofloxacin, but attributed this to extravasation due to high hydrostatic pressure, in which a total perfusate volume of 60 mL was administered. If greater drug dilution is desired, exsanguination of the limb may be indicated prior to IVRLP to allow a greater perfusate volume to be administered without complication.

Limb exsanguination prior to IVRLP is usually performed in human patients (17,20) and has been shown to reduce tourniquet leakage (19,21). In the horse, limb exsanguination can be performed by the application of an esmarch bandage proximally starting at the level of the hoof capsule or distal tourniquet where 2 tourniquets are used (12,22). Alternatively, a volume of blood equal to or greater than the perfusate volume could be drawn out of the limb following tourniquet placement. Limb exsanguination does not appear to be commonly performed in clinical practice, but may help achieve consistently higher antimicrobial concentrations during IVRLP in horses.

In this study an over-the-needle catheter was used in place of the commonly used butterfly catheter. When used, the butterfly catheter is removed following perfusate injection, which can allow perfusate leakage through the needle tract due to high intravenous pressure and has been associated with hematoma formation (10). With the over-the-needle catheter, the infusion system can remain in place for the duration of the perfusion and be removed after the tourniquet has been released and time allowed for equilibration of intravenous pressure. It is our and others (11) clinical impression that this results in decreased perfusate extravasation and also injection site swelling post perfusion. Additionally, application of 1% diclofenac sodium topical cream to the site of IVRP, as performed in this study, has also been shown to decrease inflammation associated with the procedure (23).

Aminoglycoside antibiotics are commonly used in IVRLP (3,8,11–13). Gentamicin was selected for evaluation in this study because of availability and affordability. Approximately 70% to 84.5% of bacterial isolates from septic joints are susceptible to gentamicin (24). Minimum inhibitory concentration (MIC) of gentamicin for common bacterial isolates in the horse is 2 μg/mL (25) and maximal antibacterial effect is seen at concentrations 10 times the MIC for concentration dependent antimicrobials (11). In this study 1 horse in Group 1 (16.66%) and 2 horses in both groups 2 and 3 (33.33%) did not achieve synovial fluid concentrations > 20 μg/mL. Failure to achieve desired antimicrobial concentration in target tissues (8,10,13,26) and wide variation in antimicrobial concentrations achieved following IVRLP (2,3,6,12,14,15) have been well-documented and attributed to many factors including inadequate tournquet pressure (13) or placement (6), low antimicrobial dose (13), rate and site of injection (19), individual variation in limb vasculature (8), movement of the animal and body weight discrepancies affecting dose (6).

Antimicrobial dose and perfusate volume were not calculated according to the weight of each individual animal in this study. This may have contributed to the wide variation in the results, although variation in concentration was not reduced despite determining antimicrobial dose on a mg/kg basis (13,27) nor when perfusate volume was determined on a mL/kg basis (27) in other studies.

In this study, the intent was to imitate a clinical situation in which an esmarch tourniquet would be used for vascular occlusion. The esmarch tourniquet is effective for IVRLP providing the tourniquet is sufficiently wide (2,6); however, the application of this type of tourniquet cannot be standardized. We attempted to reduce variability associated with the esmarch tourniquet application by the measuring sub-tourniquet pressure in subjects. A commercial device for this purpose could not be obtained; therefore, the device described was constructed and appeared to serve its purpose. It is possible that the tourniquet pressure used was not adequate to prevent tourniquet leakage during perfusate injection and this could have been a reason for low gentamicin concentrations in synovial fluid following IVRLP in some limbs.

Limbs were perfused with 500 mg gentamicin (approximately 14% to 18% of daily systemic dose), which is less than a published recommendation of 33% the daily systemic dose of antimicrobial when performing IVRLP (10). This recommendation came from a study in which the tourniquet was placed proximal to the carpus; therefore, we thought it appropriate to use a lower dose for performing a distal limb perfusion. As less than desired synovial fluid gentamicin concentrations were obtained in several metacarpophalangeal joints in this study, we conclude that it is advisable to use a higher antimicrobial dose even for lower limb perfusions to ensure that high antimicrobial concentrations will be achieved in target tissues.

Movement was observed in all horses in this study ranging from an occasional shift in weight to pawing, likely associated with tourniquet-related pain. Movement of the patient increases peripheral vascular resistance in the distal limb (11) which can result in intravascular pressures greater than effective tourniquet pressure and tourniquet leakage (18). It is difficult to prevent movement during IVRLP in the standing horse, but it can be reduced with adequate sedation, and although not used in this study, possibly through the use of local analgesia, either by peripheral nerve block proximal to the tourniquet (2) or by addition of local anesthetic agent to the perfusate (28). Widely varying and low overall antimicrobial concentrations have also been reported while performing IVRLP under general anesthesia (8,10,14,15), so while patient movement is not the sole cause of low antimicrobial concentration following IVRLP, it is an additional contributory factor that warrants consideration in a clinical setting.

In our opinion it is likely that a combination of these factors led to the wide variability and low gentamicin concentrations found in synovial fluid following IVRLP in some limbs in this study. Our results and those of other IVRLP studies indicate that while IVRLP usually is successful in achieving high antimicrobial concentrations in perfused tissue, the technique appears to be inherently unreliable and other techniques of local antimicrobial therapy should be considered. Other techniques include direct intra-synovial injection or continuous infusion of antimicrobials through an indwelling catheter in a synovial cavity, implantation of antimicrobial impregnated material (polymethylmethacrelate, plaster of paris, collagen, hydroxyapatite, polyanhydride, hyaluronan) into infected tissue, or regional intraosseous perfusion. The advantages, disadvantages, and comparisons of these different techniques have been described previously (3,14–16,26,18,29–31).

There are flaws in the study design reported here, in addition to the factors previously mentioned. Multiple limb perfusions were performed simultaneously, similar to other studies (3,6,8). The advantage of this over unilateral limb perfusion for research purposes is that a greater number of perfusions can be performed in a small population of horses. Alternatively, half of the perfusions could have been performed unilaterally in 9 horses, and the contralateral limbs perfused following observation of a wash-out period for gentamicin. While performing bilateral limb perfusions, tourniquet leakage could have occurred resulting in a rise in systemic gentamicin serum concentrations. While possible, we think it is unlikely that systemic concentrations of gentamicin would have increased the synovial fluid gentamicin concentration in the contralateral forelimb and metacarpophalangeal joint, as tourniquet leakage occurs due to venous pressure exceeding tourniquet pressure during injection (17,19) rather than arterial flow under the tourniquet. Although synovial fluid samples were drawn before tourniquet removal in an attempt to reduce the influence of systemic gentamicin concentrations on the synovial fluid sample, the potential still exists for systemic antimicrobial influence on metacarpophalangeal synovial fluid.

Normal horses without synovitis of the metacarpophalangeal joint were studied. The presence of synovitis has been shown to result in earlier and higher maximal concentration of amikacin following IVRLP when compared to normal joints (2).

Pre-treatment synovial fluid samples were not obtained to assess background signal for gentamicin activity on the assay used. It is our observation that penetration of the joint capsule with a hypodermic needle often causes hemorrhage within the joint. We were concerned that this could falsely increase the gentamicin concentration in synovial fluid in the post-perfusion samples. Horses used in this study had not received gentamicin sulfate or other medications within 3 months of the study; however, limitations of the fluorescence polarization immunoassay due to a high degree of variability and false positive results have been reported (3) and would not have been recognized in this study.

In conclusion, we proved the hypothesis that the use of low perfusate volumes during IVRLP can be effective in achieving synovial fluid antimicrobial concentrations greater than MIC; however, our second hypothesis that low volume IVRLP would be less effective than higher perfusate volumes was disproven. Low volume IVRLP may be beneficial in reducing antimicrobial loss through tourniquet leakage or extravasation. There does not appear to be any negative effects from this practice and it allows for faster and easier perfusate administration. Perfusate volume does not appear to significantly affect concentrations of gentamicin in synovial fluid following IVRLP. CVJ