Abstract
Objective—To evaluate the effectiveness of a novel trimethoprim-sulfadiazine oral suspension for the treatment of naturally acquired Streptococcus equi subsp zooepidemicus infection in horses.
Design—Randomized, controlled field trial.
Animals—180 horses with S equi subsp zooepidemicus infection.
Procedures—Horses with lower respiratory tract infections caused by S equi subsp zooepidemicus were treated with a new formulation of combined trimethoprim-sulfadiazine oral suspension at a dosage of 24 mg/kg (10.9 mg/lb) twice daily for 10 days (treatment group) or with an equivalent volume of saline (0.9% NaCl) solution (placebo group). Response to treatment, including clinical signs and fecal consistency scores, was assessed twice daily. Any adverse effects were recorded. The primary outcome variable was clinical response; the secondary outcome variable was eradication of S equi subsp zooepidemicus on study day 17 as determined by bacteriologic culture of repeated transtracheal-wash specimens.
Results—Of the 119 horses allocated to the treatment group, 69 (58%) had a positive clinical response. A significantly smaller proportion of horses in the placebo group (9/61 [15%]) had a positive clinical response. By day 5, 25 of 61 (41%) placebo horses had been withdrawn from the study because of negative clinical response, compared with only 10 of 119 (8.4%) treated horses. By day 10, 28 of 61 (46%) placebo horses had been withdrawn because of negative clinical response, compared with only 13 of 119 (11%) treated horses. There were few adverse events associated with the trimethoprim-sulfadiazine suspension. There were no significant differences in fecal consistency scores between treatment and placebo groups.
Conclusions and Clinical Relevance—The new oral suspension administered at 24 mg/kg twice daily effectively treated the clinical signs of S equi subsp zooepidemicus lower respiratory infection in horses and eliminated the organism from the respiratory tract. Adverse effects were minimal.
Sulfonamides, including sulfamethazine, sulfamethoxazole, and sulfadiazine, are some of the oldest antimicrobials available and are still commonly used in equine practice. Sulfonamides inhibit folate synthesis by competitive antagonism of p-aminobenzoic acid, thus inhibiting bacterial DNA synthesis.1,2 To overcome antimicrobial resistance, sulfonamides are often used in combination with a pyrimidine, in which case they are considered potentiated sulfonamides. Trimethoprim is a diaminopyrimidine that inhibits folate synthesis by binding dihydrofolate reductase, inhibiting the reduction of dihydrofolic acid to tetrahydrofolic acid. This step is downstream from the point of action of sulfonamides in the synthesis of folate, allowing the 2 antimicrobials to function synergistically.1,2 The combination of a sulfonamide with trimethoprim results in bactericidal activity with lower MICs for each drug.1
Potentiated sulfonamides are routinely used in equine practice because they have a broad spectrum of bactericidal activity.3 When administered at a dosage of 30 mg/kg (13.6 mg/lb) orally twice daily, therapeutic plasma concentrations are maintained.4 A study5 evaluating trimethoprim-sulfadiazine paste administered orally to healthy adult ponies indicated that a dosage of 30 mg/kg administered twice daily results in continuous therapeutic concentrations in subcutaneous tissue chambers for 12 hours. The combination is administered at a 1:5 ratio of trimethoprim to sulfonamide1,2 to achieve the desired in vivo plasma concentration ratio for most susceptible organisms, which is approximately 1:20.1,6
Streptococcus equi subsp zooepidemicus is one of the most common bacterial pathogens of horses. It is routinely isolated from wounds and from infections of the urogenital tract and lower respiratory tract of fetuses, foals, and adults.7–12 The organism is an opportunistic pathogen and is commensal for horses, causing infection following injury, compromise of normal urogenital tract defense mechanisms, or respiratory compromise following viral pneumonia or other stressors.13–17 In 1 study,18 S equi subsp zooepidemicus was found in 94.5% of equine respiratory tract infections. In 2009, the trimethoprim-sulfadiazine MIC90 of S equi subsp zooepidemicus isolates obtained by bacteriologic culture of transtracheal-wash fluid from the lower respiratory tract of horses was reported to be 0.12 μg/mL for trimethoprim and 2.38 μg/mL for sulfadiazine.19 A pilot efficacy studya of a novel trimethoprim-sulfadiazine oral suspension found an MIC90 of 0.12 μg of trimethoprim/mL and 2.4 μg of sulfadiazine/mL for S equi subsp zooepidemicus, agreeing with published results from previously approved formulations of the drug.19 With this new oral suspension administered at a dose of 24 mg/kg (10.9 mg/lb), the concentration exceeded the MIC90 100% of the time for sulfadiazine and 98% of the time for trimethoprim in a pilot studyb in horses with respiratory disease. The suspension is a new formulation with high bioavailabilityb that is different from previously approved formulations; therefore, a new animal drug application was indicated.
The purpose of the study reported here was to evaluate the effectiveness of this new trimethoprim-sulfadiazine oral suspensionc for the treatment of naturally acquired S equi subsp zooepidemicus infection in horses under field conditions. The null hypothesis was that the outcome variables would be equal between treatment and placebo groups.
Materials and Methods
This study was a broad field trial that was conducted and submitted to the US FDA Center for Veterinary Medicine as part of the approval process for new drugs.20 The study was conducted at 5 sites in the United States (Idaho, Iowa, Missouri, Oklahoma, and Tennessee). Horses > 1 year of age that appeared sick, stressed, or in poor condition were purchased by the investigators from public and private sources to serve as a candidate pool. Candidate horses commingled at each site and were observed frequently for signs of respiratory disease. Horses with disease were enrolled in the study after meeting prespecified clinical criteria for inclusion. The study protocol was developed with the concurrence of the US FDA Center for Veterinary Medicine and was approved by all relevant state and local institutional animal care and use committees. All veterinarians and staff participated in protocol training and review of good clinical practice for the conduct of clinical trials prior to commencement of the study.
Clinical case enrollment—To qualify for initial study enrollment horses had to exhibit the following signs of lower respiratory tract disease within an 8-hour observation period: a rectal temperature ≥ 38.33°C (101.0°F), a respiratory rate > 24 breaths/min, abnormal respiratory sounds on auscultation (without rebreathing), and an intermittent or sustained spontaneous cough. Exclusion criteria included pregnancy (determined by visual inspection) or lactation, age < 1 year, previous treatment for respiratory disease within 14 days or treatment with any antimicrobials within 30 days, liver disease (determined by serum biochemical analysis), or strangles (determined by visual inspection or presence of Streptococcus equi subsp equi on bacteriologic culture of transtracheal wash fluid). Horses refractory to handling were also excluded. Horses were enrolled individually once they met the inclusion criteria. Horses meeting all criteria for enrollment were sedated with detomidine hydrochlorided (20 μg/kg [9.1 mg/lb], IV) or xylazine hydrochloridee (1 mg/kg [0.45 mg/lb], IV) for transtracheal wash. Each transtracheal wash was performed aseptically in the midcervical region of the trachea with a commercially available devicef according to the manufacturer's directions. A 5-mL fluid sample was placed in a bacteriologic culture tubeg for transport. The transtracheal wash was used to confirm the presence of bacterial infection and for bacterial counts. A venous blood sample was collected and submitted to a laboratoryh for a CBC, serum biochemical analysis, and clotting profile. The objective of the clinicopathologic evaluation was to identify potential adverse effects of the drug. The horse's weight was estimated with a weight tape.
Clinical evaluations—Once enrolled, horses were individually housed to facilitate twice-daily observations and treatments. Treatment was initiated the day of enrollment (day 0). A veterinarian performed complete physical examinations on days 0, 5, 10, and 17 and on any other day a physical examination was indicated on the basis of daily observations. Rectal temperature, pulse rate, respiration rate, and attitude (eg, lethargy) were recorded along with an assessment of each body system. Any ocular or nasal discharge was documented but not scored. Clinical observations were performed once on day 0 and twice daily from day 1 through 17 to evaluate general health, attitude, fecal consistency, and appetite and to detect possible adverse reactions. Fecal consistency was scored during the clinical observations to determine potential antimicrobial-induced diarrhea (Appendix 1). Because this was a field study, the twice-daily observations were performed by independent personnel. All individuals recording the twice-daily results of clinical observations were blinded to study group. A scoring system was used to evaluate the parameters associated with respiratory disease (Appendix 2). For results of auscultation to be considered normal, use of a rebreathing bag was required. Any subjective observation of a decline in the health of the horse during the study as noted by the observer would trigger notification of the investigator (veterinarian), and a complete physical examination in addition to those scheduled. When a study horse was withdrawn because of negative clinical response for any reason at any time during the study, the veterinarian was allowed to use clinical judgment and any available microbiological and clinicopathologic data to provide appropriate intervention.
All initially enrolled horses that received at least 1 study intervention (treatment or placebo) were evaluated for adverse events. The occurrence of adverse events and evaluation of the clinicopathologic parameters determined the safety profile of the drug in field use. Any observation concerning the horses or the people treating them that was determined to be unfavorable or unintended was recorded, irrespective of treatment group. Humans were expected to self-report symptoms of adverse reactions.
Bacteriologic culture—All transtracheal-wash specimens were shipped by overnight courier to a commercial laboratoryi for bacterial isolation by means of semiquantitative methodology and characterization with standard laboratory methods. A loopful of the undiluted transtracheal-wash solution was streaked onto 5% sheep blood agar. The plate was cross-streaked for isolation to allow semiquantitation of the resulting pathogens. The blood agar was incubated with 5 ± 2% CO2 overnight at 36 ± 2°C. The primary isolation plates were scored according to the number of presumptive S equi subsp zooepidemicus organisms (0 = 0 colonies; 1 = 1 to 10 colonies; 2 = 11 to 100 colonies; 3 = 101 to 1,000 colonies; and 4 = > 1,000 colonies) on the basis of colony and bacterial morphology and Gram stain. Agar plates were evaluated for the presence of bacteria other than S equi subsp zooepidemicus, although these organisms were not identified or retained unless they were present with a score of 3 or 4 and were isolated from a horse in which clinical response was negative. Presumptive S equi subsp zooepidemicus and other bacteria isolated from horses withdrawn from the study because of negative clinical response were identified on bacteriologic culture and speciated by colony and cellular morphology, Gram stain reaction, and biochemical profile.j All isolates of S equi subsp zooepidemicus obtained from enrolled horses were subjected to definitive antimicrobial susceptibility testing and MIC determination by means of broth microdilution.k
Each isolate identified as S equi subsp zooepidemicus was tested to determine susceptibility to trimethoprim, sulfadiazine, and trimethoprim-sulfadiazine (1:19 ratio). Broth microdilution MIC tests were conducted with cation-adjusted Mueller-Hinton broth supplemented with 3% lysed horse blood. Minimum inhibitory concentration plates were incubated at 36 ± 2°C for 20.5 to 21.5 hours. Minimum inhibitory concentration plates were prepared with dilutions of trimethoprim (0.06 to 32 μg/mL), sulfadiazine (1 to 512 μg/mL), and a combination of trimethoprim (0.06 to 32 μg/mL) and sulfadiazine (1.2 to 608 μg/mL). The Clinical and Laboratory Standards Institute has established no antimicrobial susceptibility or resistance breakpoints for S equi subsp zooepidemicus to treatment with potentiated sulfonamides.21 The general breakpoint for antimicrobial resistance to potentiated sulfonamides in streptococci is > 4 μg of pyrimidine/mL and 76 μg of sulfonamide/mL.21
Allocation, blinding, and randomization—Once horses met the initial enrollment criteria, they were assigned to either a treatment or a placebo group at a 2:1 ratio (treatment to placebo) on the basis of a randomization schedule that was unique to each site (Figure 1). Horses were blocked within each site in the order of enrollment, with each block consisting of 3 horses. Each block was internally randomized, with 2 horses assigned to receive the novel trimethoprim-sulfadiazine oral suspensionc at a dosage of 24 mg/kg twice a day for 10 days (treatment group) and 1 assigned to receive an equivalent volume of saline (0.9% NaCl) solution (placebo group). Treatment or placebo group assignment and administration were done by personnel not performing clinical assessments, observations, or physical examinations, without the observer present to prevent unmasking attributable to visual differences between treatment and placebo. Visible residue was wiped from the animal after treatment, and clinical observations and physical examinations were performed no sooner than 1 hour after treatment as a further precaution against unintended unmasking. Blinding codes were maintained until the completion of statistical analysis. The trimethoprim-sulfadiazine oral suspension was administered at a dosage of 24 mg/kg of combined active ingredient rounded up to the nearest milliliter, twice daily for 10 days (20 treatments). The suspension contained 67 mg of trimethoprim/mL and 333 mg of sulfadiazine/mL. Saline solution was administered at an equivalent volume and timing (3.0 mL/50 kg [3 mL/110 lb], twice daily). All treatments were given orally, and any loss was recorded. There were no protocol requirements as to the administration of the treatment or placebo in relation to the time of feeding.
Study flow diagram for horses enrolled in a randomized, controlled, clinical field trial of a novel trimethoprim-sulfadiazine oral suspension for treatment of naturally acquired Streptococcus equi subsp zooepidemicus lower respiratory tract infection in horses. The study was conducted at 5 sites in the United States (Idaho, Iowa, Missouri, Oklahoma, and Tennessee). Horses > 1 year of age that appeared sick, stressed, or in poor condition were purchased by the investigators to serve as a candidate pool, commingled at each site, and observed frequently for signs of respiratory disease. Horses with disease were enrolled in the study after meeting prespecified clinical criteria for inclusion. Enrolled horses were randomized and allocated to receive either a trimethoprim-sulfadiazine oral suspension at a dosage of 24 mg/kg twice daily for 10 days (treatment group) or an equivalent volume of saline (0.9% NaCl) solution (placebo group). *A semiquantitative colony count of 101 to 1,000 colonies of S equi subsp zooepidemicus in pure culture or > 1,000 colonies in mixed culture from a single loop of undiluted day 0 transtracheal wash (TTW) fluid was required. Clinical criteria determined rescue (ie, failure; online supplement available at http://avmajournals.avma.org/toc/javma/246/12). †Data analyzed for efficacy were collected from horses on days they were withdrawn.
Citation: Journal of the American Veterinary Medical Association 246, 12; 10.2460/javma.246.12.1345
Horses were enrolled and treatment was begun before bacteriologic results were known; therefore, the number of horses enrolled and allocated was expected to exceed the number of horses that met inclusion criteria. The primary outcome variable was clinical response in the horses with an S equi subsp zooepidemicus score of 3 or 4 in pure culture or score of 4 in mixed culture for the transtracheal wash. To be considered to have had a positive clinical response, horses had to have a body temperature < 38.3°C (< 101°F), or at least 2.0°F lower than day 0, by day 5 and on all subsequent examinations, have a respiratory rate ≤ 24 breaths/min, have normal findings on thoracic auscultation, and have only an intermittent cough on days 5 and 10 with the same findings and no cough by day 17.
The secondary outcome variable was the elimination of S equi subsp zooepidemicus. To be included in the S equi subsp zooepidemicus elimination group, horses had to meet the clinical criteria and enrollment criteria for lower respiratory tract bacterial infection and have a semiquantitative colony count of 101 to 1,000 colonies of S equi subsp zooepidemicus in pure culture or > 1,000 colonies in mixed culture from a single loop of undiluted day 0 transtracheal-wash fluid. Horses in the treatment group with a microbial score of 0 for S equi subsp zooepidemicus on day 17 were considered to have elimination of S equi subsp zooepidemicus; however, effectiveness measures were based solely on clinical outcome.
Criteria for study removal and rescue treatment—If a horse had signs of severe respiratory distress, diarrhea, suspected allergic reaction (as determined on the basis of clinical judgment and signs such as urticaria or bloody nasal discharge) at any time or became severely lethargic in the first 4 days, it was withdrawn from the study and administered appropriate alternate treatment as directed by the observing veterinarian and current standards of veterinary care. Horses were also withdrawn if the clinical scores worsened at any time after the day 5 examination (Online Supplement available at http://avmajournals.avma.org/toc/javma/246/12). Horses either were withdrawn from the study because of negative clinical response or reached completion of the study on day 17. At withdrawal or completion, CBC, serum biochemical analysis, clotting profile, and transtracheal wash were repeated.
Statistical analysis—The primary outcome variable, positive clinical response at day 17 in horses with a microbial score for the transtracheal-wash specimens of 3 or 4 in pure culture or microbial score of 4 in mixed culture, was included in the statistical analysis of the effectiveness of treatment. Effectiveness was analyzed with a generalized linear mixed-model, with group (treatment or placebo) as a fixed effect. Study site and site X group interaction were included as random effects. Individual clinical sign scores recorded on days 5, 10, and 17 were evaluated in the same model with group, time, and group X time interaction as fixed effects. The clinical signs of auscultation and cough scores were included in the model by the frequency of each score. Study site and site X interaction were random effects. The secondary outcome variable, eradication of S equi subsp zooepidemicus on day 17, was compared between treatment and placebo groups by means of a Fisher exact test. Values of P ≤ 0.05 were considered significant.
For statistical analysis of the incidence of adverse events between the 2 groups, values of P ≤ 0.10 were considered significant. Adverse reactions were tabulated and compared between groups with a Fisher exact test. Fecal consistency was analyzed between groups by means of a Wilcoxon rank sum test. Vital signs after day 0, including body temperature, pulse, and respiratory rate, were analyzed with a mixed-model repeated-measures ANOVA with terms for group, time, and group X time interaction. Pretreatment data were included as a covariate. A compound symmetric covariance matrix was used to compare treatment and placebo groups. The clinicopathologic data were analyzed by means of an ANOVA with a term for the group (treatment or placebo). The day 0 data were included as a covariate. The groups were compared on the basis of linear contrasts. All statistical analyses were performed with a commercial software package.l
Results
A total of 270 horses met the initial clinical study inclusion criteria and were enrolled between June 2010 and March 2011.20 There were 39 horses enrolled in Idaho, 65 in Iowa, 5 in Missouri, 81 in Oklahoma, and 80 in Tennessee. Study animals had a median age of 3 years (range, 1 to 25 years). Seven pure breeds and 6 known breed crosses were represented among the study horses. The most common breeds represented were Quarter Horse (n = 110), American Paint Horse (37), Tennessee Walking Horse (24), and Paso Fino (9). The remaining 3 breeds (Appaloosa, Fox Trotter, and Arabian) were each represented by ≤ 5 individuals. Thirty-four horses were of known mixed breeding, and 49 were grade. There were 148 female, 57 sexually intact male, and 65 castrated male horses. Random allocation resulted in 182 horses receiving treatment and 88 receiving placebo. At the completion of the study, 180 horses were evaluated for the primary outcome variable, including 119 receiving the treatment and 61 receiving the placebo. The 90 horses not evaluated for the primary outcome variable were excluded because the pretreatment transtracheal-wash specimen did not yield the requisite number of S equi subsp zooepidemicus organisms. All the cases that met initial clinical criteria for enrollment were followed through the completion of the study for evaluation of adverse events.
On day 17, of the 119 horses receiving treatment, 69 (58%) had a positive clinical response; a significantly (P < 0.001) smaller proportion of horses in the placebo group (9/61 [15%]) had a positive clinical response, and therefore the null hypothesis of equality between groups was rejected. By day 5, 25 of 61 (41%) placebo horses had been withdrawn because of negative clinical response, compared with only 10 of 119 (8.4%) treated horses. By day 10, 28 of 61 (46%) placebo horses had been withdrawn because of negative clinical response, compared with only 13 of 119 (11%) treated horses. The remainder of the horses were withdrawn because of negative clinical response from day 11 to 17. The mean rectal temperature, respiratory rate, auscultation scores, and cough scores were significantly improved in the trimethoprim-sulfadiazine–treated horses, compared with those values in the placebo group, over the course of the study (Table 1).
Clinical variables evaluated in 180 horses with naturally acquired Streptococcus equi subsp zooepidemicus infection enrolled in a clinical field trial of a novel trimethoprim-sulfadiazine oral suspension and randomly allocated to either treatment with a novel oral suspension of trimethoprim-sulfadiazine at a dosage of 24 mg/kg twice a day for 10 days (n = 119; treatment group) or an equivalent volume of saline (0.9% NaCl) solution (61; placebo group).
| Day 0 | Day 5 | Day 10 | Day 17 | |||||
|---|---|---|---|---|---|---|---|---|
| Clinical sign | Treatment (n = 119) | Placebo (n = 61) | Treatment (n = 118) | Placebo (n = 61) | Treatment (n = 109) | Placebo (n = 36) | Treatment (n = 106) | Placebo (n = 33) |
| Rectal temperature (°C) | 39.28 ± 0.54 | 39.11 ± 0.54 | 37.78 ± 0.57 | 38.44 ± 0.87 | 37.5 ± 0.53 | 37.83 ± 0.71 | 37.61 ± 0.58 | 37.72 ± 0.94 |
| Respiratory rate (breath/min) | 34.1 ± 6.77 | 35.6 ± 8.75 | 23.4 ± 6.88 | 29.0 ± 7.56 | 21.8 ± 6.07 | 23.2 ± 5.0 | 20.4 ± 5.14 | 24.5 ± 6.0 |
| Auscultation score | ||||||||
| 0 | 0 | 0 | 84 | 23 | 91 | 21 | 95 | 20 |
| 2 | 119 | 61 | 34 | 38 | 18 | 15 | 11 | 13 |
| Coughing score | ||||||||
| 0 | 0 | 0 | 55 | 23 | 97 | 17 | 98 | 20 |
| 1 | 0 | 0 | 15 | 3 | 9 | 4 | 5 | 4 |
| 2 | 94 | 49 | 48 | 27 | 3 | 14 | 3 | 9 |
| 3 | 25 | 12 | 0 | 8 | 0 | 1 | 0 | 0 |
Values reported are means ± SD for temperature and respiratory rate and number of horses for auscultation score and coughing score. The distributions of scores for auscultation (Appendix 1) and coughing (Appendix 2) are reported. There was a significant (P = 0.002) interaction between time and rectal temperature, indicating clinical improvement of both groups over the study period; however, the treated group improved significantly faster. There was also a significant interaction between time and coughing scores with treated horses showing significant improvement on days 5 (P = 0.004), 10 (P < 0.001), and 17 (P < 0.001). Horses were evaluated for rescue on the basis of clinical variables (online supplement available at http://avmajournals.avma.org/toc/javma/246/12); data are reported for horses that were not rescued.
Of the 180 horses that were evaluable for the primary outcome variable of clinical response, 172 were evaluable for the secondary outcome variable of elimination of the organism. One horse was excluded because of incorrect data sampling, and 7 were excluded as a result of delays in shipping the day 17 sample to the laboratory for bacteriologic culture. This resulted in culture of S equi subsp zooepidemicus at levels high enough to evaluate for elimination of infection and evaluable posttreatment transtracheal washes in 116 trimethoprim-sulfadiazine–treated horses and 56 placebo horses. Microbial elimination occurred in a significantly (P < 0.001) greater proportion (76/116 [66%]) of the trimethoprim-sulfadiazine–treated horses than placebo horses (12/56 [21%]). The pretreatment MIC90 was 0.25 μg of trimethoprim/mL (MIC range, 0.12 to 0.5 μg/mL) and 4.75 μg of sulfadiazine/mL (MIC range, 2.4 to 9.5 μg/mL) for all horses with positive clinical outcome. For horses withdrawn because of negative clinical response, the MIC90 was also 0.25 μg of trimethoprim/mL (MIC range, 0.12 to 0.5 μg/mL) and 4.75 μg of sulfadiazine/mL (MIC range, 2.4 to 9.5 μg/mL).
Four hundred eighty-five transtracheal-wash samples were submitted for microbial testing, with S equi subsp zooepidemicus recovered from 300 (62%). Of the 270 day 0 cultures, 208 (77%) had a score of at least 1 for S equi subsp zooepidemicus. In the 215 cultures obtained at any day after day 0, 86 (40%) had a score of at least 1 for S equi subsp zooepidemicus. Other bacterial species were found in 343 of the 485 (71%) samples. Of the 485 transtracheal-wash samples, 42 (12%) mixed cultures were isolated from horses withdrawn because of negative clinical response, and these bacterial species were identified. The organisms included in this group were Pasteurella spp (10 isolates), Escherichia coli and Streptococcus spp other than S equi subsp zooepidemicus (5), Mannheimia haemolytica (4), Staphylococcus spp (3), and Bordetella bronchiseptica (2), with a variety of organisms in only 1 sample (9) and 4 organisms that could not be identified.
The MIC90 for all S equi subsp zooepidemicus colonies isolated during the study was 0.25 μg of trimethoprim/mL and 4.75 μg of sulfadiazine/mL. Of the 300 S equi subsp zooepidemicus transtracheal-wash isolates, only 1 (0.3%) had an MIC ≤ 0.06 μg of trimethoprim/mL and 1.2 μg of sulfadiazine/mL, whereas 112 (37.3%) were susceptible at 0.12 μg of trimethoprim/mL and 2.4 μg of sulfadiazine/mL, 185 (61.7%) at 0.25 μg of trimethoprim/mL and 4.75 μg of sulfadiazine/mL, and the remaining 2 (0.7%) at 0.5 μg of trimethoprim/mL and 9.5 μg of sulfadiazine/mL.
Seventeen adverse events were recorded during the study in 16 horses; 10 occurred in treated horses and 7 in placebo horses (Table 2; online supplement available at http://avmajournals.avma.org/toc/javma/246/12). No adverse events occurred in humans. There were no significant differences in the overall occurrence of adverse events between groups. The incidence of individual types of events was too low to allow statistical analysis. In the treatment group, colic was seen in 3 horses on days 1, 4 (removed because of colic), and 7; diarrhea in 2 on days 4 and 5 (removed because of diarrhea); abscess or swelling in 2 on days 5 and 14; lameness in 1 on day 8; swollen lymph node in 1 on day 10; and strangles (S equi subsp equi upper respiratory tract infection) in 1 on day 4 (removed because of strangles). In the placebo group, colic was seen in 2 horses on days 7 and 11 (both were treated with administration of mineral oil via nasogastric tube and remained in the study), abscess or swelling in 1 on day 5, swollen lymph node in 1 on day 5, strangles in 1 on day 3 (removed because of strangles), abortion in 1 on day 13 (removed because of abortion), and swollen muzzle in 1 on day 14. All animals recovered by the end of the study whether they were removed because of the adverse event or not.
Number (%) of adverse events recorded for 270 horses with naturally acquired S equi subsp zooepidemicus lower respiratory tract infection enrolled in a clinical field trial of a novel trimethoprim-sulfadiazine oral suspension.
| Adverse event | Treatment (n = 182) | Placebo (n = 88) |
|---|---|---|
| Colic | 3 (1.6) | 2 (2.3) |
| Diarrhea | 2 (1.1) | 0 (0) |
| Abscess or swelling | 2 (1.1) | 1 (1.1) |
| Lameness | 1 (0.5) | 0 (0) |
| Swollen lymph nodes | 1 (0.5) | 1 (1.1) |
| Strangles | 1 (0.5) | 1 (1.1) |
| Abortion | 0 (0) | 1 (1.1) |
| Swollen muzzle | 0 (0) | 1 (1.1) |
See Table 1 for key.
The fecal consistency scores assessed twice daily throughout the study period were not significantly different between groups. The treated and placebo groups had statistically similar clinicopathologic data on day 0. There were small but significant differences in the clinicopathologic data between groups on day 17 (Table 3).
Clinicopathologic findings on day 17 in 180 horses with naturally acquired S equi subsp zooepidemicus infection enrolled in a clinical field trial of a novel trimethoprim-sulfadiazine oral suspension that were treated with either a novel trimethoprim-sulfadiazine oral suspension (n = 119; treatment group) or an equivalent volume of saline solution (61; placebo group).
| Variable | Reference range | Treatment (n = 119)* | Placebo (n = 61)* | P value |
|---|---|---|---|---|
| Absolute lymphocyte count (X 103 lymphocytes/μL) | 1.4–8.8 | 3.58 ± 1.43 | 3.16 ± 1.45 | < 0.001 |
| Relative lymphocyte count (%) | 25–70 | 33.08 ± 11.3 | 26.86 ± 10.5 | < 0.001 |
| Absolute monocyte count (X 103 monocytes/μL) | 0.0–0.9 | 0.49 ± 0.24 | 0.62 ± 0.32 | < 0.001 |
| Relative monocyte count (%) | 0.0–7.0 | 4.57 ± 2.01 | 5.45 ± 2.98 | 0.003 |
| Absolute neutrophil count (X 103 neutrophils/μL) | 1.7–8.1 | 6.36 ± 2.65 | 7.63 ± 2.96 | 0.013 |
| Relative neutrophil count (%) | 30–65 | 56.59 ± 12 | 62.65 ± 11 | < 0.001 |
| Hct (%) | 32–52 | 32.37 ± 5.94 | 30.95 ± 6.58 | 0.071 |
| Mean corpuscular volume (fL) | 34–58 | 43.67 ± 4.24 | 43.26 ± 4.56 | 0.064 |
| Fibrinogen (mg/dL) | 100–400 | 300.5 ± 86.7 | 372.9 ± 119 | < 0.001 |
| Prothrombin time (s) | 10.4–12.2 | 11.58 ± 0.53 | 11.96 ± 0.84 | < 0.001 |
| Albumin (g/dL) | 2.7–4.2 | 3.08 ± 0.34 | 2.98 ± 0.3 | < 0.001 |
| Globulin (g/dL) | 3.0–5.1 | 4.31 ± 0.77 | 4.47 ± 0.65 | 0.007 |
| Albumin-to-globulin ratio | 0.7–1.5 | 0.75 ± 0.19 | 0.69 ± 0.14 | < 0.001 |
| BUN (mg/dL) | 8–25 | 19.46 ± 3.84 | 17.53 ± 4.39 | < 0.001 |
| Calcium (mg/dL) | 11.2–13.6 | 11.32 ± 0.55 | 10.98 ± 0.59 | < 0.001 |
| Potassium (mmol/dL) | 3.0–5.1 | 4.41 ± 0.74 | 4.27 ± 0.58 | 0.098 |
| Sodium (mmol/L) | 132–146 | 135.2 ± 2.77 | 134.3 ± 3.43 | 0.028 |
Values reported are mean ± SD.
Discussion
Results of the present clinical field trial indicated that the new trimethoprim-sulfadiazine oral suspension was an effective treatment for S equi subsp zooepidemicus lower respiratory tract infection in horses. Evidence of efficacy was based on clinical signs and elimination of S equi subsp zooepidemicus from the respiratory tract with a drug dosage of 24 mg/kg administered orally twice daily for 10 days. There were few adverse events associated with trimethoprim-sulfadiazine administration. The fecal consistency scores assessed twice daily throughout the study period were not significantly different between the treatment and placebo groups.
For S equi subsp zooepidemicus isolated from lower respiratory tract infections in horses, the MIC90 of potentiated sulfonamides did not increase from 1989 to 2008, indicating that these remain valuable antimicrobials in the treatment of lower respiratory disease in horses.19 The data in the present study indicated that this novel trimethoprim-sulfadiazine oral suspension was effective in a field trial for use in horses with S equi subsp zooepidemicus respiratory tract infection. In this study, the suspension was found to be safe, with few adverse events noted over the 10-day treatment period. Horses were followed to day 17 to identify any relapses after treatment was stopped.
There is a potential risk of gastrointestinal flora disturbance in horses during antimicrobial treatment. The administration of potentiated sulfonamides in horses has been associated with diarrhea, but its prevalence has not been significantly different than in horses receiving other antimicrobials.22 Adverse events noted with oral use of potentiated sulfonamides in horses are diarrhea, which can be severe, and colitis.23 Daily administration of a potentiated sulfonamide paste at 30 mg/kg twice daily for 5 days has been found to reduce the number of colony-forming units of coliforms in horses in 1 study,4 but numbers returned to normal by the end of the study, and no change was seen in fecal consistency. Effective absorption of orally administered antimicrobials is important to limit exposure of the luminal flora to the antimicrobial. The trimethoprim-sulfadiazine oral suspension used in this study is well absorbed,b and the total amount administered was reduced by 20% from standard dosing recommendations, to 24 mg/kg twice daily. Furthermore, with a suspension, it is possible to calculate and more accurately dose horses to the nearest milliliter, compared with administration of paste or tablets.
The low number of adverse events seen with the trimethoprim-sulfadiazine combination used in this study was expected on the basis of a previous safety study.24 A blinded, randomized, controlled margin-of-safety study24 was performed with 32 mature horses that used the same trimethoprim-sulfadiazine suspensionc at 0, 1, 3, or 5 times the recommended dose of 24 mg/kg. Doses were administered at 12-hour intervals for 30 days, 3 times the recommended treatment duration of 10 days. The twice-daily treatment was well tolerated at up to 5 times the dose of 24 mg/kg. All clinical adverse effects were mild and self-limiting. Loose feces were the most common observation that was likely related to trimethoprim-sulfadiazine treatment. The incidence of loose feces was greater in groups treated with higher dosages of trimethoprim-sulfadiazine. However, all episodes were self-limiting, and in no case did the feces have a mostly liquid consistency (ie, watery diarrhea).
Adverse events were uncommon and similar between treatment and placebo groups in the present study. Diarrhea was seen in 2 of 182 treated horses (online supplement available at http://avmajournals.avma.org/toc/javma/246/12). Given the presence of respiratory disease and stress from commingling and shipping, this was not unexpected. The diarrhea noted in 2 horses was self-limiting, with no additional treatment required. All horses had fecal consistency scored twice daily, and statistical analysis showed no significant difference between the treatment and placebo groups. Three treatment group horses and 2 placebo group horses had colic noted as an adverse event. All 5 of these horses were at the same study site, and the episodes occurred at nearly the same time. The episodes were therefore attributed to management changes.
There were some significant differences in clinicopathologic data between the groups at the end of the study (day 17). Fibrinogen concentration was lower in the treated group, a difference attributed to reduced inflammation due to antimicrobial activity. There was a small but significant decrease in prothrombin time in the treated horses. We suggest that this difference was likely due to normal biological variation, versus the suggestion25 that administration of a potentiated sulfonamide may result in excessive hemorrhage from minor surgical procedures. A single case of immune-mediated hemolytic anemia in a horse associated with administration of a potentiated sulfonamide has been reported.26 There was no unexplained or excessive hemorrhage in any of the 182 horses receiving trimethoprim-sulfadiazine in the present study. We found small but significant differences in electrolyte concentrations between groups. Although treatment with potentiated sulfonamides has been associated with hyperkalemia in human patients,27,28 significant changes in serum potassium concentration in horses were not found in a prior study,24 including when 5 times the recommended dose of 24 mg/kg was administered over 30 consecutive days. The mean potassium concentrations on day 17 for both groups in this study were within the reference range. We suggest that the small difference seen was likely attributable to biological variation or associated with improved feed intake among the treated horses. Some of the changes may also have resulted from the more rapid clinical improvement observed for horses in the treatment group.
van Duijkeren et al29 compared the pharmacokinetics of 3 oral formulations of trimethoprim-sulfadiazine (dosed at 5 mg of trimethoprim/kg [2.3 mg/lb] and 25 mg of sulfadiazine/kg [11.4 mg/lb]). All formulations were administered to horses from which food had been withheld. Mean plasma concentrations of trimethoprim and sulfadiazine peaked within 2 hours after drug administration and decreased steadily thereafter. The mean peak plasma concentrations ranged from 1.31 to 1.72 μg/mL for trimethoprim and from 12.1 to 15.4 μg/mL for sulfadiazine. Peak concentrations of both drugs were well above that required for antimicrobial activity against S equi subsp zooepidemicus, and the area under the curve ranged from 6.32 to 6.87 μg·h/mL for trimethoprim and 137.6 to 156.6 μg·h/mL for sulfadiazine. The elimination halflife for trimethoprim was between 2 and 3 hours, and that of sulfadiazine was between 6.4 and 8.2 hours, indicating that a twice-daily dosing schedule was therapeutically preferable to the once-daily labeled dosage of the commercially available paste preparation. The conclusion was that a total dose of 30 mg of the combined active ingredients (5 mg trimethoprim and 25 mg sulfadiazine)/kg administered at 12-hour intervals in the form of the commercially available paste preparation was sufficient for treatment of most susceptible infections.
A bioequivalence studyb was conducted, comparing the suspension used in this studyc administered via nasogastric intubation versus a commercially available paste preparationm given orally. The bioequivalence studyb was a randomized, 2-period, 2-sequence crossover study with 2 treatments and 16 horses/crossover group. That studyb found that the bioavailability of the oral suspension was not the same as the paste. For sulfadiazine, the geometric mean of the peak plasma concentration for the suspension was 176% that of the reference paste (19.4 vs 11.0 μg/mL), and the area under the curve from time 0 to the limit of quantitation for the suspension was 136% that of the paste (205.6 vs 153.2 μg·h/mL). For trimethoprim, the geometric mean of the peak plasma concentration for the suspension was 127% that of the paste (2.4 vs 1.9 μg/mL), and the area under the curve from time 0 to the limit of quantitation for the suspension was 118% that of the paste (205.6 vs 153.2 μg·h/mL). In the pharmacokinetic study,30 the bioavailability was similar for all 3 oral combinations tested. In a bioequivalence study31 of another potentiated sulfonamide powder,n the bioequivalences of the paste and powder were similar. The bioavailability of the suspension tested in that study23 exceeded that of previously tested potentiated sulfonamides.
On the basis of results of prior studies, it does not appear that administration of oral potentiated sulfonamides is affected by feeding. Sigel et al32 administered trimethoprim and sulfadiazine oral paste in a single daily dose of 35 mg/kg [15.9 mg/lb] to 2 horses for 3 days in both food-withholding and fed states, and determined serum concentration-versus-time profiles for each active ingredient. Absorption of both drugs was slower in the fed horses on the first day of treatment, but there was no apparent difference between the rates of absorption in fed horses and horses from which food had been withheld on subsequent days.
The comparatively increased bioavailabilityb of the trimethoprim-sulfadiazine oral suspension used in this study allowed the use of a lower dose than the 30 mg/kg of the commercial paste recommended by van Duijkeren et al.29 A preliminary studyb of similar design to the present study used the trimethoprim-sulfadiazine oral suspension to treat naturally occurring lower respiratory disease; that study compared clinical response among 3 treatment groups (20, 24, and 30 mg/kg, twice a day) and 1 placebo group. Positive clinical response was determined on the basis of the absence of S equi subsp zooepidemicus in the posttreatment transtracheal-wash specimens. The 24 mg/kg dosage was effective in eliminating the organism and clinical signs of lower respiratory tract infection. No clinical advantage was seen with the use of the higher 30 mg/kg dosage. Further, the plasma concentration of sulfadiazine was not linear with increasing dosage, and the higher dosage did not result in a significant increase in the time the plasma concentration exceeded the MIC90. The lower dosage of 24 mg/kg was subsequently chosen for the present study.
The criteria for positive clinical response in the present study required improvement by day 5. Once a horse was withdrawn because of negative clinical response, subsequent improvement was not considered a positive clinical response. These criteria exceeded the expectations in most clinical situations. Of the 119 horses receiving treatment, 69 (58%) had a positive clinical response; a significantly (P < 0.001) smaller proportion of horses in the placebo group (9/61 [15%]) had a positive clinical response.
Because this was a study under field conditions, with horses being treated for naturally occurring disease, horses were randomized and allocated prior to determination for study eligibility. According to rescue criteria, some horses were then removed, and this removal affected the randomization scheme and may have introduced bias. Details of the horses that were removed and the rescue protocol are provided (online supplement available at http://avmajournals.avma.org/toc/javma/246/12). Additionally, a potential limitation of the FDA-guided study design was that the study antimicrobial was compared with saline solution rather than another accepted treatment. In a comparable study30 with some variations in outcome parameters and timing, 32% of the horses administered saline solution improved, and 67% of horses given ceftiofur improved. Because of changes in the FDA approval process, older trimethoprim-sulfonamide formulations have not been subjected to similar field trials.
ABBREVIATIONS
| MIC | Minimum inhibitory concentration |
| MIC90 | Minimum concentration to inhibit 90% of isolates |
Bade DJ, Microbial Research Inc, Fort Collins, Colo: Unpublished data, 2012.
Stroebel M, Aurora Pharmaceutical LLC, Northfield, Minn: Unpublished data, 2012.
Equisul-SDT (sulfadiazine/trimethoprim), Aurora Pharmaceutical LLC, Northfield, Minn.
Dormosedan, Pfizer Animal Health, Kalamazoo, Mich.
Anased 100, Lloyd Inc, Shenandoah, Iowa.
TW1228, Mila International Inc, Erlanger, Ky.
Port-A-Cul collection vial 221625, BD Diagnostics, Franklin Lakes, NJ.
PRL Central Laboratory Services, Overland Park, Kan.
Microbial Research Inc, Fort Collins, Colo.
Microlog 3, BIOLOG, Hayward, Calif.
Trek Diagnostic Systems Inc, Sensititre Division, Cleveland, Ohio.
SAS software, version 9.1.3, SAS Institute Inc, Cary, NC.
Tribrissen 400 Oral Paste, Schering Plough, Kenilworth, NJ.
Uniprim Powder, Macleod Pharmaceuticals Inc, Fort Collins, Colo.
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Appendix 1
Four-point scoring system used to evaluate cough and fecal consistency in 180 horses with naturally acquired Streptococcus equi subsp zooepidemicus infection enrolled in a clinical field trial of a novel trimethoprim-sulfadiazine oral suspension and randomly allocated to either treatment at a dosage of 24 mg/kg twice a day for 10 days (n = 119; treatment group) or an equivalent volume of saline (0.9% NaCl) solution (61; placebo group).
| Score | Coughing | Fecal consistency |
|---|---|---|
| 0 | None elicited by laryngeal palpation | Formed |
| 1 | Cough elicited by laryngeal palpation | Mixed fecal balls and liquid |
| 2 | Intermittent spontaneous | Unformed |
| 3 | Sustained without stimulus | Watery diarrhea |
Appendix 2
Scoring system used to evaluate variables associated with respiratory disease in 180 horses with naturally acquired S equi subsp zooepidemicus lower respiratory tract infection enrolled in a clinical field trial of a novel trimethoprim-sulfadiazine oral suspension and randomly allocated to either treatment at a dosage of 24 mg/kg twice a day for 10 days (n = 119; treatment group) or an equivalent volume of saline (0.9% NaCl) solution (61; placebo group).
| Score | Rectal temperature (°C) | Respiratory rate (breaths/min) | Auscultation score |
|---|---|---|---|
| 0 | < 38.33 | ≤ 24 | Normal* |
| 2 | ≥ 38.33 | > 24 | Abnormal |
To be considered normal, use of a rebreathing bag was required.
